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HB ki V a ts n w dH ith C CE V About ® EliGene Respiratory Infections Sexually Transmitted Urinary Infections Viral Infections Elisabeth Pharmacon 2014 Bacterial Infections Human Genetics DNA/RNA Isolation & Spectral Calibration Instruments ZEPHYRUS® elisabeth pharmacon group EliGene® ZEPHYRUS B I O S A F E T Y B OX ® ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O Content Pages: 1. Introduction 02 - 06 About ® EliGene 07 - 10 Respiratory Infections 11 - 14 Sexually Transmitted Urinary Infections Product Guarantee Policy Shipping EliGene Kits and RealTime PCR Instruments Sample Processing - Sampling, Transport, Storage, DNA Isolation 2. Diagnostics of Respiratory Infections Legionella pneumophila (CE) Mycoplasma pneumoniae (CE) Chlamydophila pneumoniae (CE) Influenza A/B/pandemic (CE) 3. Diagnostics of Sexually Transmitted Urinary Infections Chlamydia trachomatis (CE 1023) Neisseria gonorrhoeae (CE) Ureaplasma sp. (CE) Mycoplasma hominis/genitalium (CE) 15 - 24 4. Viral Diagnostics Adenovirus (CE) Norovirus (CE) Enterovirus (CE) Herpes simplex virus 1 (CE) Herpes simplex virus 2 (CE) Varicella zoster virus (CE) Hepatitis B virus (CE 1023) Hepatitis C virus (CE 1023) 5. Bacterial Diagnostics 25 - 27 Bacterial Infections 28 - 30 Human Genetics 31 - 33 DNA/RNA Isolation & Spectral Calibration 34 - 35 Instruments ZEPHYRUS® Mycobacterium tuberculosis (CE) Borrelia (CE) Anaplasma (CE) 6. Diagnostics for Human Genetics Coeliac disease (CE) Lactose intolerance (CE) Spondylitis HLA-B27 (CE) 7. DNA Isolation and Spectral Calibration Kits ® EliGene MTB Isolation kit (CE) EliGene® Urine Isolation kit (CE) ® EliGene Spectral Calibration kit LC EliGene® Spectral Calibration kit LC2 EliGene® Spectral Calibration plate 8. Instruments ZEPHYRUS ® ZEPHYRUS® Magneto and isolation kits MAGNETO ® ZEPHYRUS Bio Safety Box ZEPHYRUS® PCR Box Harmony www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com Viral Infections 01 1 - Introduction 1. ELISABETH PHARMACON About ® EliGene ELISABETH PHARMACON was founded in Brno, Czech Republic in 2001 by Czech biologist Omar Šerý and businessman Petr Čermák. In its early days the company was known for research, development and producing DNA diagnostics under the brand name EliGene®. ELISABETH PHARMACON cooperates with many research institutes and clinics. Since its beginnings, the company has cooperated with the Faculty of Science, Masaryk University. In 2008, it became the first Czech company to produce DNA diagnostics under EN ISO 13485:2003 certification. Since 2012 company develops and manufactures laboratory instruments, e.g. biohazard boxes. The quality systems EN ISO 13485:2012 and EN ISO 9001:2008 cover all activities such as designing, developing, manufacturing, purchasing, storage and selling of in-vitro diagnostic medical devices and molecular biology testing, measuring and analysis. The aim is to offer the best quality products at the best available price on the market. By 2013, the company had invested more than 2,4 million EUR in laboratory instruments and the research. The company's research is continually supported by grant projects of the Investment and Business Development Agency (CzechInvest) and the Ministry of Industry and Business of the Czech Republic. In 2012, ELISABETH PHARMACON company was founded also in United Kingdom for the distribution of products. Both companies are members of ELISABETH PHARMACON Group. 2. Product guarantee policy Product quality is our highest priority at ELISABETH PHARMACON. Clinical studies are available for all CE certified kits. As manufacturing, testing, packaging and delivery are carried out under the EN ISO 13485:2003 quality system, we guarantee batch-to-batch consistency. All EliGene products are supplied with an Certificate of Analysis, summarizing the conditions and results of quality assurance tests. 3. Shipping EliGene diagnostic kits must be shipped frozen on dry ice, for short journeys, kits can be shipped packed on blue ice. Freeze-thaw cycles lower the sensitivity of diagnostic kits. Diagnostic kits are packed in polystyrene boxes to protect them from thawing during shipment and laboratory work. 02 elisabeth pharmacon group EliGene® ZEPHYRUS B I O S A F E T Y B OX ® ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 4. EliGene kits and RealTime PCR instruments EliGene® kits are divided according to detection chemistry to basic lines RT, LC and UNI, respectively. About ® EliGene In RT line, the TaqMan chemistry using hydrolyzing fluorescence probes is used in the combination with hotstart polymerase and ROX dye as a passive reference (see Figure 1A). The total reaction volume is 25ul when 5ul of isolated DNA is added directly to ready-to use mastermix containing all necessary components for reaction. In RT kits, the fluorescence dyes with excitation wavelengths of 495, 520 nm and emission wavelengths of 520, 550 nm together with non-fluorescence quenchers are used. These kits are basically designed for ABI instruments of 7000 line and they are optimized on LightCycler480 instrument but can be used with other instruments that meet the spectral characteristics of used fluorescence dyes. The typical time of the run for RT kits is about 2 hours. The list of compatible instruments you can find in Table 1. In LC line, the Molecular Beacons chemistry using hybridization fluorescence probes is used in combination with hot-start polymerase (see Figure 1B). The total reaction volume is 20 ul when 5 ul of isolated DNA is added to mastermix created by mixing of polymerase with reaction mix containing all necessary components for the reaction. The fluorescence dyes with excitation wavelengths of 495, 460 and emission wavelengths of 515, 705 together with non-fluorescence quenchers are used. The LC kits are basically designed for LightCycler 1.2 and 2.0 instruments but they can be used with other instruments, that meets on the one hand the spectral characteristics of used fluorescence dyes in individual versions and on the other hand rapid heating/cooling rate. The typical time of run for LC kits is about 1 hours. The list of compatible instruments you can find in Table 1. In UNI line, the Molecular Beacons chemistry using hybridization fluorescence probes is used in combination with hot-start polymerase (see Figure 1B). The total reaction volume is 20 ul when 5 ul of isolated DNA is added directly to ready-to use mastermix containing all necessary components for reaction. The usage of uracil in the reaction mix enables UNG treatment to prevent the re-amplification of carryoverPCR products by removing any uracil incorporated into single- or double-stranded DNA. In UNI kits the fluorescence dyes with excitation wavelengths of 495, 520 and emission wavelengths of 520, 550 together with non-fluorescence quenchers are used. These kits are designed for almost all real-time instruments on the market. The typical time of run for UNI kits is about 50 minutes. The list of compatible instruments you can find in Table 1. Figure 1. Basic principle of TaqMan hydrolization probes (A) and Molecular Beacons hybridization probes (B) action. Annealing Annealing The TaqMan probe hybridizes between the primers in annealing step and during elongation step probe is cleaved by exonuclease activity of Taq Polymerase. The Molecular Beacon probe forms beacon-like structure that opens to specific sequence during the hybridization in annealing step (B). “TaqMan” is registered trademark of Roche Molecular Systems Inc., “Molecular Beacon” is registered trademark of Public Health Research Institute Properties, Inc. (PHRI) www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 03 1 - Introduction Table 1. Kit / Instrument Compatibility Chart About ® EliGene The gray ticks mean that the compatibility was not tested and it is based only on technical specification of individual instruments. Instrument RT kits Uni kits LC kits Compatibility tested with individual kits (*not tested) ABI 7300 (Applied Biosystems) * ABI 7500FAST (Applied Biosystems) LightCycler 2.0 (Roche) LightCycler 480 (Roche) LightCycler Nano (Roche) Smart cycler (Cepheid) RotorGene 6000 (Qiagen) RotorGeneQ RotorGene 3000 (Qiagen) Compatibility on the basis of filter configuration (not tested) Illumina Eco (Illumina) Mastercycler® ep realplex (Eppendorf) Mx3000P QPCR System (Agilent) Mx3005P QPCR System (Agilent) iQ5 & MyiQ Cycler (Bio-Rad) CFX96 & CFX384 (Bio-Rad) SLAN® Real-Time PCR (Shanghai Hongshi Medical Technology Co., Ltd.) ExiCycler™ 96 (Bioneer) Advantages of EliGene® isolation kits 04 Isolation on columns enables robust and rapid isolation of DNA Special composition of provided reagents and columns guarantees high yields and minimize risk of inhibition during PCR Isolation procedures tested directly with variety of clinical samples covering blood, plasma, serum, sputum, urine, sperm and buccal, cervical, eye, rectal and urethral swabs Kits are CE certified IVD New fully automatic isolation of DNA/RNA from all kinds of clinical samples by ZEPHYRUS® Magneto instrument CE certified kits for automatic isolation fully compatible with EliGene® Real Time PCR kits. elisabeth pharmacon group EliGene® ZEPHYRUS B I O S A F E T Y B OX ® ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O Advantages of EliGene® detection kits The usage of the Internal Control allows to monitor the extraction procedure and to check possible PCR inhibition Kits are supplied with positive control allowing quality control of the analysis Simultaneous amplification (multiplex) of DNA/RNA from pathogen and internal control in one PCR tube The usage of Hot-Start technology minimize the risk of non-specific reaction and provide ultimate sensitivity 1-10 copies of nucleic acid/reaction The usage of Ready to use Mastermixes containing all reaction components for easier reaction setup and handling The usage of uracil in the Mastermixes of UNI kits enables UNG treatment to prevent the reamplification of carryover-PCR products by removing any uracil incorporated into single- or doublestranded DNA To ensure the highest quality of our kits, the row material from the leading providers is used Quantitative Standards in the most of the kits enables to calculate precisely the NA load from 102 to 106 copies/reaction High stability of used reagents The usage of EliGene® UNI kits enables complete detection in 50 minutes Kits are CE certified IVD Kits are validated on LightCycler® 1.2, 2.0, Nano and 480, ABI® 7300, 7500 and 7500FAST, RotorGene-Q (RotorGene 6000) Compatibility of the kits with broad range of instruments - RotorGene®, SmartCycler®, Mx3000P® and 3005P® QPCR systems or iQ5®, CFX96® and other cyclers (see details in Table 1) About ® EliGene LightCycler is registered trademark of Roche Molecular Systems Inc., "ABI" is a registered trademark of Applied Biosystems. "iQ5", "MyiQ" and " CFX96 & CFX384 " are registered trademarks of Bio-Rad Laboratories. "SmartCycler" is registered trademark of Cepheid. "Rotor-Gene" is a trademark of Corbett Research. "MX 3000P" and "MX3000P" are registered trademarks of Stratagene, Inc. 5. Sample processing - sampling, transport, storage, DNA isolation The amplification success depends mostly on the quality of the purified NA. The highest sensitivity in PCR detection can only be achieved when you have first-rate sample preparation. The quality of sample preparation depends not only on isolation procedure but also on timing of collection (clinical relevancy), transport and storage conditions. The presence of conservation agents in transport media could lead to PCR inhibition as well. Taking together all these facts, the isolation procedure must be rigorously tested to avoid false negative results. EliGene® and MAGNETO isolation kits offer to customer optimized solution with the high quality reagents for manual and automatic preparation. EliGene® and MAGNETO isolation kits are rigorously tested in order to exclude the presence of contaminants and PCR inhibitors. Together with EliGene® detection kits including Internal Control, EliGene® or MAGNETO isolation kits, we provide optimal solution for accurate and high sensitive diagnostics. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 05 1 - Introduction 6. Catalog numbers of products About ® EliGene Respiratory Infections Sexually Transmitted Urinary Infections Viral Infections Bacterial Infections Human Genetics DNA/RNA Isolation & Spectral Calibration Instruments ® ZEPHYRUS Standards TM EliDNA Name of kit RT line LC line UNI line EliGene® Chlamydophila pneumoniae (CE) None None 90057-UNI EliGene® Legionella pneumophila (CE) None None 90056-UNI EliGene® Mycoplasma pneumoniae (CE) None None 90055-UNI EliGene® Influenza A/B/pandemic (CE) None 90058-LC* None EliGene® Chlamydia trachomatis (CE 1023) None 90046-LC 90046-UNI EliGene® Neisseria (CE) None 90047-LC 90047-UNI EliGene® Ureaplasma (CE) None 90049-LC 90049-UNI EliGene® Mycoplasma hom/gen (CE) None 90052-LC 90052-UNI EliGene® Adenovirus (CE) 90036-RT None None EliGene® Enterovirus (CE) None 90053-LC None EliGene® Norovirus (CE) None 90054-LC None EliGene® HSV1 (CE) 90035-RT None None EliGene® HSV2 (CE) 90038-RT None None EliGene® VZV (CE) 90040-RT None None EliGene® HBV (CE 1023) 90037-RT None 90037-UNI EliGene® HCV (CE 1023) None 90059-LC* None EliGene® MTB (CE) 90030-RT 90030-LC 90030-UNI EliGene® Borrelia (CE) 90024-RT 90024-LC 90024-UNI EliGene® Anaplasma phagocytophilum (CE) None None 90061-UNI EliGene® Coeliac (DQ2, DQ8, DRB4 | CE) 90048-RT None None EliGene® Coeliac (DQ2, DQ8 | CE) 90048-RT28 None None EliGene® Lactose Intolerance C-13910T (CE) None 90044-LC None EliGene® Lactose Intolerance G-22018A (CE) None 90045-LC None EliGene® Spondylitis HLA-B27 (CE) 90060-RT None None EliGene® MTB Isolation kit 50 isolations 90043-50 EliGene® MTB Isolation Supplement 50 isolations 1220-MTB-IZO EliGene® Urine Isolation Kit 50 isolations 90051-50 EliGene® Sperm Isolation Supplement 50 isolations 90051-50SPO Magneto BodyFluid DNA/RNA isolation kit 180 isolations M-7001-180 Magneto 1ml Serum/Plasma DNA/RNA isolation kit 180 isolations M-7002-180 EliGene® Spectral calibration kit 1 run 90099-SC EliGene® Spectral calibration kit2 1 run 90099-SC2 EliGene® Spectral calibration plate 1 x 96 well plate 90099-SCP ZEPHYRUS® PCR Box Harmony (Basic) 668 x 670 x 760 Z-PCR-600 ZEPHYRUS® PCR Box Harmony (Eco) 668 x 670 x 760 Z-PCR-600-E ZEPHYRUS® PCR Box Harmony (Active) 668 x 670 x 760 Z-PCR-600-A ZEPHYRUS® Bio Safety Box (120) 1207 x 1530 x 855 Z-BSB-120 ZEPHYRUS® Bio Safety Box (150) 1507 x 1530 x 855 Z-BSB-150 ZEPHYRUS® Bio Safety Box (180) 1807 x 1530 x 855 Z-BSB-180 ZEPHYRUS® Magneto 620 x 520 x 550 Z-MAG-12 EliDNA™ Adenovirus QRT standard 5 reactions 90036-CL.QRT EliDNA™ HSV1 QRT standard 5 reactions 90035-CL.QRT EliDNA™ HSV2 QRT standard 5 reactions 90038-CL.QRT EliDNA™ VZV QRT standard 5 reactions 90040-CL.QRT EliDNA™ Lactose Intolerance C-13910T LC standard 20 reactions 90044-CL.LC EliDNA™ Lactose Intolerance G-22018A LC standard 20 reactions 90045-CL.LC * kit is compatible with LightCycler® 480, LightCycler® Nano, LightCycler® 2.0 and also with ABI 7300, ABI 7500, RotorGene 6000/Q 06 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 2.1 Legionella pneumophila Kit EliGene® Legionella pneumophila UNI Kit 90056-UNI Package size: Analytical specificity: Specimens: 50 reactions Legionella pneumophila sputum, bronchoalveolar lavage, nasopharyngeal swab, nasopharyngeal aspirate Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480, LightCycler® Nano, LightCycler® 2.0, fully compatible with RotorGene instruments. CE certification: Detection technology: Clinical study description and results Yes, CE Molecular Beacons new kit, clinical study will be finished in 2014 Respiratory Infections Description: Legionella pneumophila is a thin, aerobic, gram-negative, non-encapsulated, aerobic bacillus of the genus Legionella containing more than 50 species. It is the best-characterized member of the genus and major causative agent of Legionnaires' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen when the bacteria invades and replicates in alveolar macrophages and epithelial cells. Macrolides (azithromycin) or fluoroquinolones (moxifloxacin) are the standard treatment for L. pneumophila in humans. About 3 to 8% of all community-acquired pneumonias are caused by L. pneumophilla when the case-fatality rate for patients with legionellosis is between to 30% with elderly and immunocompromised patients at greater risk of death. References: Heuner K, Swanson M. 2008. Legionella: Molecular Microbiology. Caister Academic Press. ISBN 978-1-904455-26-4. Newton HJ, Ang DK, van Driel IR, Hartland EL. 2010. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 23(2):274-98 www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 07 2. Diagnostics of Respiratory Infections 2.2 Chlamydophila pneumoniae Respiratory Infections Kit EliGene® Chlamydophila pneumoniae UNI Kit 90057-UNI Package size: Analytical specificity: Specimens: 50 reactions Chlamydophila pneumoniae sputum, bronchoalveolar lavage, nasopharyngeal swab, nasopharyngeal aspirate Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480, LightCycler® Nano, LightCycler® 2.0, fully compatible with RotorGene instruments. CE certification: Detection technology: Clinical study description and results Yes, CE Molecular Beacons new kit, clinical study will be finished in 2014 Description: Chlamydophila pneumoniae is an obligate intracellular pathogen. It is a member of the Chlamydiae, a diverse range of obligate intracellular bacteria that includes parasites of amoebae, fish, reptiles, mammals, and humans. Today C. pneumoniae is associated with a remarkably wide range of chronic diseases as meningoencephalitis, arthritis or myocarditis and it is the second most often cause of community-acquired pneumonia. C. pneumoniae spreads by drop infection but in the form of infection elementary bodies (EB) resistant to environmental stresses, it can persist on the surfaces or be spreaded by the aerosol. During the life about 80% of population is infected by C. pneumoniae however in majority of cases the infection is asymptomatic. Some forms of pneumonia can be very serious but in these cases is not clear if C. pneumoniae is not only co-pathogen. References: Pignanelli S, Shurdhi A, Delucca F, et al. 2009. Simultaneous use of direct and indirect diagnostic techniques in atypical respiratory infections from Chlamydophila pneumoniae and Mycoplasma pneumoniae. J Clin Lab Anal. 23 (4): 206–9. Roulis E, Polkinghorne A, Timms P. 2013. Chlamydia pneumoniae: modern insights into an ancient pathogen. Trends Microbiol. 21(3):120-8. 08 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 2.3 Mycoplasma pneumoniae Kit EliGene® Mycoplasma pneumoniae UNI 90055-UNI Package size: Analytical specificity: Specimens: 50 reactions Mycoplasma pneumoniae sputum, bronchoalveolar lavage, nasopharyngeal swab, nasopharyngeal aspirate Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480, LightCycler® Nano, LightCycler® 2.0, fully compatible with RotorGene instruments. CE certification: Detection technology: Clinical study description and results Yes, CE Molecular Beacons new kit, clinical study will be finished in 2014 Respiratory Infections Description: Mycoplasma pneumoniae belonging to class Mollicutes has one of the smallest known genomes. It lacks a peptidoglycan cell wall and thus it is resistant to the effects of penicillins and other beta-lactam antibiotics. In humans it causes atypical pneumonia. M. pneumoniae infects the upper and lower respiratory tracts, leading to upper respiratory tract infection, tracheobronchitis, bronchitis and community-acquired pneumonia. Infections of the lower respiratory tract generally manifest with a cough, sometimes with dyspnoea, adenopathy, wheezing and, rarely, with respiratory failure. Although M. pneumoniae infections are usually mild, and many are asymptomatic, they are not always selflimiting. M. pneumoniae causes up to 40% of cases of community-acquired pneumonia, but M. pneumoniae respiratory tract infections are also associated with a wide range of extra-pulmonary manifestations, including neurological, cardiac, dermatological, musculoskeletal, haematological and gastrointestinal symptoms. References: Sánchez-Vargas FM, Gómez-Duarte OG. 2008. Mycoplasma pneumoniae-an emerging extra-pulmonary pathogen. Clin Microbiol Infect. 14(2):105-17. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 09 2. Diagnostics of Respiratory Infections 2.4 Influenza virus Respiratory Infections Kit EliGene® Influenza A/B/pandemic LC 90058-LC Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions YES Influenza virus A, B, A/H1N1 1 – 10 viruses in the amplified sample serum, plasma, sputum, bronchoalveolar lavage, nasopharyngeal swab, nasopharyngeal aspirate, tissue Compatible instruments: ABI 7000, 7300, 7500 (Applied Biosystems), LightCycler® 480 and LightCycler® Nano, LightCycler® 2.0 (Roche), RotorGene 6000 or RotorGene Q (Qiagen) CE certification: Detected DNA region: Detection technology: Clinical study description and results Yes, CE genome Molecular Beacons probes new kit, clinical study will be finished in 2013 Detection technology description: The EliGene® Influenzavirus A/B/H1N1 kit is intended for detection and quantification of Influenza A, B and H1N1 virus RNA. Kit contains reversion transcription in one mix with RealTime detection. Results of analyzes distinguishes Influenza A, B and H1N1 virus types. Pathogen description: Influenza is an infectious disease of birds and mammals caused by RNA viruses of the family Orthomyxoviridae, the influenza viruses. The most common symptoms are chills, fever, runny nose, sore throat, muscle pains, headache, coughing, weakness and general discomfort. Approximately 33% of people with influenza are asymptomatic. Flu can occasionally lead to pneumonia, either direct viral pneumonia or secondary bacterial pneumonia, even for persons who are usually very healthy. Typically, influenza is transmitted through the air by coughs or sneezes, creating aerosols containing the virus. Influenza can also be transmitted by direct contact with bird droppings or nasal secretions, or through contact with contaminated surfaces. Influenza viruses can be inactivated by sunlight, disinfectants and detergents. Often, new influenza strains appear when an existing flu virus spreads to humans from another animal species, or when an existing human strain picks up new genes from a virus that usually infects birds or pigs. An avian strain named H5N1 raised the concern of a new influenza pandemic after it emerged in Asia in the 1990s, but it has not evolved to a form that spreads easily between people. In April 2009 a novel flu strain evolved that combined genes from human, pig, and bird flu. Initially dubbed "swine flu" and also known as influenza A/H1N1. Influenza B almost exclusively infects humans. This type of influenza mutates at a rate 2–3 times slower than type A. References: Rose N, Hervé S, Eveno E, Barbier N, Eono F, Dorenlor V, Andraud M, Camsusou C, Madec F, Simon G. 2013. Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events. Vet Res. 44(1):72. Labella AM, Merel SE. 2013. Influenza. Med Clin North Am. 97(4):621-45. Cha RM, Smith D, Shepherd E, Davis CT, Donis R, Nguyen T, Nguyen HD, Do HT, Inui K, Suarez DL, Swayne DE, PantinJackwood M. 2013. Suboptimal protection against highly pathogenic avian influenza (H5N1) viruses from Vietnam in ducks vaccinated with commercial poultry vaccines. Vaccine. S0264-410X(13)01137-7. 10 elisabeth pharmacon group EliGene® ZEPHYRUS B I O S A F E T Y B OX ® ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 3.1 Chlamydia trachomatis Kits EliGene® Chlamydia trachomatis LC Kit 90046-LC EliGene® Chlamydia trachomatis UNI Kit 90046-UNI Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions On demand Chlamydia trachomatis, including “Sweden variant“ 1 – 10 copies of bacterial DNA in the amplified sample urine, swabs (cervical, urethral, conjunctival, rectal), sperm Compatible instruments: LC Kit is intended for LightCycler® 2.0, LightCycler® 480 instruments UNI Kit is intended for ABI7500FAST, SmartCycler, RotorGene 6000 and for all LightCycler® instruments CE certification: Detected DNA region: Detection technology: yes, certified by Notified Body No. 1023, ITC Zlín Chlamydia cryptic plasmid and genome (ompA gene) Molecular Beacons Sexually Transmitted Urinary Infections Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® Chlamydia trachomatis LC kit overall 120 clinical specimens were analyzed and results were verified by Cobas® Amplicor® Chlamydia trachomatis Test and by DNA sequencing of 16S ribosomal RNA gene. From 120 clinical samples, 81 samples were C. trachomatis positive and 36 specimens were right determined by EliGene® Chlamydia trachomatis LC kit as C. trachomatis negative. Sensitivity: 98,78% Specificity: 100% Pathogen description: Chlamydias are gram-negative bacteria that parasitize inside the susceptible cells due their disability to synthesise ATP. Genus Chlamydia covers four known species: C. trachomatis, C. psittaci, C. pecorum and C. pneumoniae when C. psittaci and C. pecorum primary belongs to animal parasites. Chlamydia trachomatis is the most frequently reported sexually transmitted pathogen when up to 10% of population is infected. References: Jurstrand M, Christerson L, Klint M, Fredlund H, Unemo M, Herrmann B. 2010. Characterisation of Chlamydia trachomatis by ompA sequencing and multilocus sequence typing in a Swedish county before and after identification of the new variant. Sex Transm Infect. 86(1): 56-60. Kalwij S, Macintosh M, Baraitser P. 2010. Screening and treatment of Chlamydia trachomatis infections. BMJ. 21: 340:c1915. Figure 2. Analysis of Positive sample by EliGene® Chlamydia trachomatis LC kit on LightCycler® 2.0 instrument showing results in 530 channel (Chlamydia detection) and 705 channel (Internal Control detection) www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 11 3. Diagnostics of Sexually Transmited Urinal Infections 3.2 Neisseria gonorrhoeae Sexually Transmitted Urinary Infections Kits EliGene® Neisseria LC Kit 90047-LC EliGene® Neisseria UNI Kit 90047-UNI Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions On demand Neisseria gonorrhoeae 1 – 10 copies of bacterial DNA in the amplified sample urine, swabs (cervical, urethral, conjunctival, rectal), sperm Compatible instruments: LC Kit is intended for LightCycler® 2.0, LightCycler® 480 instruments UNI Kit is intended for ABI7500FAST, SmartCycler, RotorGene 6000 and for all LightCycler® instruments CE certification: Detected DNA region: Detection technology: yes genome (porA gene) Molecular Beacons Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® Neisseria LC Kit, totally 88 clinical specimens were analyzed and the results were determined by Cobas® Amplicor® CT/NG Test. The EliGene® Neisseria LC Kit diagnosed as Neisseria gonorrhoeae positive all 35 specimens. Totally 53 specimens were rightly determined by EliGene® Neisseria LC kit as Neisseria gonorrhoeae negative. Sensitivity: 100% Specificity: 100% Pathogen description: Neisseria gonorrhoeae (gonococci) are gram-negative, non-motile and non-sporulated diplococci. Of the eleven species of Neisseria that colonize humans, only two are pathogens, N. gonorrhoeae and N. meningitis. N. gonorrhoeae is the causative agent of sexually transmitted disease gonorrhoea that is the third most common sexual disease. Worldwide, about 62,2 millions cases of gonorrhoea are registered. The simultaneous infection by Chlamydia trachomatis is common. References: Whiley DM, Buda PP, Freeman K, Pattle NI, Bates J, Sloots TP. 2005. A real-time PCR assay for the detection of Neisseria gonorrhoeae in genital and extragenital specimens. Diagn Microbiol Infect Dis. 52(1):1-5. Whiley DM, Garland SM, Harnett G, Lum G, Smith DW, Tabrizi SN, Sloots TP, Tapsall JW. 2008. Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae. Sex Health. 5(1):17-23. 12 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 3.3 Ureaplasma sp. Kits EliGene® Ureaplasma LC Kit 90049-LC EliGene® Ureaplasma UNI Kit 90049-UNI Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions On demand Ureaplasma urealyticum, Ureaplasma parvum 1 – 10 copies of bacterial DNA in the amplified sample urine, swabs (cervical, urethral, conjunctival, rectal), sperm Compatible instruments: LC Kit is intended for LightCycler® 2.0, LightCycler® 480 instruments UNI Kit is intended for ABI7500FAST, SmartCycler, RotorGene 6000 and for all LightCycler® instruments CE certification: Detected DNA region: Detection technology: yes genome (ureA gene) Molecular Beacons Sexually Transmitted Urinary Infections Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® Ureaplasma LC Kit, totally 57 clinical specimens (urine, swabs) were analyzed. The EliGene® Ureaplasma LC Kit diagnosed correctly 9 specimens as Ureaplasma urealyticum or Ureaplasma parvum positive. Totally 48 specimens were rightly determined by EliGene® Ureaplasma LC kit as Ureaplasma urealyticum or Ureaplasma parvum negative. Sensitivity: 100% Specificity: 100% Pathogen description: Ureaplasma urealyticum is bacterium belonging to the family Mycoplasmataceae. U. urealyticum consists of 14 serovars that can be divided into two biovars, biovars 1 and biovars 2, on the basis of genotypic characteristics. Evidence has been presented that the species currently known as U. urealyticum should be separated into two species, namely, U. parvum (previously, U. urealyticum biovar 1) and U. urealyticum (previously, U. urealyticum biovar 2). The majority of human Ureaplasma isolates belong to the new species U. parvum. Ureaplasma sp. is the main cause of non-gonococcal, non-chlamydial urethritis, acute prostatitis and acquired arthritis in men. In pregnant and non-pregnant women, Ureaplasma can cause chorioamnionitis and pre-term delivery, abortion, pre-term birth, vaginitis and cervicitis. Clinical studies have demonstrated that infants born to infected mothers become infected with these bacteria, and colonization of the respiratory tract of infants has been associated with pneumonia, respiratory distress and meningitis. References: Allam AB, Alvarez S, Brown MB, Reyes L. 2011. Ureaplasma parvum infection alters filamin A dynamics in host cells. BMC Infect Dis. 11:101. Pinna GS, Skevaki CL, Kafetzis DA. 2006. The significance of Ureaplasma urealyticum as a pathogenic agent in the paediatric population. Curr Opin Infect Dis. 19(3):283-9. Figure 3. Analysis of Clinical specimens by EliGene® Ureaplasm LC kit on LightCycler® 2.0 instrument including Positive control (PC) www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 13 3. Diagnostics of Sexually Transmited Urinal Infections 3.4 Mycoplasma hominis / genitalium Sexually Transmitted Urinary Infections Kits EliGene® Mycoplasma hom/gen LC Kit 90052-LC EliGene® Mycoplasma hom/gen UNI Kit 90052-UNI Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions On demand Mycoplasma hominis, Mycoplasma genitalium 1 – 10 copies of bacterial DNA in the amplified sample urine, swabs (cervical, urethral, conjunctival, rectal), sperm Compatible instruments: LC Kit is intended for LightCycler® 2.0, LightCycler® 480 instruments UNI Kit is intended for ABI7500FAST, SmartCycler, RotorGene 6000 and for all LightCycler® instruments CE certification: Detected DNA region: Detection technology: yes genome (gap gene – M. hominis; mgpB gene – M. genitalium) Molecular Beacons, multiplex PCR distinguishes between M. hominis and M. genitalium in LC kit Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® Mycoplasma hom/gen LC Kit, totally 60 clinical specimens (urine, swabs) were analyzed. The EliGene® Ureaplasma LC Kit diagnosed correctly 8 specimens as Mycoplasma hominis and 5 specimens as Mycoplasma genitalium positive. Totally 47 specimens were rightly determined by EliGene® Mycoplasma hom/gen LC Kit as Mycoplasma hominis or Mycoplasma genitalium negative. Sensitivity: 100% Specificity: 100% Pathogen description: Mycoplasma sp. are bacteria belonging to the family Mycoplasmataceae that are a group of microorganisms that can involve the respiratory and genital tracts in man to produce clinical disease. They are found in about 70% of sexually active humans. Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginitis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. In case of Mycoplasma genitalium there is strong evidence of having a causative role in non-chlamydia, non-gonococcal urethritis (NCNGU) in men and cervicitis in women. M. genitalium infection has also been associated with pelvic inflammatory disease but the exact role has not yet been determined. References: Edberg A, Jurstrand M, Johansson E, Wikander E,Höög A, Ahlqvist T, Falk L, Skov J, Fredlund H. 2008. A comparative study of three different PCR assays for detection of Mycoplasma genitalium in urogenital specimens from men and women. Journal of Medical Microbiology,57,304–309. Baczynska A, Svenstrup HF, Fedder J, Birkelund S, Christiansen G. 2004. Development of real-time PCR for detection of Mycoplasma hominis. BMC Microbiology, 4:35. Simpson T, Oh MK. 2004. Urethritis and cervicitis in adolescents. Adolesc Med Clin. 15(2):253-71. 14 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 4.1 Adenovirus Kit EliGene® Adenovirus RT Kit 90036-RT Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions EliDNA ADV QRT standard all 51 types of Adenoviruses 1 – 10 copies of viral DNA in the amplified sample urine, CSF, feces, swabs, serum, plasma Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480 and NANO fully compatible with RotorGene instruments. CE certification: Detected DNA region: Detection technology: yes genome (37 hexon gene) TaqMan probes Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® Adenovirus RT kit overall 350 clinical specimens were analyzed. From these 350 clinical specimens were 20 positive. EliGene® Adenovirus RT kit analyzed 20 samples as positive. Totally 330 clinical specimens were properly by EliGene® Adenovirus RT kit analyzed as negative. Sensitivity: 100% Specificity: 100% Viral Infections Pathogen description: Adenoviruses represent the largest nonenveloped viruses. They are able to be transported through the endosome. They are about 70 to 90 nm in size, nonenveloped icosahedral viruses that are made from up to 252 capsomeres containing double-stranded DNA. Infection is usually transmitted by droplets of respiratory or ocular secretions, by alimentary transfer or sexual intercourse and by the contact with contaminated things or water (virus is able to live out of body at temperature 20°C and lower for a few weeks). The places of the first reproduction are the most often cells of epithelial conjunctiva, nasopharinx and intestine. Adenovirus infection is clinically manifested by the fever, upper respiratory tract infections, tonsillitis, laryngitis, bronchitis and pneumonia. References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Jothikumar N, Cromeans TL, Hill VR, Lu X, Sobsey MD, Erdman DD. 2005. Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41. Appl Environ Microbiol. 71(6):3131-6. Hlinomazová Z, Loukotová V, Horáčková M, Serý O. 2010. The treatment of HSV1 ocular infections using quantitative real-time PCR results. Acta Ophthalmol. 10: 1755-3768. Figure 4: Analysis of EliGene® ADV QRT Standards of 102 to 106 cop/reaction on ABI®7300 Real-time PCR cycler with resulted Standard curve www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 15 4. Viral Diagnostics 4.2 Norovirus Kit EliGene® Norovirus LC Kit 90054-LC Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions YES Norovirus genogroup 1 and 2 1 – 10 viruses in the amplified sample serum, plasma, feces, water, CSF Compatible instruments: ABI 7500 (Applied Biosystems), LightCycler® 480 and LightCycler® Nano, LightCycler® 2.0 (Roche), RotorGene 6000 or RotorGene Q (Qiagen). CE certification: Detected DNA region: Detection technology: yes genome Molecular Beacons probes Detection technology description: The EliGene® Norovirus LC kit is intended for detection and quantification of Norovirus RNA. Kit contains reversion transcription in one mix with RealTime detection. Viral Infections Clinical study description and results: Within the frame of testing functional characteristics of the EliGene® Norovirus LC kit overall 100 clinical specimens were analyzed. From these specimens 100 blind specimens were verified by reference method (La Rosa et al. 2010). From these 100 samples 56 samples were Norovirus RNA positive. The EliGene® Norovirus LC kit diagnosed as Norovirus RNA positive all 56 specimens. There were no discrepant results. Totally 44 specimens were right determined by the EliGene® Norovirus LC kit as Norovirus RNA negative. Sensitivity: 100% Specificity: 100% Pathogen description: Noroviruses are a genetically diverse group of single-stranded RNA, non enveloped viruses in the Caliciviridae family. The viruses are transmitted by fecally contaminated food or water, by person-to-person contact, and via aerosolization of the virus and subsequent contamination of surfaces. Noroviruses are the most common cause of viral gastroenteritis in humans. Norovirus affects people of all ages. Outbreaks of norovirus infection often occur in closed or semiclosed communities, such as long-term care facilities, overnight camps, hospitals, prisons, dormitories, and cruise ships, where the infection spreads very rapidly either by person-to-person transmission or through contaminated food. Norovirus is rapidly inactivated by either sufficient heating or by chlorine-based disinfectants, but the virus is less susceptible to alcohols and detergents, as it does not have a lipid envelope. References: BannisterBA, BeggNT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. 1999. Molecular evolution of the human Noroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification". J. Virol. 73 (3):1941–8 16 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 4.3 Enterovirus Kit EliGene® Enterovirus LC Kit 90053-LC Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions Yes Enteroviruses (Coxsackie A, Coxsackie B and Echovirus) 1 – 10 copies of viral RNA in the amplified sample CSF, feces, respiratory samples, pericardial fluid, blood Compatible instruments: LightCycler® 2.0, LightCycler® 480, LightCycler® NANO, ABI7500 and RotorGene 6000 CE certification: Detected DNA region: Detection technology: yes genome Molecular Beacon Clinical study description and results: Clinical study will be finished in 2013 Pathogen description: Human Enteroviruses are a genus of (+)ssRNA viruses associated with several human and mammalian diseases. Until now, 66 serotypes are known of which there are 62 non-polio enteroviruses that can cause disease in humans: 23 Coxsackie A viruses, 6 Coxsackie B viruses, 28 echoviruses, and 5 other enteroviruses. Enteroviruses are ubiquitous pathogens with a high incidence worldwide (ca. 500 million infections/year) and are often found in the respiratory secretions (e.g., saliva, sputum, or nasal mucus) and stool of an infected person. Enteroviruses may cause lifethreatening infections, especially among children when diseases such as myocarditis, paralysis, multiple organ failure, meningitis and encephalitis may be associated with infections. Viral Infections References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. 1999. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification". J. Virol. 73 (3): 1941–8. Santti, Juhana; Heli Harvala, Leena Kinnunen, and Timo Hyypia. 2000. "Molecular epidemiology and evolution of coxsackievirus A9. Journal of General Virology 81 (5): 1361–1372. Figure 5: Analysis of Positive sample by EliGene® Enterovirus LC kit on LightCycler® 2.0 instrument showing results in 530 channel (Enterovirus detection) www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 17 4. Viral Diagnostics 4.4 Herpes simplex virus 1 Kit EliGene® HSV1 RT Kit 90035-RT Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions EliDNA HSV1 QRT standard Herpes simplex virus 1 1 – 10 copies of viral DNA in the amplified sample CSF, biopsy, swabs (skin, mucosa), blood Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480 and NANO, fully compatible with RotorGene instruments. CE certification: Detected DNA region: Detection technology: yes genome (US4 gene) TaqMan probes Clinical study description and results: Viral Infections Within the frame of testing the functional characteristics of EliGene® HSV1 RT kit overall 636 clinical specimens were analyzed. From these 636 clinical specimens 201 were positive. EliGene® HSV1 RT kit analyzed 197 samples as positive. Totally 435 clinical specimens were properly by EliGene® HSV1 RT kit analyzed as negative. Sensitivity: 98,04% Specificity: 100% Pathogen description: HSV1 is enveloped DNA virus. To date, at least eight different herpesvirus species are known to infect man. A primary infection typically occurs during the childhood. The primary infection goes often through symptomless. The HSV1 infection can manifest as an undifferentiational catarrh of respiratory system of adults or older children. The infection of the conjunctiva and cornea manifests as keratoconjunctivitis. HSV1 is a neurotropic virus. References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Hlinomazová Z, Loukotová V, Horáčková M, Serý O. 2010. The treatment of HSV1 ocular infections using quantitative real-time PCR results. Acta Ophthalmol. 10: 1755-3768. 18 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 4.5 Herpes simplex virus 2 Kit EliGene® HSV2 RT Kit 90038-RT Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions EliDNA HSV2 QRT standard Herpes virus 2 1 – 10 copies of viral DNA in the amplified sample CSF, biopsy, swabs (skin, mucosa), blood Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480 and NANO, fully compatible with RotorGene instruments. CE certification: Detected DNA region: Detection technology: yes genome (US4 gene) TaqMan probes Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® HSV2 RT kit overall 211 clinical specimens were analyzed. From these 211 clinical specimens 24 were positive. EliGene® HSV2 RT kit analyzed all 24 samples as positive. Totally 187 clinical specimens were properly by EliGene® HSV2 RT kit analyzed as negative. Sensitivity: 100% Specificity: 100% Viral Infections Pathogen description: Herpesvirus is enveloped DNA virus. To date, at least eight different herpesvirus species are known to infect man. HSV2 causes genital herpes, characterized by sores in the area of the genitalia. Genital herpes is a sexually transmitted disease. The main symptoms are herpes ulcers in glans penis in men. Lesions in woman are localized in cervix, vagina or perianal area. The activation of the latent infection can produce asymptomatic releasing of virus in cervical secret. HSV2 can cause infection of the brain (encephalitis) in the case of severely defective or compromised immune system. References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Hlinomazová Z, Loukotová V, Horáčková M, Serý O. 2010. The treatment of HSV1 ocular infections using quantitative real-time PCR results. Acta Ophthalmol. 10: 1755-3768. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 19 4. Viral Diagnostics 4.6 Varicella zoster virus Kit EliGene® VZV RT Kit 90040-RT Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions EliDNA VZV QRT standard Varicella zoster virus 1 – 10 copies of viral DNA in the amplified sample CSF, biopsy (skin, liver), swabs Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480 and NANO, fully compatible with RotorGene instruments. CE certification: Detected DNA region: Detection technology: yes genome TaqMan probes Clinical study description and results: Viral Infections Within the frame of testing the functional characteristics of EliGene® VZV RT kit overall 481 clinical specimens were analyzed. From these 481 clinical specimens 51 were positive. EliGene® VZV RT kit analyzed all 51 samples as positive. Totally 430 clinical specimens were properly by EliGene® VZV RT kit analyzed as negative Sensitivity: 100% Specificity: 100% Pathogen description: VZV is a double stranded DNA virus of the Herpes group. VZV infection can have two different manifestations of the disease - chickenpox or shingles. The manifestations include rash, low grade fever and malaise. Varicella may rarely result in complications including VZV encephalitis or pneumonia. VZV is certainly transmissible through the airborne route and does not require close personal contact. The skin lesions are certainly full of infectious virus particles. After an incubation period of 14 days, the virus arrives at its main target organ, the skin. The virus can survive in the human body in the latent form for a long time. References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Dwyer DE, Cunningham AL. 2002. Herpes simplex and varicella-zoster virus infections. Med J Aust. 177(5):267-73. Hlinomazová Z, Loukotová V, Horáčková M, Serý O. 2010. The treatment of HSV1 ocular infections using quantitative real-time PCR results. Acta Ophthalmol. 10: 1755-3768. 20 elisabeth pharmacon group EliGene® ZEPHYRUS B I O S A F E T Y B OX ® ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 4.7 Hepatitis B virus Kits EliGene® HBV RT Kit 90037-RT EliGene® HBV UNI kit 90037-UNI Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions Yes Hepatitis B virus 0.1 IU/ml of isolated specimen serum, plasma Compatible instruments: RT Kit intended for ABI 7000, 7300, 7500 (Applied Biosystems), LightCycler® 480 and LightCycler® Nano (Roche), RotorGene 6000 or RotorGene Q (Qiagen). UNI Kit intended for LightCycler® 2.0 CE certification: Detected DNA region: Detection technology: Yes, CE 1023 genome (core protein) TaqMan probes (RT version), Moleular Beacons (UNI version) Clinical study description and results: Viral Infections Within the frame of testing the functional characteristics of EliGene® HBV RT Kit and EliGene® HBV UNI Kit overall 500 clinical specimens were analyzed. From these specimens, 300 HBV positive specimens and 200 HBV negative specimens were confirmed by artus® HBV TM PCR Kit. The EliGene® HBV RT Kit and EliGene® HBV UNI Kit diagnosed as HBV positive 299 specimens. 1 specimen from Nigeria was determined by the EliGene® HBV RT Kit and EliGene® HBV UNI Kit as negative. The sensitivity and specificity of EliGene® HBV RT Kit and the EliGene® HBV UNI Kit is 98.78% and 100%, respectively. EliGene® HBV RT Kit and the EliGene® HBV UNI Kit shows 99,8% (499 from 500 specimens) match in detection of HBV DNA compared to results determined by artus® HBV TM PCR Kit. 100% sensitivity can be declared for both kits for the concentration of 5 IU/ml of serum corresponding to concentration of 0.1 IU/ml of isolated specimen by Magneto 1ml Serum/Plasma DNA/RNA isolation kit (see page 34) - extraction volume: 1.0 ml, elution volume: 50 μl. The linear range of the EliGene® HBV RT Kit and EliGene® HBV UNI Kit has been determined to cover concentrations from 0.1 IU/μl to at least 1 x 105 IU/μl. Precision data of the EliGene® HBV RT Kit were calculated on basis of the Cp values of the amplification curves using the Quantification standard 4 (10 000 IU/ml) and Internal Control (Figure 7). Pathogen description: Hepatitis B virus is a small DNA virus divided into Hepadnaviridae family. Eight different genotypes signed by alphabetical letters A to H, have been previously described. Since the proposal of the four described genotypes (A–D), four others (E–H) have been characterized during the two last decades. Recently, a ninth “genotype” evidenced in North-West China, India, Lao and Vietnam and tentatively termed “I” was suggested, although it is still subject to debate and as being a recombinant strain with a genotype C backbone. Finally, very recently, a tenth genotype provisionally assigned to genotype “J” was proposed for a Japanese patient's HBV isolate. Hepatitis B (HB) is transmitted by the exchange of body fluids e.g. blood, semen, breast milk and saliva. The virus spread between people who have unprotected sexual intercourse, drug users who share needles and syringes and health care workers in contact with potentially contaminated blood or body fluids. It is estimated that one third of the world's population has serological evidence of past or present infection with HBV and that 350 to 400 million people are still chronically infected, of whom 78 % lived in Asia, 16 % in Africa, 3 % in South America and the remaining 3% in Europe, North America and Oceania. HBV infection has a broad spectrum of clinical diseases, ranging from acute hepatitis (including fulminant hepatic failure) to a low viraemic asymptomatic “inactive” carrier state or to progressive chronic hepatitis that may lead to cirrhosis with an annual rate of 2 to 5 % in HBe-positive patients and hepatocellular carcinoma (HCC) with a cumulative 5-year incidence of 15 to 20 %. Both HBV-related end-stage liver disease and HCC are responsible for around 1 million deaths per year. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 21 4. Viral Diagnostics 4.7 Hepatitis B virus (continued) References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. Ho SKN, Yam W-C, Leung ETK, Wong L-P, Leung JKH, Lai K-N, Chan TM. 2003. Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes. J Med Microbiol. 52(5): 397-402. Viral Infections Figure 6. Calculation of the linear range. The straight was determined by linear regression of the log10 calculated concentrations with the log10 nominal concentrations. The equation of the regression line in included in the figure. Figure 7: Precision data on the basis of the Cp values. Figure 8: Analysis of specimens with different concentration of HBV from 0.5 IU/ml to 5000 IU/ml with EliGene® HBV RT kit. A – amplification curves for FAM channel (HBV detection), B - amplification curves for HEX channel (Internal control) 22 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 4.8 Hepatitis C virus Kit EliGene® HCV LC Kit 90059-LC Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions Yes Hepatitis C virus 1 – 10 viruses in the amplified sample serum, plasma Compatible instruments: ABI 7000, 7300, 7500 (Applied Biosystems), LightCycler® 480 and LightCycler® Nano, LightCycler® 2.0 (Roche), RotorGene 6000 or RotorGene Q (Qiagen). CE certification: Detected DNA region: Detection technology: Yes, CE 1023 genome (core protein) Molecular Beacons probes Clinical study description and results: The EliGene® HCV LC kit is intended for detection and quantification of hepatitis C virus RNA. Kit contains reversion transcription in one mix with RealTime detection. Clinical study description and results: Viral Infections Within the frame of testing the functional characteristics of EliGene® HCV LC Kit overall 500 clinical specimens were analyzed. From these specimens, 300 HCV positive specimens and 200 HCV negative specimens were verified by COBAS AMPLICOR HCV MONITOR Test, version 2.0 (v2.0). The EliGene® HCV LC Kit diagnosed as HCV positive 299 specimens. 1 specimen was determined by the EliGene® HCV LC Kit as negative. The sensitivity and specificity of EliGene® HCV LC Kit is 98,78% and 100%, respectively. The correlation of the quantitative results of EliGene® HCV LC Kit and COBAS AMPLICOR HCV MONITOR Test, version 2.0 (v2.0) was analysed by linear regression, when the logarithmised results of both kits were plotted as scatterplots against each other (Figure 9). Correlation coefficient was calculated to R2=0,81. 100% sensitivity can be declared for the concentration of 50 IU/ml of serum isolated by Magneto 1ml Serum/Plasma DNA/RNA isolation kit (see page 34) - extraction volume: 1.0 ml, elution volume: 50 μl. The linear range of the EliGene® HCV LC Kit has been determined to cover concentrations from 50 IU/μl to at least 1 x 105 IU/ml (Figure 10). Pathogen description: Hepatitis C virus is enveloped RNA virus classified to Flaviviridae family. Virus genome consists of 9379 nucleotides. HCV represents heterogeneous group of viruses divided into 6 genotypes (groups) and more than 40 sub-types. EliGene® HCV LC Kit gives the positive results with genotypes 1a, 1b, 2a, 2b, 3a, 4a, 6 and 6b. Today, the word prevalence about 175 milions persons infected by hepatitis C virus is assessed. Virus is disseminating mainly by parenteral way, the sexual transfer is observed rare. In our conditions, significantly risk group is represented by the injection drug consumers. References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 23 4. Viral Diagnostics 4.8 Hepatitis C virus (continued) Figure 9. Comparison of the COBAS AMPLICOR HCV MONITOR Test (v.2.0) against the EliGene® HCV LC Kit Viral Infections Figure 10. Calculation of the linear range. The straight was determined by linear regression of the log10 calculated concentrations with the log10 nominal concentrations. The equation of the regression line is included in the figure. Figure 11. Analysis of specimens with different concentration of HCV from 126 IU/ml to 21000 IU/ml with EliGene® HCV LC kit. A – amplification curves for five HCV standards, B - amplification curves for HCV positive specimens. 24 elisabeth pharmacon group EliGene® ZEPHYRUS B I O S A F E T Y B OX ® ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 5.1 Mycobaterium tuberculosis Kits EliGene® MTB LC Kit 90030-LC EliGene® MTB RT Kit 90030-RT EliGene® MTB UNI Kit 90030-UNI Package size: Possibility of quantification: Analytical specificity: 50 reactions On demand Mycobacterium tuberculosis, Mycobacterium bovis, negative result for Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium avium and Mycobacterium marinum 1 – 10 copies of bacterial DNA in the amplified sample urine, sputum, BAL, exudates, tissue, paraffin embedded tissue Analytical sensitivity: Specimens: Compatible instruments: LC Kit intended for LightCycler® 2.0, LightCycler 480 instruments RT Kit intended for ABI7000, ABI7300, ABI7500 instruments, fully compatible with RotorGene instruments UNI Kit is intended for ABI7500FAST, SmartCycler, RotorGene 6000 and for all LightCycler® instruments CE certification: Detected DNA region: Detection technology: yes genome (insertion sequence IS6110) Molecular Beacons (LC, UNI versions), TaqMan probes (RT version) Clinical study description and results: Totally 559 clinical samples were tested by EliGene® MTB RT Kit, 518 of them were blind samples with unknown status and 41 MTB positive samples verified by cultivation from Brno region. EliGene® MTB RT Kit detected correctly all 52 positive samples. Totally 481 samples were correctly diagnosed by EliGene® MTB RT Kit as MTB negative. Sensitivity: 100% Specificity: 100% Bacterial Infections Pathogen description: Genus Mycobacterium comprises more than 50 species when among them obligatory pathogenic etiological agents of tuberculosis and leprosy and on the other hand also facultative pathogenic and non-pathogenic species representing important part of natural biotopes can be found. M. tuberculosis comprises to the one complex with genetically close related M. bovis and it is etiological agents of human tuberculosis. M. bovis is etiological agent of cattle tuberculosis and some others pets and wild beast and it is rarely detected in humans. References: Kriz P, Kralik P, Slany M, Slana I, Svobodova J, Parmova I, Barnet V, Jurek V, Pavlik I. 2011. Mycobacterium pinnipedii in a captive Southern sea lion (Otaria flavescens): a case report. Veterinarni Medicina, 56:307-313. Mendes A.C., Fernandes S.J., Ferreira L.C., Pereira M., Ramos H., Cabeda J.M. 2009. Evaluation of MTB Q Alert kit for detection of Mycobacterium tuberculosis directly on patient samples Clinical Microbiology and Infection, Suppl. 19, P1321. Figure 12. Sensitivity test of EliGene® MTB LC kit on LightCycler® 2.0 instrument shows the detection of 2 DNA copies in reaction mix. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 25 5. Bacterial Diagnostics 5.2 Borrelia Kits EliGene® Borrelia LC Kit 90024-LC EliGene® Borrelia RT Kit 90024-RT EliGene® Borrelia UNI Kit 90024-UNI Package size: Possibility of quantification: Analytical specificity: Analytical sensitivity: Specimens: 50 reactions On demand Borrelia garinii, Borrelia afzelii and Borrelia burgdorferi sensu stricto 1 – 10 copies of genomic DNA in the amplified sample blood, urine, CSF, synovial fluid, tissue Compatible instruments: LC Kit intended for LightCycler® 2.0, LightCycler 480 instruments RT Kit intended for ABI7000, ABI7300, ABI7500 instruments, fully compatible with RotorGene instruments UNI Kit is intended for ABI7500FAST, SmartCycler, RotorGene 6000 and for all LightCycler® instruments CE certification: Detected DNA region: Detection technology: yes genome (fla gene) Molecular Beacons (LC, UNI versions), TaqMan probes (RT version) Clinical study description and results: Totally 486 clinical samples were tested by EliGene® Borrelia RT Kit. EliGene® Borrelia RT Kit detected correctly all 9 positive samples. Totally 477 samples were correctly diagnosed by EliGene® Borrelia RT Kit as Borrelia negative. Sensitivity: 100% Specificity: 100% Bacterial Infections Pathogen description: Lyme borreliosis is a disease caused by spirochete Borrelia burgdorferi. The detection of this bacterium could be made from any clinical material using the DNA diagnostics in Europaen Union. There can be found three pathogenic species of Borrelia genus: B. b. sensu stricto, B. afzelii and B. garinii. EliGene® Borrelia Kits contain mix of primers and probes that are specific to all three mentioned Borrelia species. DNA diagnostics is useful in cases of Borrelia suspicion (anamnesis of tick or insect that suck the blood). References: Hytönen J, Hartiala P, Oksi J, Viljanen MK. 2008. Borreliosis: recent research, diagnosis, and management. Scand J Rheumatol. 37(3):161-172. Priem S, Rittig MG, Kamradt T, Burmester GR, Krause A. 1997. An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis. J Clin Microbiol. 35(3): 685–690. Figure 13. Detection of pathogenic Borrelia sp. in DNA samples (isolated from ticks) by EliGene® Borelia RT kit on ABI®7500FAST instrument. 26 Figure 14. Detection of pathogenic Borrelia sp. in DNA samples (isolated from ticks) by EliGene® Borelia RT kit on ABI®7500FAST instrument. elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 5.3 Anaplasma phagocytophilum Kit EliGene® Anaplasma phagocytophilum UNI Kit 90061-UNI Package size: Analytical specificity: Specimens: 50 reactions Anaplasma phagocytophilum blood, urine, CSF, synovial fluid, tissue Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480, LightCycler® Nano, Light Cycler 2.0, fully compatible with RotorGene instruments. CE certification: Detection technology: Clinical study description and results: yes Molecular Beacons new kit, clinical study will be finished in 2013 Description: Anaplasma phagocytophilum is obligate intracellular, gram-negative rickettsial organisms infecting human leukocytes. Anaplasma is transmitted to humans by tick bites (Ixodes scapularis, Ixodes ricinus and Ixodes pacificus). It causes human granulocytic anaplasmosis (HGA) previously known as human granulocytic ehrlichiosis (HGE). Typical symptoms include fever, headache, chills, and muscle aches and they usually occur within 1-2 weeks of a tick bite. The epidemiology of this infection is very much like that of Lyme disease (caused by Borrelia burgdorferi) and babesiosis (caused primarily by Babesia microti), which all have the same tick vector. References: Gary P. Wormser et al. 2006. The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 43 (9): 1089-1134. Johan S. Bakken, MD, Stephen Dumler. 2008. Human Granulocytic Anaplasmosis. Clin Infect Dis. 22 (3): 433–448 www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com Bacterial Infections 27 6. Diagnostics for Human Genetics 6.2 Lactose Intolerance Kits EliGene® Lactose Intolerance C-13910T LC Kit 90044-LC EliGene® Lactose Intolerance G-22018A LC Kit 90045-LC Package size: 50 reactions Standards: EliDNA Lactose Intolerance C-13910T Standard (Ref. 90044-CL.LC) TM EliDNA Lactose Intolerance G-22018A Standard (Ref. 90045-CL.LC) Analytical specificity: Specimens: C-13910T polymorphism (EliGene® Lactose Intolerance C-13910T LC Kit) G-22018A polymorphism (EliGene® Lactose Intolerance G-22018A LC Kit) blood, buccal swabs Compatible instruments: LightCycler® 2.0, LightCycler 480, RotorGene 6000 CE certification: Detected DNA region: Detection technology: yes genome (core protein) Molecular Beacons TM Clinical study description and results: EliGene® Lactose Intolerance C-13910T LC Kit and EliGene® Lactose Intolerance G-22018A LC Kit specificity were tested on 50 samples of human DNA with genotypes determined by DNA sequencing. The specificity of the method was validated by searching in the DNA databases also. Specificity: 100% Description: Patients presenting with symptoms of lactose intolerance are in some laboratories routinely tested for a singlenucleotide polymorphism C-13910T and G-22018A that are located upstream of the lactase gene (LCT) and are tightly associated with genetically determined lactase persistence/non-persistence. The genotyping of these polymorphisms enables differential diagnosis of genetic versus secondary causes of lactose intolerance. Several PCR-based methods have been established as tests for this SNP. In particular, automated genotyping assays conducted on LightCycler platforms provide a rapid method for routine high-throughput analysis of this variant. Human Genetics References: Ridefelt P, Håkansson LD. 2005. Lactose intolerance: lactose tolerance test versus genotyping. Scand J Gastroenterol. 40(7):822-6. Bulhões AC, Goldani HA, Oliveira FS, Matte US, Mazzuca RB, Silveira TR. 2007. Correlation between lactose absorption and the C/T-13910 and G/A-22018 mutations of the lactase-phlorizin hydrolase (LCT) gene in adult-type hypolactasia. Braz J Med Biol Res. 40(11):1441-6. Figure 15. Analysis of samples with -13910 CC and TT genotypes by EliGene® Lactose Intolerance C-13910T LC kit on LightCycler® 2.0 instrument 28 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 6.1 Coeliac disease Kits EliGene® Coeliac RT Kit 90048-RT EliGene® Coeliac RT28 Kit 90048-RT28 Package size: Analytical specificity: 50 reactions EliGene® Coeliac RT Kit - HLA-DQ2, DQ8 and DRB1*04 EliGene® Coeliac RT28 Kit - HLA-DQ2 and DQ8 blood, buccal swab Specimens: Compatible instruments: Real-Time Systems ABI 7000, 7300, 7500 (Applied Biosystems), LightCycler® 480 and Nano (Roche), fully compatible with RotorGene and Q- instruments. CE certification: Detection technology: yes TaqMan probes Clinical study description and results: EliGene® Coeliac RT kit specificity was tested on 50 samples of human DNA with genotypes determined by DNA sequencing. The specificity of the method was validated by searching in the DNA databases also. It is necessary to use recommended concentration of human DNA for the genotypization. Specificity: 100% Pathogen description: Coeliac disease, also called coeliac sprue is one of the most common enteropathogenic disorders and is characterized by a lifelong hypersensitivity to gluten proteins found in wheat, rye, oat and barley. Until recently, coeliac disease was considered relatively uncommon with an estimated prevalence rate ranging from 1 in 1000 to 1:4000. However, the availability of new serologic tests have led to the observation that coeliac disease is much more common, affecting about 1 of 100-400 persons in Europe, the majority of patients showing little clinical symptoms. Considerable evidence now indicates that coeliac disease has a strong genetic component. Coeliac disease is a multifactorial disorder associated with HLA-DQ2 (DQA1*05/DQB1*02) or DQ8 (DQA1*0301/DQB1*0302) or HLA-DRB1*04. HLA DQ2 is expressed in the majority (>90%) of those with coeliac disease and DQ8 in about 8%. The expression of these HLA-DQ2 or DQ8 molecules is necessary but not sufficient to develop coeliac disease and accounts for only about 50% of the genetic component of the disease. Studies in sibling (sib recurrence risk for coeliac disease of 10%) and of identical twins (concordance of 70%) suggest that the contribution of HLA genes in coeliac disease is less than 50%. About 95% of all coeliac patients possess this particular genotype compared to approximately 20% of the normal population. From this point of view absence of these alleles is useful in excluding coeliac disease. However, at least one other non-HLA gene and some environmental factors are also likely to be involved in the disease. On the basis of recent results the analysis of HLA-DQ2 (DQA1*05/DQB1*02) and DQ8 (DQA1*0301/DQB1*0302) alleles is sufficient to exclude coeliac disease. Human Genetics References: Troncone R, Jabri B. 2011. Coeliac disease and gluten sensitivity. J Intern Med. 269(6):582-90. Trynka G, Wijmenga C, van Heel DA. 2010. A genetic perspective on coeliac disease. Trends Mol Med. 6(11):537-50. Bourgey M. et al. 2007. HLA related genetic risk for coeliac disease. Gut 2007;56:1054–1059. Margaritte-Jeannin P. et al. 2004. HLA-DQ relative risks for coeliac disease in European populations: a study of the European Genetics Cluster on Coeliac Disease. Tissue Antigens 63: 562–567. Figure 16. Tables used for evaluation of results obtained by EliGene®Coeliac RT kit (A) and EliGene®Coeliac RT28 kit (B). www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 29 6. Diagnostics for Human Genetics 6.3 Spondylitis HLA-B27 Kit EliGene® Spondylitis HLA-B27 RT Kit 90060-RT Package size: Analytical specificity: Specimens: 50 reactions allele HLA-B27 blood, buccal swabs Compatible instruments: Real-Time Systems 7000, 7300, 7500 Applied Biosystems, LightCycler® 480, LightCycler® Nano, fully compatible with RotorGene instruments. CE certification: Detection technology: Yes, CE TaqMan probes Clinical study description and results: EliGene® Spondylitis HLA-B27 RT Kit specificity were tested on 100 samples of human DNA with genotypes determined by DNA sequencing. The specificity of the method was validated by searching in the DNA databases as well. Specificity: 100% Description: Human Genetics Human Leukocyte Antigen (HLA) B27 (subtypes B*2701-2759) is an HLA class I surface antigen that is encoded in the B References: locus in the major histocompatibility complex (MHC) on the short arm of chromosome 6. In HLA-B27 antigen a strong association with ankylosing spondylitis (AS) and spondyloarthropathies (SpA) was found. Moreover the increased incidence of this antigen in other diseases as Reiter´s syndrome or uveitis was found. Association studies found an association between HLA-B27 antigen and AS in all ethnic and racial groups worldwide however the prevalence of HLAB27 antigen and the strength of its association with AS does vary. For example, the prevalence of HLA-B27 antigen is about 8% in Caucasians, 4% in North Africans, 2-9% in Chinese, and 0.1-0.5% in Japanese. Further, among northern Europeans only 8% of the general population possesses HLA-B27, but more than 90% of the patients with AS possess this gene. In contrast, among African Americans 2% to 4% of the general population and only 50% to 60% of patients with AS possess this gene. From this reasons HLA-B27 test cannot be used to screen an asymptomatic population to detect AS but the test provides a statement of increased probability of the existence of AS in the symptomatic patient. Also, the presence of HLA-B27 antigen influences the clinical manifestations of AS disesase, because HLA-B27–positive patients have a significantly younger age at onset of their disease and a higher prevalence of episodes of eye inflammation (acute anterior uveitis) and hip joint involvement. References: Khan MA. Remarkable Polymorphism of HLA-B27: An Ongoing Saga. Curr Rheumatol Report. 2010; 12: 337-41 Robinson PC, Brown MA. 2012. The genetics of ankylosing spondylitis and axial spondyloarthritis. Rheum Dis Clin North Am. 38(3):539-53 Thomas GP, Brown MA. Genetics and Genomics of Ankylosing Spondylitis. Immunol Rev. 2010; 233:162-180. 30 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 7.1 Isolation of DNA from MTB and other bacteria Kits EliGene® MTB Isolation Kit 90043-50 EliGene® MTB Isolation Supplement 12200-MTB-IZO Package size: Specimens: 50 isolations sputum, decontaminated sputum, cultures, BAL, exudates, urine, swabs Intended use: EliGene® MTB Isolation Kit is intended for the isolation of bacterial DNA from clinical material EliGene® MTB Isolation Supplement is intended for DNA isolation from M. tuberculosis and eventually futher microorganisms (G+) from sputum or cultures by using Maxwell®* 16 FFPE Tissue LEV DNA Purification Kit (Promega). CE certification: Isolation technology: yes silica columns Clinical study description and results: Within the frame of testing the functional characteristics of EliGene® MTB Isolation Kit overall 559 clinical specimens were analyzed. From these specimens, 518 blind specimens from Prague region and 41 MTB positive specimens verified by cultivation form Brno region were analyzed. The isolation kit showed 100% sensitivity in isolation of DNA from clinical decontaminated material which means that this procedure for isolation of MTB DNA gives in 100% sufficient amount of MTB DNA for the next DNA diagnostic method. The effectiveness of these isolation procedures is 95,34% which means that from total amount of isolated specimens were isolated 95,34% of specimens without inhibition. Description: Many problems like inhibitions or low sensitivity occur when one make the isolation of DNA from Mycobacterium species. EliGene® MTB Isolation Kit is designed to avoid all problems with PCR inhibitors and low yields of DNA. The total time of isolation process is about 2 hours including two incubations, each for 30 minutes. Kit contains all components necessary for the DNA isolation including microtubes. No addition of ethanol or other chemicals is needed. References: Bannister BA, Begg NT, Gillespie SH. 2000. Infectious Disease. Blackwell Science, 2th Ed. DNA/RNA Isolation & Spectral Calibration www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 31 7. DNA Isolation and Spectral Clibration Kits 7.2 Isolation of total DNA (including bacterial) from clinical specimens Kits EliGene® Urine Isolation Kit 90051-50 EliGene® Sperm Isolation Supplement 90051-50SPO Package size: Specimens: 50 isolations Intended use: EliGene® Urine Isolation Kit is intended for the isolation of DNA from clinical material. EliGene® Sperm Isolation Supplement is intended for DNA isolation from the sperm. CE certification: Isolation technology: yes columns blood, buccal tissue samples, sperm, urine, swabs (cervical, urethral) Clinical study description and results: Within the frame of testing, the functional characteristics of EliGene® Chlamydia trachomatis LC kit overall 120 clinical specimens of urine were analyzed. Before the detection by EliGene® Chlamydia trachomatis LC, DNA from all samples was isolated by the EliGene® Urine Isolation Kit. No inhibition was observed. The effectiveness of this isolation procedure was 100%. It means that from total amount of isolated specimens were isolated 100% of specimens without inhibition. Description: Many problems like inhibitions, low sensitivity etc. occur when one make the isolation of DNA from clinical samples. EliGene® Urine Isolation Kit is designed to avoid all problems with PCR inhibitors and low yields of DNA. The total time of isolation process is about 1 – 2 hour including incubation. Kit contains all components necessary for the DNA isolation including microtubes. No addition of ethanol or other chemicals is needed. DNA/RNA Isolation & Spectral Calibration 32 elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 7.3 Spectral Calibration Kit Kits EliGene® LC Spectral Calibration Kit 90099-SC EliGene® LC Spectral Calibration Kit 2 90099-SC2 Package size: For one Spectral calibration run on LightCycler® 1.2 (ref: 90099-SC) and LightCycler® 2.0 instruments (ref: 90099-SC2) Description: EliGene® LC Spectral Calibration Kit is intended for spectral calibration of instruments LightCycler® 1.2 and 2.0 for the EliGene® LC kits using 530 and 705 channels for detection. EliGene® LC Spectral Calibration Kit 2 is intended for spectral calibration of instruments LightCycler® 2.0 for the EliGene® LC kits using 530, 550 and 705 channels for detection. Before the usage of EliGene® LC kits, it is necessary to perform spectral calibration with EliGene® LC Spectral Calibration Kit or EliGene® LC Spectral Calibration Kit 2. Once the spectral calibration was done; it is valid until the instrument is upgraded or optics is changed. Kit EliGene® LC Spectral Calibration Plate 90099-SCP Package size: 96-well plate for Spectral calibration run on LightCycler® 480 Description: EliGene® LC Spectral Calibration Plate is intended for spectral calibration of instruments LightCycler® 480 for the kits EliGene® LC and UNI using 530, 560 and 705 channels for detection. Before the using of EliGene® LC kits it is necessary to perform spectral calibration with EliGene® LC Spectral Calibration Plate. Once the spectral calibration was done, it is valid until the instrument is upgrading or optics is changed. DNA/RNA Isolation & Spectral Calibration www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com 33 8. Instruments ZEPHYRUS 8.1 ZEPHYRUS® Magneto (CE IVD ) ZEPHYRUS® M A G N E T O ZEPHYRUS® Magneto represents high quality sample preparation instrument based on proven m a g n et i c p a r t i c l e s e p a rat i o n technology. It utilizes many years experience in automated nucleic acid isolation and in combination with EliGene CE IVD kits it provides optimal solution for sensitive, accurate and cost effective diagnostic applications. Reagents are delivered by automated dispensing system instead of using expensive prefilled cartridges. The total hand on time is minimized with processing time about 50 minutes for 1 or up to 12 samples per batch. ZEPHYRUS® Magneto use metal rods for magnetic separation that are magnetised by an external magnet. To enable the often complicated re-suspension step of once separated magnetic particle pellets e.g. in wash or elution buffers, the magnet can be switched off. Re-suspension is then realized by rotation of the rods guaranteeing the efficient, complete and smooth re-suspension of the bead pellet. Due to this solution this step becomes quick and thorough, resulting in isolation products with both high yields and purities respectively. Parameters: DNA/RNA Isolation & Spectral Calibration Instruments ZEPHYRUS® 34 1 – 12 samples per run sample sizes up to 1000 µl Magneto kits and protocols for a huge variety of sample materials (Body Fluid, Blood, Tissue, Serum/Plasma, Swabs) certified according to Annex 3 of the EU IVD Directive 98/79/EC integrated buffer dispensing revolutionary resuspension technology standard plastic devices touch panel software USB slot for data transfer Ref. Name / description Z-MAG-12 ZEPHYRUS® Magneto Available isolation kits for ZPEHYRUS® Magneto Ref. Name / description EZ-7001-50 M-7001-180 Magneto BodyFluid DNA/RNA isolation kit / for DNA/RNA isolation from 200 microliters of blood, serum, plasma, buccal swabs, tissues, sperm, urine, synovial fluid, CSF etc. EZ-7001-50 M-7002-180 Magneto 1ml Serum/Plasma DNA/RNA isolation kit / for DNA/RNA isolation from HIV, HCV, HBV and other viruses from 1000 microliters of serum or plasma elisabeth pharmacon group EliGene® ZEPHYRUS ® B I O S A F E T Y B OX ZEPHYRUS® ZEPHYRUS® P C R B O X H A R M O NY M A G N E T O 8.2 ZEPHYRUS® PCR Box Harmony (PCR workstation) 400.0µm First PCR workstation with ZEPHYRUS® P C R B O X H A R M O NY nano-filtration ZEPHYRUS® PCR Box Harmony (PCR workstation) brings several advantages: Greater mobility of the arms in the workzone; the samples are better protected against DNA contamination; automatic and programmable UVC light decontamination; replaceable plexiglass box at the end of its life; unique filtration of air by nanofilter, security sensors for turn off the UVC light when you open the cover; enough space for pipettes and plastic in the PCR workstation etc. Equipment of individual variants BASICsize External ECO size External ACTIVEsize External Ref.: x Z-PCR-600 x Z-PCR-600-E x Z-PCR-600-A Upper part with buttons and electronics x x x x x x x x x x x x x x x x x x Replaceable plexiglass part (580 x 580 x 520 mm) 1 Manual start of immediate decontamination Manual start of scheduled decontamination 2 3 Automatically started scheduled decontamination Electronics for power-saving operation 4 Electronically monitored function of UV lamps Active decontamination x 5 x 6 Special air nano-filtration x x 7,8 x - x x - x x OPTION Stainless base USB interface for export of operation log to PC 9 Accessories x x x x x x Notes: Ref. Description Z-PCR-601 ZEPHYRUS® Plexiglass Z-PCR-602 ZEPHYRUS® Stainless base Z-PCR-603 ZEPHYRUS® UVC lamp 15 W Z-PCR-604 ZEPHYRUS® Y light 15 W Z-PCR-605 ZEPHYRUS® W Light 15 W Z-PCR-606 ZEPHYRUS® UVC AD Z-PCR-607 ZEPHYRUS® Ventilator AD Z-PCR-608 ZEPHYRUS® Nanofilters AD (10 pcs) Z-PCR-609 ZEPHYRUS® Nanofilters AD (50 pcs) Z-PCR-610 ZEPHYRUS® USB interface 1) UV decontamination starts immediately after a longer press of the UV button. 2) Scheduled UV decontamination in preset time starts after a short press of the UV button. 3) Scheduled UV decontamination starts automatically in preset time after closing cover. 4) Usage of special electronics helps to decrease power consumption by more than 30 % in comparison with the basic version. Furthermore, the service life of the lamp is extended. 5) UV lamp malfunction can be detected by the electronics, information of malfunction is showed to the operator. 6) Active decontamination of air in the workplace is achieved by a germicide UV lamp placed in a closed decontamination tunnel. Thus decontamination can be running even while working inside the PCR Box. Air circulation through decontamination tunnel is provided by two ventilators. 7) At the output of the decontamination tunnel there is a mounted filter made of specially designed nanomaterial, which is capable of catching DNA fragments longer than 100bp, bacteria, viruses and all particles larger than 25nm. 8) A variant available only in connection with active decontamination. 9) Logged operation data can be exported via USB cable into PC. It is necessary to install a USB interface driver and communication SW, which can organize received data for example into an excel table or create regular decontamination protocol. www.elisabethgroup.com | www.elisabeth.cz | www.zephyrusbsb.com Instruments ZEPHYRUS® 35 8. Instruments ZEPHYRUS 8.3 ZEPHYRUS® Bio Safety Box ZEPHYRUS® B I O S A F E T Y B OX Biohazard box new generation... Class II Type A2 Bio safety cabinet 30% / 70% Zephyrus® bio safety box (Class II Type A2) can be either exhausted back into the room or connected to a facility exhaust air duct system. The cabinet's airflow is 30% exhausted / 70% recirculated to minimize cross-contamination of low to moderate risk biological materials in the absence of volatile toxic chemicals. Standard Features Germicidal UV light Touch screen Automatic correction of airflow Air pressure control on both HEPA filters Emblem diagnostic signalization One eletrical outlet and gas valve Economical Features Common steel body of cabinet Box without emblem diagnostics signalization Optional Features and Accessories Remote control of cabinet's performance - remote service support Control of light intensity Stainless steel stand Simple carbon steel stand Different size of box on request available Up to 4 eletrical outlets Up to 4 gas valves LCD screen in work zone Technical specifications: Instruments ZEPHYRUS® Style of cabinet: Cabinet construction: Eletrical: Viewing window: Standard inflow velocity: Bench top / base stand Pressure tight welded stainless steel 304 230 V / 50 Hz, 10 A Fully closed to fully open 480 mm 0,5 m/s Ref. External size (mm) Work zone (mm) Weight Z-BSB-120 1207 x 1530 x 855* 920 x 660 x 620 175 kg Z-BSB-150 1507 x 1530 x 855* 1220 x 660 x 620 210 kg Z-BSB-180 1807 x 1530 x 855* 1520 x 660 x 620 245 kg * Transport through 800mm doors possible after front cover removal. 36 elisabeth pharmacon group EliGene® Manufacturer: ELISABETH PHARMACON, spol. s r.o. Namesti Svobody 18, 602 00 Brno phone: +420 542 213 851 e-mail: [email protected] www.elisabeth.cz ® ZEPHYRUS® ZEPHYRUS® ZEPHYRUS B I O S A F E T Y B OX P C R B O X H A R M O NY M A G N E T O Distributor:
