Download DNA / RNA Extraction - q

Work package 7 - Progress summary
On-Site Confirmation and Monitoring
Partners involved: PRI, Fera, ACW, NIB, UNIBO, CIP, CAIQ, Qlinea and Optisense
WP Coordinator: PRI
12th June, 2012 Q-detect
Overview of work for Task 7
Task 7.1
DNA extractions
Task 7.2
Generation of protocols for isothermal, singleplex and multiplex
amplification of targets
Task 7.3
Generation of devices for multiplex detection on-site
Task 7.4
Evaluation of promising on-site detection systems for
monitoring
Task 7.5
Implementation of procedure on user-friendly detection system
Confirmation and Monitoring
DNA/RNA extractions on different substrates performed in
the field and/or on-site are:
White flies viruses from traps
Bacterial pathogens from plant material
Potato pathogens from tuber and leaves
Phytophthora and plant pathogenic Fungi
DNA/RNA Extraction Criteria:
-
Easy
Fast
Efficient
Cheap
DNA/RNA extractions (summary):
 Qiagen DNeasy/RNeasy extraction kids have been
successfully used for:
 Viruses (ssRNA) and (ssDNA), bacteria, fungi and nematodes
 Boiling of infected plant tissue (leafs, shoots, blossoms)
 Epicentre DNA/RNA extraction procedure
 Applicable for ssRNA and ssDNA viruses
 Efficient recovery
 LFD based DNA/RNA extractions:
 Applicable for different substrates
(RNA and DNA viruses has to be tested)
 Recovery has to be improved
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
Bacterial pathogens from plant material
Potato pathogens from tuber and leaves
Phytophthora and plant pathogenic Fungi
Vector of Q-viruses:
 Bemisia tabaci
 Trialeurodes vaporariorum
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
Bacterial pathogens from plant material
Potato pathogens from tuber and leaves
Phytophthora and plant pathogenic Fungi
Whitefly vectored viruses
Crini viruses (ssRNA):
Tomato chlorosis virus, (TOCV)
Tomato infectious chlorosis virus, (TICV)
Cucurbit yellow stunting disorder virus, (CYSDV)
Potato yellow vein virus, (PYVV)
Begomovirus (ssDNA):
Tomato yellow leaf curl virus, (TYLCV)
Cotton leaf curl virus (CLCuV)
Field validation can be performed
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
Bacterial pathogens from plant material
Potato pathogens from tuber and leaves
Phytophthora and plant pathogenic Fungi
Erwinia amylovora
Fire blight of pome fruit (Spiraeoidea)
Xanthomonas arboricola pv. pruni
Bacterial spot of stone fruit (Prunus)
Pseudomonas syringae
Symptoms of Psa
Confirmation and Monitoring
XAP Ring test
ACW Wädenswil, CITA Zaragoza, IVIA Valencia, APR Lisse
Inter- Laboratory concordance
Washes DIR.
Washes1:10
Washes 1:100
Comminuted DIR.
Comminuted 1:10
Comminuted 1:100
Cohen´s
kappa index
Agreement
(Concordance)
<0,00
Less than chance
0,00–0,20
Slight
0,21–0,40
Fair
0,41–0,60
Moderate
0,61–0,80
Substantial
0,81–1,00
Almost perfect
Global concordance index inter laboratories
REAL-TIME PCR
LAMP
0.875±0.248 - 1±0.25
1±0.25
(almost perfect)
(almost perfect)
1±0.25
1±0.25
(almost perfect)
(almost perfect)
0.75±0.2421 - 1±0.25
0.4783±0.2133 –
(substantial-almost perfect)
0.8462±0.247
(moderate-almost perfect)
0±0 - 1±0.25
0±0 - 1±0.25
(slight-almost perfect)
(slight-almost perfect)
0.875±0.248 - 1±0.25
0.875±0.248 - 1±0.25
(almost perfect)
(almost perfect)
0.875±0.248 - 1±0.25
0.875±0.248 - 1±0.25
(almost perfect)
(almost perfect)
Confirmation and Monitoring
 DNA/RNA extractions on different substrates to be
performed in the field and/or on-site
White flies viruses from traps
Bacterial pathogens from plant material
Potato pathogens from tuber and leaves
Phytophthora and plant pathogenic Fungi
Ralstonia solanacearum
Clavibacter michiganensis ssp. Sepedonicus
PSTVd (Potato Spindle Tuber Viroid)
Confirmation and Monitoring
LAMP target genes for Ralstonia solanacearum: fliC. and 16S rRNA
Strain
Nr
LAMP fliC
LAMP 16S
rRNA
Real time PCR
(16s rRNA)
R. solanacearum
27
25
27
27
13
/
1 (late signal)
1
56
4 positive out of 56
18 positive out of 28
3 positive out of 20
Other negative control
strains
Negative potato extracts
(real samples)
16S rRNA: good sensitivity and specificity on control strains, but shows cross
reactivity with some potato extracts (soil bacteria?).
Egl (endogluconase): in silico covers all RS isolates, work in progress.
Confirmation and Monitoring
RT-LAMP on PSTVd
Viroid (Specificity results)
Potato Spindle Tuber Viroid
Number of
Isolates
(PSTVd)
NIB RTLAMP
Tsutsumi
RT-LAMP
RT-PCR-RČ
16
16
14
16
Tomato Planta Macho Viroid (TPMVd)
3
3
/
3
Tomato Chlorotic Dwarf Viroid (TCDVd)
12
3
2
12
Chrysanthemum Stunt Viroid (CSVd)
4
1
/
/
Tomato Apical Stunt Viroid
(TASVd)
7
/
/
3
Citrus Exocortis Viroid
(CEVd)
3
/
/
2
Columnea Latent Viroid
(CLVd)
13
1
/
1
Peach Latent Mosaic Viroid
(PLMVd)
3
/
/
/
Eggplant Latent Viroid
(ELVd)
1
/
/
/
Avocado Sunblotch Viroid
(ASBVd)
1
/
/
/
Hop Latent Viroid
(HLVd)
1
/
/
/
• Specificity for PSTVd; TPMVd, CSVd and CLVd discriminated by Tm
Confirmation and Monitoring
Bursaphelenchus xylophilus Pine Wood Nematode (PWN)
 DNA/RNA extractions on different substrates
to be performed in the field and/or on-site
 LAMP assay design on ITS region of B. xylophilus
Confirmation and Monitoring
LAMP assay design for the vector insect
LAMP assay design on COI gene of Monochamus
galloprovincialis (positive control for LAMP assay on PWN)
Confirmation and Monitoring
Detection of blue-stain fungi
(Ophiostoma clavatum or
Ophiostoma brunneo-ciliatum)
associated to Ips acuminatus using
LAMP technology
Ips acuminatus on
Pinus sylvestris
Confirmation and Monitoring
 LAMP assay development:

O. clavatum and O. brunneo-ciliatum primers
were designed on the β-tubulin gene

Ips acuminatus primers were designed in the COX I
gene
 The blue-stain fungus O. clavatum is associated to Ips
acuminatus in the Italian Alps
 LAMP technology can be useful also for ecological and
biological studies
Monitoring of LAMP products (first line screening)
Multiplex and simplex amplification
• Easy
• Fast
• Sensitive
• Applicable for DNA and RNA
• On-site
Detection
The detection system needs to be adjusted for field
inspectors who might not necessarily have laboratory-work
experiences, while the given result has to be unambigous.
The most promising detection systems include:
1) Turbidity
2) Fluorescence
Positive reaction is green
Positive reaction is turbid
Detection
3) LFD
4) Array-tubes
On-site diagnostics device
(Forsite Diagnostics Ltd.).
Micro reaction vial with
integrated biochip
(Alere Technologies GmbH).
Combined amplification and detection
To simplify the procedure, amplification and detection can be
combined in a single system:
Genie II/III
- 16/8-well device with touch screen to follow DNA amplification in real-time
- Stand-alone operation without PC
- Internal rechargeable Li-Po battery
(OptiSense Ltd.)
Combined amplification and detection
Master Mix and Enzymes
•
•
•
•
ISO-001 standard mastermix (GspSSD)
GspM and GspM2.0 polymerases
TIN-001 DNA polymerase
‘Lyse & LAMP’
•
•
•
•
•
Lyse in Potassium Hydroxide solution (0.5 Molar)
Dispense directly into reaction tube
Mastermix buffer modified with lower pH
Method being used by researchers in crop science
Plant leaves used directly
Licensing
•
Q-Detect assays discussed at meeting held with Eiken
Combined amplification and detection
To simplify the procedure, amplification and detection can be
combined in a single system:
Microfluidics
Microfluidics allow multiple reactions combined with detection (PRI)
Extraction
DNA / RNA
First line screening
(monitoring via fast
semi specific method)
+
Χ
Second line screening
(confirmation via target
specific detection
methods)
Specific target detection
Combination of TICV, ToCV and TYLCV LAMP primer sets
Multiplex TYLCV, TOCV
and TICV

Multiplex LAMP with biotinylated nucleotides can be used for
First and Second line screening
Ligation based Univ-LAMP

After ligation biotinylated LAMP amplicon can efficiently and
specifically be detected with Luminex
Developed Isothermal methods
LAMP
• LAMP method for Ralstonia solanacearum
• RT-LAMP for PSTVd detection
• LAMP method for E. amylovora and X. arboricola. prun. are
developed and validated
• LAMP assay for B. xylophilus developed and validated
• LAMP assays in white flies for TICV, TOCV, CYSDV and PYVV
(Crini viruses) and TYLCV, CLCuV (Begomoviruses) have
been developed. Validation is in progress.
• LAMP assays designed for quarantine L. huidobrensis, L.
sativae, L. trifolii (vegetable leafminer). Also assays in
development for TCDVd, CSNV
Contact end users
Engage with end users to ensure your deliverables are
fit for purpose and work with other WP to ensure
synergies are created.
 PWN assays developed by University of Padova utilised on
trapped monchamus (linking WP7 and WP4). Assays also
requested by Australian Inspector who attended EPPO
workshop
 PRI developed suite of assays for whitefly viruses to enable
testing of trapped whitefly (linking WP7 and WP4)
 PRI have planned a demonstration for Dutch NPPO.
 ACW talking to Swiss NPPO about needs (list of pest
elaborated), they plan to implement LAMP pipeline
 Fera have an implementation project with UK inspectors, plan to
implement LAMP/Genie at Heathrow airport.
Dissemination
Develop and implement a dissemination plan for
information and experience gained from WP7.

EPPO workshop should be more hands on – need to develop a
programme.

Work with CIP/CAIQ to deliver training in China and Peru

Good dissemination already achieved with papers, TV shows,
videos, newspapers, web site etc.

ACW working with AQIS in Melbourne have validated assays
on Australian isolates and AQIS plan to implement LAMP
Follow up
How you plan for the deliverables and knowledge
gained in the q-detect project to be utilised after the
project finishes.
 Exploitation plan for simplex LAMP well established
 Optisense already started negotiation of license with Eiken
 Partners starting to validate assays to EPPO standard
 Large number of other assays under development.