Detection of pospiviroid in pepper

REFERENCE PROTOCOL NAKTUINBOUW
Real-time RT-PCR (RT TaqMan PCR) for pospiviroids
(CEVd, CLVd, MPVd, PCFVd, PSTVd, TASVd, TCDVd and
TPMVd) on seeds of pepper (Capsicum annuum)
Protocol number:
Version:
Date:
Validation:
SPN-V044e
v1.1
01-08-2014
Under validation
This protocol is a translation of the Dutch protocol SPN-V044. In case of discrepancies between the
English and Dutch text, the Dutch text prevails. This protocol is made available without any warranty.
Naktuinbouw cannot guarantee that the results obtained by laboratories that follow this protocol are
accurate and representative. Many factors (e.g. personnel skills, lab conditions, quality of reagents,
sampling methods etc.) can influence the results. Consequently, Naktuinbouw will not accept any liability
with respect to the use of this protocol.
1.
Objective
To detect the absence or presence of potentially relevant pospiviroidae (CEVd, CLVd, MPVd,
PCFVd, PSTVd, TASVd, TCDVd and TPMVd) in pepper seeds by isolation of RNA followed by
TaqMan RT PCR.
2.
Principle
RNA from seed extract of pepper is isolated and purified with a Qiagen RNeasy mini plant kit. The
possible presence of viroid RNA is demonstrated by means of RT TaqMan PCR using selective
sets of primers and labeled TaqMan probes. Each subsample is spiked with DLVd as an internal
amplification control (IAC) to monitor the performance of RNA extraction and RT Taqman PCR.
3.
Abbreviations
cDNA
CEVd
CLVd
Ct-value
DLVd
GH+ buffer
IAC
MPVd
NC seed
PC seed
PCFVd
PSTVd
RT Taqman
TASVd
TCDVd
TPMVd
4.
Complementary DNA
Citrus exocortis viroid
Columnea latent viroid
“Cycle threshold” value (number of PCR cycli till reaching the threshold)
Dahlia latent viroid (spike viroid)
Guanidine–hydrochloride extraction buffer
Internal amplification control
Mexican papita viroid
Negative control pepper seeds (process control)
Positive control TASVd contaminated pepper seed lot (process control)
Pepper chat fruit viroid
Potato spindle tuber viroid
Reverse transcriptase Taqman
Tomato apical stunt viroid
Tomato chlorotic dwarf viroid
Tomato planta macho viroid
Materials
Biorad CFX 96 PCR apparatus
DLVd stock
GH+ buffer (see appendix)
Grinding bags 100 ml (Interscience BagPage)
Hard Shell PCR plates (Biorad, Catalog no. HSP9645)
Interscience BagMixer 100
Positive RNA controls
RNase-free water
RNeasy plant mini kit (Qiagen)
Taqman RNA-to-Ct 1 step kit (Applied Biosystems, productno. 4392938/4392656)
Thermoshaker
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5.
Method
5.1
Safety and warnings
 Disinfection of seeds with hypochlorite and / or trisodium phosphate strongly decreases the
sensitivity of this assay.
 Viroids can be present in very high concentrations in plant tissue and cross-contamination is a
possibility. Accuracy of operations is important to reduce the chance of cross-contamination.
Wear gloves and use pipets with filter tips at all times.
5.2 Execution
5.2.1
Preparation of pepper seed samples
1. Weigh from each sample (3.000 or 20.000 seeds) 3 subsamples of 1000 or 50 subsamples of
400 seeds and transfer each subsample to a grinding bag (Interscience BagPage 100ml).
2. Calculate the required amount of GH+ extraction buffer based on the total number of
subsamples. Dilute the DLVd spike 10x. Add 10 μl diluted DLV spike for each 100 ml
GH+ extraction buffer as internal amplification control (IAC).
3. Add to each bag with 1,000 or 400 seeds respectively 20 or 12 ml GH+ extraction buffer
including the DLVd IAC.
4. Soak the seeds for 30-60 minutes at room temperature.
5. Extract the subsamples for 4 minutes using the Interscience BagMixer (position 4).
6. Preheat thermoshaker (setting: 65 °C, 850 rpm).
7. Transfer 1.5 ml of seed extract per subsample gently into 1.5 ml tube.
8. Include a TASVd-contaminated subsample (PC pepper seed with a Ct of about 20-23) and a
negative control (NC pepper seed with a Ct > 37) per sample series.
9. Incubate in thermoshaker for 15 minutes (setting: 65 °C, 850 rpm).
10. Centrifuge the tubes for 10 minutes at 16,000 g.
NB. Handle the PC seed as the last subsample in order to minimize the chance of cross-contamination.
5.2.2
RNA isolation
1. Use “RNeasy plant mini kit” for the isolation of RNA.
2. Transfer 750 µl supernatant into QIAshredder-spin-column (purple).
3. Centrifuge tubes for 2 minutes at 16,000 g.
4. Store leftover of supernatant at -20°C until the test is completed.
5. Transfer 600 µl supernatant (avoid pellet) to new 1,5 ml tube with 300 µl ethanol (96-100%).
6. Mix well by pipetting up en down.
7. Transfer 700 µl of extract into RNeasy-mini-column (pink).
8. Centrifuge 1 minute at 16,000 g.
9. Transfer filter to new collection tube.
10. Add 700 μl RW1-wash buffer to RNeasy-mini-column.
11. Centrifuge 30 seconds at 16,000 g.
12. Place RNeasy-mini-column in new collection tube.
13. Transfer 500 μl RPE-wash buffer (including ethanol) to RNeasy-mini-column.
14. Centrifuge 30 seconds at 16.000 g.
15. Transfer filter to new collection tube.
16. Transfer 500 μl RPE-wasbuffer (including ethanol) to RNeasy-mini-column.
17. Centrifuge 2 minutes at 16,000 g to dry RNeasy-silicagel-membrane.
18. Place RNeasy-mini-column in new collection tube.
19. Transfer 75 µl RNase-free water to RNeasy-mini-column.
20. Incubate 1 minute.
21. Centrifuge 1 minute at 8,000 g.
22. Continue directly with PCR or store purified RNA at -20 °C.
5.2.3
Taqman RT-PCR
1. Prepare Taqman RT-PCR mix (Table 4a, 4b, 4c and 4d).
NB. Work on ice as much as possible and prevent prolonged exposure of probes to light. Wear clean lab
coat and gloves to minimize the risk of cross-contamination.
2. Calculate the required amount of reaction mix based on the number of subsamples & controls
+ 2.
3. Transfer 19 µl mix into a strip of white PCR tubes or PCR plates with white bottom / green
border.
4. Pipette 6 µl of the purified RNA sample in 19 µl mix.
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5.
6.
Cover the strip / plate after adding the RNA.
In each run, include a no-template control and positive RNA controls (Table 3) giving a Ct
value of approximately 30. 7. Run the Taqman RT PCRs according to the following program
(Table 5).
Table 3. Positive RNA controls per mix
Mix
Relevant RNA controls
A
PSTVd, TCDVd, MPVd, PCFVd and DLVd (IAC)
B
CEVd, CLVd and DLVd (IAC)
C
TPMVd and Nad5
D
TASVd
Table 4a. PCR mix PSTVd, TCDVd, MPVd (FAM), PCFVd (VIC) and DLVd (IAC)(Texas red)
PCR mix
1 reaction
RNase-free water
4,625
μl
TaqMan RT-PCR Mix (2x) (Applied biosystems)
12,5
μl
10 µM Pospi A primer mix (351a)
0,75
μl
10 µM Pospi A probe mix (351b)
0,5
μl
TaqMan RT Enzym Mix (40x) (Applied biosystems)
0,625
μl
Subtotal
19
μl
Sample (RNA)
6
μl
Total
25
μl
Table 4b. PCR mix CEVd, CLVd (FAM) and DLVd (IAC)(Texas red)
PCR mix
RNase-free water
TaqMan RT-PCR Mix (2x) (Applied biosystems)
10 µM Pospi B primer mix (352a)
10 µM Pospi B probe mix (352b)
TaqMan RT Enzym Mix (40x) (Applied biosystems)
Subtotal
Sample (RNA)
Total
1 reaction
4,625
12,5
0,75
0,5
0,625
19
6
25
μl
μl
μl
μl
μl
μl
μl
μl
Table 4c. PCR mix TPMVd (FAM) and Nad5 (IAC)(Texas red)
PCR mix
RNase-free water
TaqMan RT-PCR Mix (2x) (Applied biosystems)
10 µM Pospi C primer mix (353a)
10 µM Pospi C probe mix (353b)
TaqMan RT Enzym Mix (40x) (Applied biosystems)
Subtotal
Sample (RNA)
Total
1 reaction
4,625
12,5
0,75
0,5
0,625
19
6
25
μl
μl
μl
μl
μl
μl
μl
μl
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Table 4d. PCR mix TASVd (FAM)
PCR mix
RNase-free water
TaqMan RT-PCR Mix (2x) (Applied biosystems)
10 µM Pospi TASVd-F2-200 (281a)
10 µM Pospi TASVd-R2-269 (281b)
10 µM Pospi TASVd-P2-228 (281c)
TaqMan RT Enzym Mix (40x) (Applied biosystems)
Subtotal
Sample (RNA)
Totaal
1 reaction
3,875
12,5
0,75
0,75
0,5
0,625
19
6
25
μl
μl
μl
μl
μl
μl
μl
μl
μl
Table 5. RT-Taqman program CFX96 (version 2 or higher) for mixes Table 4a, 4b, 4c and 4d
temperature time
hold
48°C 15' 00"
hold
95°C 10' 00"
40 cycli
95°C 0' 15"
60°C 1' 00"
6.
Evaluation and interpretation
6.1
Evaluation test result
 Turn on FAM, VIC and TR signal for all 96 wells.
 Use single threshold setting (RFU 200).
 Turn on option "fluorescent drift correction".
 Check shape of curve (S-shape) for positive samples. Compare, if necessary, with PC seed.
Please note that the TPMVd Taqman RT occasionally gives atypical planar curves (no S-shaped
curve) that should be considered as noise. In case of doubt always check with responsible
person.
 Monitor the performance of PCs to maintain the reliability of the test list and note the RFU value
used per data set.
 See decision matrix (Table 6a, b, c, and d) for interpretation of the results of samples. Based on
the signals in different channels, an indication of the identity of a viroid can be obtained.
6.2
Validity of test result
 Results may only be issued if the positive controls give a clear signal (Ct seed PC and RNA PC
between 18-25 and 28-32, respectively).
 Results may only be issued if Ct in the negative control samples > 35.
 The Ct DLVd must be < 32. For the matrix pepper seeds the Ct values for the Nad5 IAC are less
important since the reproducibility is not optimal. Also Nad5 degradation is much faster than viroid
degradation and the quantity of Nad5 is variable per seed lot. For a subsample Nad5 Ct > 32 can
be acceptable provided that the IAC DLVd is < 32 and the TPMVd PC is clearly positive.
 Contact the responsible person when the results differ from the expectations.
 Contact the responsible person when a seed sample is suspect.
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6.3 Decision matrix (subsample level)
Table 6a. Decision matrix PCR mix 4A: PSTVd, TCDVd, MPVd (FAM), PCFVd (VIC) and DLVd (IAC)(TR)
Ct FAM
Ct VIC
Ct TR (IAC)
>32
>32
<32
PSTVd, TCDVd, MPVd and PCFVd not detected
PSTVd, TCDVd and MPVd not detected
>32
<32
<32
PCFVd detected
PSTVd and/or TCDVd and/or MPVd detected*
<32
>32
n.a.
PCFVd not detected
PSTVd and or TCDVd and/or MPVd detected*
<32
<32
n.a.
PCFVd detected
Not valid/repeat (check other IACs)
>32
>32
>32
*if desired, perform sequence analysis to identify the specific pospiviroid
Table 6b. Decision matrix for PCR mix 4B: CEVd, CLVd (FAM) and DLVd (IAC)(TR)
Ct FAM
Ct TR (IAC)
CEVd and CLVd not detected
>32
<32
<32
n.a.
>32
>32
CEVd and/or CLVd detected
Not valid/repeat (check other IACs)
Table 6c. Decision matrix for PCR mix 4C: TPMVd (FAM) and Nad5 (TR)
Ct FAM
Ct TR (IAC)
TPMVd not detected
>32
<32
<32
n.a.
>32
>32
TPMVd detected
Check DLVd IACs (mix A and mix B) since IAC
DLVd is more relevant. Check with responsible
person.
Tabel 6d. Decision matrix for PCR mix 4D: TASVd (FAM)
Ct FAM
TASVd not detected
>32
TASVd detected
<32
7.
Literature and instructions
Literature:

Boonham, N., L.. González-Pérez, ,M.S. Mendez, E. Lilia Peralta, A. Blockley, K. Walsh, I. Barker & R.A. Mumford
(2004) Development of a real-time RT-PCR assay for the detection of Potato spindle tuber viroid. Journal of Virological
Methods 116:139-146.

Botermans, M., van de Vossenberg, B. T. L. H., Verhoeven, J. Th. J.,Roenhorst, J. W., Hooftman, M., Dekter, R. &
Meekes, E. T. M. (2013) Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids.
J. Virol. Methods 187, 43–50.

Koenraadt, H., Jodlowska, A., van Vliet, A. & Verhoeven, K. (2009) Detection of TCDVd and PSTVd in seeds of tomato.
Phytopathology 99, S66.

Menzel, W., Jelkmann, W., and Maiss, E. (2002) Detection of four apple viruses by multiplex RT-PCR assays with
coamplification of plant mRNA as internal control. J. Virol. Methods 99: 81-92.

Monger, W., Tomlinson, J., Boonham, N., Marn, M.V., Plesko, I.M., Molinero-Demilly,V., Tassus, X., Meekes, E.,
Toonen, M., Papayiannis, L., Perez-Egusquiza, Z., Mehle, N., Jansen, C., Nielsen, S.L., (2010) Development and interlaboratory evaluation of real-time PCR assays for the detection of pospiviroids. J. Virol. Methods 169, 207–210.
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
Mumford, R.A., A.L. Skelton, N. Boonham, K.I. Posthuma, M.J. Kirby, & A.N. Adams (2001) The Improved Detection of
Strawberry Crinkle Virus Using Real-Time RT-PCR (TaqMan®). Acta Horticulturae 656: 81-86.
Manuals:
8.

TaqMan® RNA-to-CT™ 1-Step Kit Protocol

RNeasy plant mini kit (Qiagen)
History and revisions

20-12-2013 Version 1.0 - Protocol based on ISO17025 accredited PSTVd/ TCDVd seed assay (SPN-V003, version 3.1)
for tomato.

Overnight soaking of seeds was reduced to 30-60 minutes to limit break down of viroid.

Introduction of DLVd spike as an internal amplification control (IAC) in multiplex RT Taqman to replace endogenous
Nad5 target since amount of Nad5 RNA is highly variable (14-40) in the seed matrix.

Use of GH+ extraction buffer instead of PN1 extraction buffer (LGC) since degradation in GH+ RNA extraction buffer is

Use of several new multiplex RT Taqmans to detect additional Pospiviroidae as CEVd, CLVD, PCFVd, TASVd and

03-02-2014 Version 1.1 - Correction numbering primer sets in appendices. 201d instead 201c, at 349 / mc instead of
much less than in PN1 extraction buffer leading to a higher PSTVd sensitivity.
TPMVd.
249 at / c and 350a, 350b instead 250a, 250b.
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9.
Appendices
GH+ extraction buffer (6M)
guanidine-hydrochloride (harmful)
NaAC-buffer (4M)
EDTA (di-natrium)
PVP-10
Adjust to 1 liter with water
NaAc buffer (4M)
Adjust to 1 liter with water
573
50
9.3
25
NaAC (CH3COONa)
Check/adjust pH to 5.2
328
g
ml
g
g
g
DLVd spike
Take a leaf from a DLVd infected Dahlia plant and make an leaf
extract. Dilute the extract experimentally to a Ct value of 25. Store
aliquots at -80 °C.
After thawing store aliquot at -20 ˚C for up to one week.
Primer
No.
Sequence
Naktuinbouw
primer collection
Lit.
ref.
PSTV-231F1
PSTV-296R
PSTV-251T-probe
DaVd1-FT
DaVd1-RT
DaVd1-P
TPMVd-F1
TPMVd-R
pUCCR2
CLVd-F
CLVd-F2
CLVd-R
CLVd-P
CEVd-F2-304
CEVd-R2-399
CEVd-P2-337
TASVd-F2-200
TASVd-R2-269
TASVd-P2-228
PCFVd-F
PCFVd-R
PCFVd-Probe
nad5 F
nad5 R
nad5-Probe
201a
201b
201d
320a
320b
320c
350a
350b
307i
279a
279b
279c
279d
280a
280b
280c
281a
281b
281c
349a
349b
349c
151a
151b
151d
1
GCCCCCTTTGCGCTGT
AAGCGGTTCTCGGGAGCTT
6FAM-CAGTTGTTTCCACCGGGTAGTAGCCGA-BHQ1
GCTCCGCTCCTTGTAGCTTT
AGGAGGTGGAGACCTCTTGG
Texas red-CTGACTCGAGGACGCGACCG-BHQ2
AAAAAAGAATTGCGGCCAAA
GCGACTCCTTCGCCAGTTC
6FAM-CCGGGGAAACCTGGA-NFQ-MGB
GGTTCACACCTGACCCTGCAG
AAACTCGTGGTTCCTGTGGTT
CGCTCGGTCTGAGTTGCC
6FAM-AGCGGTCTCAGGAGCCCCGG-BHQ1
CTCCACATCCGRTCGTCGCTGA
TGGGGTTGAAGCTTCAGTTGT
6FAM-CCCTCGCCCGGAGCTTCTCTCTG-BHQ1
CKGGTTTCCWTCCTCTCGC
CGGGTAGTCTCCAGAGAGAAG
6FAM-TCTTCGGCCCTCGCCCGR-BHQ
TCTTCTAAGGGTGCCTGTGG
GCTTGCTTCCCCTTTCTTTT
VIC-CTCCCCCGAAGCCCGCTTAG-BHQ1
GATGCTTCTTGGGGCTTCTTGTT
CTCCAGTCACCAACATTGGCATAA
Texas red-AGGATCCGCATAGCCCTCGATTTATGTG-BHQ2
1
1
2
2
2
3
3
4
5
5
5
5
6
6
6
7
7
7
8
8
8
9
9
9
1: Boonham et al. (2004), 2, 3, 8 and 9: R&D Naktuinbouw, 4: Botermans et al. (2011), 5: Monger et al. (2010), 6 and 7: FERA.
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Composition primer and probe mixes A, B and C (10 µM each)
351a
Pospi primermix A 201a, 201b, 349a, 349b, 320a, 320b
351b
Pospi primermix A 201d, 349c, 320c
352a
Pospi primermix B 280a, 280b, 279a, 279b, 279c, 320a, 320b
352b
Pospi primermix B 280c, 279d, 320c
353a
Pospi primermix C 350a, 350b, 151a, 151b
353b
Pospi primermix C 307i, 151d
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