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18 Diagnostic Microbiology Using Real-Time PCR Based on FRET Technology XUAN QIN Introduction Molecular amplification of specific nucleic acid–based targets associated with microbial organisms has advanced our existing tools in infectious diseases diagnosis (Fredricks, 1999; Louie, 2000; Peruski, 2003; Yang, 2004). Laboratory diagnosis of infectious diseases in the past has relied on cultivation of the microorganisms in vitro. Hence the viability of the organisms and the laboratory conditions used to mimic the in vivo environment ultimately dictates the successfulness of in vitro amplification of the intact organism(s) outside of the infected host. Nucleic acid–based technologies allow detection by amplification of specific microbial genetic material irrespective of viability or integrity of the organism (Nissen, 2002; Gulliken, 2004; Mackay, 2004). Fluorescence resonance energy transfer (FRET)-based nucleic acid amplification technology has emerged from the marriage between polymerase chain reaction (PCR) and real-time monitoring of fluorescent chemistry. Historically, fluorescence-based detection is well established in the biosciences and has successfully replaced radioactive isotope labeling. Fluorescent dyes have an “environmental advantage”: they have a longer shelf life, are inexpensive to discard, and safer to handle. Fluorescence detection of nucleic acid molecules itself is not more sensitive than immunological or radioactive isotope detection. In fact, in most nano-detection systems, radioactive labeling displays 10- to a 1000-fold higher sensitivity. There are two major factors that make fluorescent real-time PCR advantageous. One is the potential of multiple parallel measurements by using different-colored dyes and the other is the potential of time-resolved continuous data acquisition. The increase of fluorescent signal is detectable in a closed system during amplification cycles. Therefore, the absolute sensitivity is no longer a decisive factor for these detection methods, particularly because most applications are based on nucleic acid amplification. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon accumulated by every PCR cycle as opposed to the end-point analysis (Higuchi, 1992, 1993). Various FRET systems and instrumentation that 291 292 X. Qin allow real-time monitoring of fluorescence within PCR vessels have shown great promise in infectious diseases diagnosis. The significance of real-time PCR as a diagnostic tool can be summarized in two important aspects. First, determination of amplification products of the targets by probes and melting analysis is highly accurate compared with size analysis by post-PCR gel electrophoresis that is prone to amplicon carry-over contamination. Second, quantitative analysis of a wide range of concentration levels of the initial target material is made possible by progressive monitoring of the dynamic accumulation of FRET signals over time, provided that the appropriate standards are available. Several real-time PCR platforms have been employed and marketed to meet various demands of assays designed for specific analysis. Principles of FRET Technique The real-time PCR system is based on the detection and quantitative measurement of fluorescent reporter molecules either intercalated between DNA double helix or covalently attached to specific probes (Lee, 1993; Livak, 1995). Fluorescent signal increases in direct proportion to the amount of PCR product in a reaction. The time or PCR cycle where the fluorescence signal significantly increases above background is in proportion to the initial amount of starting material in the sample well. By monitoring the amount of fluorescence emitted at the end of each cycle, it is possible to capture the PCR reaction during its exponential phase. The increase of fluorescence in the logarithmic phase can be extrapolated from a common trapezoidal curve (Fig. 18.1) where the first significant increase in the amount of PCR product correlates to the initial amount of template materials. The higher the starting copy number of the nucleic acid target, the fewer cycles needed to register a significant increase in fluorescence (Ct : cycle time at (b) 5E+6 1E+8 1E+7 4E+6 copies/100 ng DNA Relative fluorescence intensity (a) 1E+6 1E+7 1E+6 3E+6 1E+5 1E+4 2E+6 1E+3 1E+2 2E+1 1E+1 1E+0 1E-1 1E+1 1E+2 H2O 1E+0 1E+6 0E+6 10 30 20 Ct 40 2 R = 0.9919 1E+5 1E+4 1E+3 1E+2 45 40 35 30 25 20 15 10 Ct CT FIGURE 18.1. Quantitative PCR. (a) CT, cycle at which the observed fluorescence is 10-fold above backround (10× amplification). (b) Generating a standard curve using known amounts of target DNA and measuring the CT for each reaction. 5 0 293 Quantitative and qualitative measurement. Quantitative and qualitative measurement. Quantitative and qualitative measurement in conjunction with mutational analysis. Quantitative and qualitative measurement with an emphasis on mutation detection. TaqMan probes Dual hybridization probes Molecular beacons Application Intercalating dyes (SYBR Green) FRET real-time PCR Mutational analysis made easy by real-time quantitative measurement and multiplexing. target sequences r Useful for mutational analysis r Multiplexing r Very specific for the target sequences r Good for viral load analysis r Very specific for the polymorphism internal to amplicon design/optimize r Inexpensive r Accommodate r Sensitive r Easy to Advantages The mutational base(s) has to be known or known to reside in the region. The size of the amplicon needs to be long enough to accommodate the two probes. Do not tolerate polymorphism in the probe region. Signal measurement may not be specific without melt-peak analysis Disadvantages Choose target sequences with knowledge of the hot spot mutations covered by the loop region of the probe. Choose target sequences with known mismatches for mutational analysis. Choose non-polymorphic targets and design probes with knowledge. Melt-peak analysis. Additional considerations TABLE 18.1. Comparison of four fluorescence resonance energy transfer (FRET) real-time PCR applications. The rifampin resistance mutations in rpoB of M. tuberculosis and drug-resistant hepatitis B viral variants. Viral load as well as point mutation analysis in CMV and HIV diagnosis. Viral load analysis and assays that need copy number precision. Bacterial and viral detection (qualitative or quantitative) when more than one target is analyzed with melt-peak analysis. Bacterial resistance gene detection such as mecA, vanA, and vanB. Examples 294 X. Qin which the observed fluorescence is 10-fold above background). Different detection chemistries are developed concerning various diagnostic preferences, which may include DNA intercalating dyes, hydrolysis probes, hybridization probes, and molecular beacons (Table 18.1). Intercalating Dyes A simple and cheaper detection method in real-time PCR requires a dye that emits fluorescent light when intercalated into double-stranded DNA (dsDNA). The light unit of the fluorescence signal is proportional to the amount of all dsDNA present in the reaction, including specific, nonspecific amplification products and primer–dimer complex. Therefore, this method is not a sequence-specific fluorescent measurement. However, the dye employed does not bind to single-stranded DNA (ssDNA). SYBR Green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA (Morrison, 1998). Because these dyes do not make a distinction between the various dsDNA molecules in a PCR reaction, the production of nonspecific amplicons must be prevented. Therefore, primer design and optimization of the reaction conditions require extensive pilot testing. Melt-curve analysis after completion of a PCR reaction can provide additional specificity with standard controls (Ririe, 1997). Hydrolysis Probes (TaqMan Probes) The hydrolysis or TaqMan probe chemistry depends on the 5 –3 exonuclease activity of the engineered Thermus aquaticus DNA-polymerase (Fig.18.2a). A DNA probe, labeled with a reporter dye and a quencher dye at opposite ends of the sequence, is designed to hybridize internal to the amplicon (Hiyoshi, 1994; Chen, 1997). When irradiated in the absence of a specific amplicon, the excited fluorescent dye transfers energy to the nearby quenching dye molecule (this is called FRET) rather than fluorescing. Thus, the close proximity of the reporter and quencher prevents emission of any fluorescence while the probe is intact. In the presence of specific amplicon, as the polymerase replicates a template on which a TaqMan probe is bound, its 5 exonuclease activity cleaves the probe (Holland, 1991). Departuring from the activity of quencher (no FRET), the reporter dye starts to emit fluorescence that increases in each cycle growing at the rate of probe cleavage. Accumulation of PCR products is detected by monitoring the increase in fluorescence of the reporter dye (note that primers are not labeled). The probe is usually longer than the primers (20–30 bases long with a Tm value of 10◦ C higher) that contain a fluorescent dye preferred on the 5 base and a quenching dye typically on the 3 base. FAM (6-carboxyfluorescein) and TAMRA (6-carboxy-tetramethyl-rhodamin) are most frequently used as reporter and as quencher, respectively. The process of hybridization and cleavage does not interfere with the exponential accumulation of the amplification product (the probe and primer sites do not overlap). One specific requirement for fluorogenic probes is 18. Real-Time PCR Based on FRET R = Reporter Q = Quencher Polymerization 5' 3' Foreward Primer R Probe Q 3' 5' 3' 5' 5' Reverse Primer Strand Displacement R Q 3' 5' 3' 5' 3' 5' 5' R Cleavage Q 3' 5' 3' 5' 3' 5' 5' R Polymerization Q 3' 5' 3' 5' 3' 5' 5' (a) hv P hυ hv hυ P (b) (c) FIGURE 18.2. (a) TaqMan PCR probes. (b) FRET Probes. (c) Molecular beacons. 295 296 X. Qin that there would be no G at the 5 end. A “G” adjacent to the reporter dye quenches reporter fluorescence even after cleavage. TaqMan probes are relatively sensitive to single base variations (mismatch). This could be extremely important when amplifying biological samples, where such a genetic variability could be present that a successful amplification may fail to result in a positive signal. Unfortunately, this sensitivity may render TaqMan probes inappropriate for genotyping, because a “nonsignal” will have to be attributed to an unknown genotype. Dual Hybridization Probes This detection method relies on FRET of two adjacent oligonucleotide probes (Fig. 18.2b). When both probes are specifically bound to the target amplicon, the energy emitted by the donor dyes excites the acceptor dye of the second probe, which then emits fluorescent light at a longer wavelength. One probe is labeled with a donor fluorochrome (fluorescein) at the 3 end, and the other probe is labeled with an acceptor dye (Cy5, LC Red 640) at the 5 end. Both probes can hybridize to the target sequences, and the two probes are usually no more than 3 bases apart (4 to 25 Å molecular distance). The first dye (fluorescein) is excited by the LED (light emitting diode) filtered light source and emits green fluorescent light at a slightly longer wavelength. When the two dyes are in close proximity as the probes simultaneously hybridize to their target, the emitted energy excites the acceptor (i.e., LC Red 640) attached to the second hybridization probe that subsequently emits red fluorescent light at a longer wavelength. The occurrence of FRET is characterized by a decrease in observed donor emission and a simultaneously increased acceptor emission. The ratio between donor fluorescence and acceptor fluorescence increases during the PCR and is proportional to the amount of target DNA generated (Wittwer, 1997; Nitsche, 1999). An advantage hybridization probes thus have over the hydrolysis probes is their relative tolerance to single base variations; therefore their suitability for genotyping in combination with melt-peak analysis. A disadvantage is the need for a larger sequence area necessary to accommodate two adjacent probes. Molecular Beacons Molecular beacons are stem-loop (hairpin) shaped hybridization probes with a fluorescent dye and a quencher dye on the opposite extremities brought to their proximity by the complementary stem (Fig. 18.2c). The commonly used fluorescent dyes are FAM, TAMRA, TET, and ROX paired with a quenching dye, typically DABCYL. While in the absence of amplicon target, the FRET between the fluorescent dye and the quencher prevents light excitation and emission. In the presence of amplicon target, the complementary loop fragment of the probe is able to hybridize to the template sequence and stretches out the two ends, thus diminishing the quenching effect and resulting in detectable fluorescence (FRET does not occur). 18. Real-Time PCR Based on FRET 297 Because the hybrid hairpin configuration is very thermostable, molecular beacons have a high specificity to hybridize to a target, which are used to distinguish single nucleotide differences. Therefore, molecular beacons are suitable for mutation analysis and single nucleotide polymorphism detection when specific mutations are known (McKilli, 2000; Szuhai, 2001; Abravaya, 2003; Bustamante, 2004; Petersen, 2004; Vet, 2005). All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair. All of the above can be used in conjunction to melting curve analysis or when SYBR Green is used only. By multiplexing, the target(s) and endogenous control can be amplified in a single tube. (Bernard, 1998; Lee, 1999; Vet, 1999; Elnifro, 2000; Read, 2001; Grace, 2003; Rickert, 2004) Real-Time PCR in Infectious Disease Diagnosis Molecular diagnostic tools and detection methods such as nucleic acid amplification are being used increasingly in the clinical microbiology laboratory to enhance the diagnosis of microbial pathogens (Lanciotti, 2001; Mackay, 2004). Nucleic acid–based technology is also used to assess drug resistance and epidemiological surveillance (Piatek, 1998; Makinen, 2001; Huletsky, 2004; Sloan, 2004). The principle of the real-time PCR is primarily used to detect and amplify a unique gene or a signature sequence of the microorganism. Quantitative measurements of viral load can also be made simple. Sensitive detection and accurate identification can speed up reporting of microbial pathogens without reliance on their phenotypic characteristics or viability after antibiotic treatment. The application of real-time PCR in infectious diseases enables the diagnosis of microbial pathogens both with accuracy and expediency. The clinical significance of using molecular diagnosis of infectious agents can be characterized by the following aspects. (1) Pathogens that show fastidious slow growth or inability to grow in vitro: Mycobacterium, Legionella, Bartonella, Leptospira, Borrelia, Bordetella, Mycoplasma, and Tropheryma whippelii may require days or weeks of incubation under specific conditions; (2) obligatory intracellular organisms (Chlamydia, Rickettsia, Coxiella, Ehrlichia, DNA and RNA viruses); (3) prior antibiotic use; (4) biochemically inert for phenotypic characterization; (5) additional waiting time for drug-resistance determination, (6) diagnostic speed from bench to bedside. Qualitative real-time amplification has outpaced conventional culture methods in detection of a long list of specific pathogens that are difficult to cultivate: Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Coxiella burnetii, Ehrlichia spp., Legionella spp., Mycoplasma pneumoniae, Chlamydia trachomatis, Rickettsia, Toxoplasma gondii, Microsporidium, Cryptosporidium, Tropheryma whippelii, Mycobacterium tuberculosis and its drug-resistant determinants (Franzen, 1999; Pretorius, 2000; Hammerschlag, 2001; Bell, 2002; Fournier, 2002; Gerard, 2002; Kovacova, 2002; Exner, 2003; Templeton, 2003; Wang, 2003; Fenollar, 2004; Koenig, 2004; Simon, 2004; Wada, 2004; Khanna, 298 X. Qin 2005). Furthermore, the rapid turn-around time supported by real-time PCR may directly benefit the patient care and reduce mortality in areas of invasive infections caused by common pathogens. The examples are infective meningitis of bacterial or viral etiology, such as Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, enteroviruses, herpes simplex viruses (HSV), and so forth (Corless, 2001; van Haeften, 2003; Archimbaud, 2004; Bryant, 2004; Guarner, 2004; Mengelle, 2004; Mohamed, 2004; Picard, 2004; Uzuka, 2004; Aberle, 2005). Quantitative measurement of viral load using real-time PCR is another significant methodology improvement, and its diagnostic implication is infinite. First of all, HIV viral copy numbers in blood and body fluids are important disease and treatment markers directly tied into actions of clinical management. RNA reverse transcription and PCR (RT-PCR) can be established in a single-tube reaction, and copy numbers can be extrapolated from a standard curve in a single run (Kostrikis, 2002; Erikkson, 2003; Lee, 2004; Watzinger, 2004). Similarly, other viral etiology such as cytomegalovirus (CMV; Jebbink, 2003), HSV-1, HSV-2, varicella-zoster virus (VZV), Epstein–Barr virus (EBV; Legoff, 2004), parvovirus B19 (Hokynar, 2004; Plentz, 2004; Liefeldt, 2005), human polyomaviruses of BK and JC, and human herpesviruses 6, 7, and 8 can be measured both qualitatively and quantitatively according to clinical needs (Whiley, 2001; Beck, 2004; Watzinger, 2004). RT-PCR can be performed to detect and quantify hepatitis A virus (HAV Costa-Mattioli, 2002), hepatitis B (HBV; Payungporn, 2004; Sum, 2004; Yeh, 2004; Pas, 2005; Zhao, 2005), and hepatitis C (HCV; Candotti, 2004; Castelain, 2004; Cook, 2004; Koidl, 2004; Walkins-Riedel, 2004) in whole-blood samples. Real-time RT-PCR panels are increasingly becoming commonplace for respiratory viral diagnosis of influenza A and B viruses, parainfluenza viruses, human adenoviruses, human metapneumovirus, and respiratory syncytial virus, respectively (Kahn, 2003; Boivin, 2004; Cattoli, 2004; Daum, 2004; Frisbie, 2004; Moore, 2004; O’shea, 2004; Stone, 2004; Templeton, 2004; Ward, 2004). Despite apparent high sensitivity and specificity, molecular amplification techniques are not error-free. Contamination as a result of amplicon carry-over is a top concern of its practice in clinical diagnostics. Physical and chemical control of amplified products has to be designed and implemented before the tests are validated. Strict separation of pre- and post-PCR (negative-pressure room for post-PCR analysis) environments through laboratory design and personnel training has to be the first step. Amplification chemistry employing uracil and uracil-N -glycosylase is an effective end-product degradation control (Pennings, 2001; Pierce, 2004). Second, microbial DNA extraction is a rate-limiting step deciding the ultimate test sensitivity. The wide spectrum of cell wall makeup pertaining to specific microorganisms makes it impossible to limit the method of extraction to any single standard approach. Specific emphasis has to be made to optimally recover DNA materials from certain species of bacteria or parasites. Sonication and/or freeze–thaw methods can be used in conjunction with enzyme digestion for DNA extraction from mycobacteria and cyst-forming protozoa parasites (Harris, 1999; Kostrzynska, 1999; Lanigan, 2004). Third, sampling error is intrinsic to PCR-based approach 18. Real-Time PCR Based on FRET 299 due to its small specimen input nature. 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