Detection of PepMV and ringtest results

Detection of PepMV and ringtest results HJ (Joe) Vetten (on behalf of the PEPEIRA consortium) JKI, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany www.jki.bund.de

Outline Ringtest preparations What type of diagnostic methods (or tests)? Which samples? Ringtest results Bioassay ELISA End-point (EP, conv.) RT-PCR qrt-pcr (TaqMan) Conclusions

PEPEIRA: Work package 4 Ringtesting of methods Assessment of available methods Selection of methods ELISA, RT-PCR, TaqMan Important for EPPO diagnostic protocol

Objectives of the ring (or method performance) tests To evaluate some detection methods in various labs robustness of the tests To provide information on their reliability [sensitivity (incl. risks of false negatives), specificity (incl. risks of false positives) and reproducibility] To obtain validated methods for the DP

Detection methods Which detection methods were considered for the EPPO Diagnostic Protocol? Bioassay on test plants Lateral-flow immunoassay Immunoelectron microscopy DAS-ELISA ringtest (all) ringtest (all) (conventional) RT-PCR ringtest (14) Real-time (q) RT-PCR ringtest (11)

Lateral-flow immunoassay (LFIA) LFIAs (or ImmunoStrips) are commercially available and intended for rapid (within minutes) detection of pathogens LFIA can be performed by unskilled operators and is recommended for on-site detection of PepMV in leaf and fruit tissue.

Immunoelectron microscopy (IEM) requires considerable expertise and access to a transmission EM For a small number of samples it can be done in a short period of time (< 1 h). Since not only the virus particles but also the reaction of the antibodies with the virions are visible, it can be an accurate means of diagnosis. recommended as a confirmatory test Adsorption Trapping Decoration

Ringtest samples Leaf tissue or seeds?

2004/200/EC: Commission Decision of 27 Feb 2004 PepMV is not only on the EPPO Alert List but also - because of 2004/200/EC - a regulated pest on tomato seeds (only) in EU 2004/200/EC: Introduction into, and movement within, the EU of tomato seeds contaminated with PepMV is prohibited.

2004/200/EC: Commission Decision of 27 Feb 2004 Within-EU movement - [and introduction into the EU] - of tomato seeds is only allowed when they have been obtained by an appropriate acid extraction method and meet one of three further requirements: proof of PepMV absence in the area (survey) or in the seed crop (inspection) or on the seeds (lab tests) Need for validated seed-testing methods

Current seed testing method (ISF) Principle: Detection of infectious PepMV on seeds Minimum sample size per seed lot : 3000 seeds (12 x 250 seeds) Grinding of sub-samples for preparing seed extracts Mechanical inoculation of N. benthamiana followed (two weeks later) by ELISA of the inoculated test plants Optional: Pre-screen seed extracts by ELISA - If ELISA is negative, test is finished; - If sub-samples are ELISA-positive, conduct bioassay: inoculate them onto 2 N. benthamiana followed by ELISA

Methods to be ringtested With reference to the ISF method: ELISA RT-PCR qrt-pcr + bioassay* followed by ELISA + bioassay* followed by ELISA + bioassay* followed by ELISA the same number and types of samples to be analysed * each sub-sample to be inoculated on 2 N. benth./occid.

Method development DAS-ELISA: already established (selecting a good AS!) (conventional) RT-PCR: has been developed [Ling et al. 2008] qrt-pcr: has been developed [Ling et al. 2007]

Genetic variability of PepMV Phylogenetic tree of complete genome (nt) sequences of 13 PepMV isolates EU (tomato) Peru US1 Ch2 recombinant isolate

Ringtest Samples Tomato seeds contaminated with the individual PepMV strains were produced just in time for the ringtests to obtain freshly harvested (and pectinex treated) seeds (provided by Martijn Schenk, Bleiswijk, NL) ~100,000 clean seeds were kindly supplied by Bert Woudt, Syngenta Seeds (checked by qrt-pcr at FERA, UK)

Ringtest Samples Freshly harvested seeds contaminated with PepMV strains were used for spiking of clean seeds Spiking ratio* Seeds contaminated with PepMV strains EU Ch2 US1 Peru Clean seeds PepMVinfected leaves buffer 1: 50 [5 + 245] + + + + 1 : 250 + + + + 1 : 1,250 + + + + 1 : 6,250 + + + + + (4x) + + 8 x 250 x 2 (sets, replicates) = 4000 seeds [x ~20 labs] Each lab: 20 x 2 (replicates) = 40 samples (+ controls)

Preparation of seed extracts Overnight soaking of seeds in extraction buffer Grinding of seeds using a homogenizer (hand model) RNA extraction kit: RNeasy Plant Mini Kit (Qiagen)

Method performance tests (= ringtests) Different steps 1. Draft final test protocol(s) 2. Design test samples 3. Stability of test material 4. Homogeneity testing of test material 5. Design spread sheets for collecting data 6. Pre-trial in >3 labs 7. Ringtest with all available laboratories 8. Data analysis

Homogeneity of test material Isolate ELISA value PepMV pos/neg Isolate ELISA value PepMV pos/neg Isolate ELISA value PepMV pos/neg EU 0.228 pos US1 0.242 pos CH2 2.252 Pos EU 0.230 pos US1 0.246 pos CH2 2.488 Pos EU 0.794 pos US1 0.405 Pos CH2 1.201 Pos EU 0.272 pos US1 0.401 Pos CH2 0.823 Pos EU 0.585 pos US1 1.044 Pos CH2 2.362 Pos EU 0.358 pos US1 1.101 Pos CH2 2.122 Pos EU 0.177? US1 0.488 Pos CH2 1.848 Pos EU 0.199? US1 1.110 Pos CH2 2.490 Pos EU 0.236 pos US1 0.511 Pos CH2 2.150 Pos EU 0.195? US1 0.623 Pos CH2 1.925 Pos Clean seeds: 0.143, 0.149, 0.150 & 0.157 Buffer control: 0.133 & 0.139

Ringtest participants majority of PEPEIRA consortium labs (combinations of methods, e.g., ELISA and qrt-pcr) Seed industry (< 5 labs) invited to participate Total: ~20 labs

Ringtest results Bioassay ELISA End-point (EP, conv.) RT-PCR qrt-pcr (TaqMan)

Bioassay: overview 20 laboratories participated 17 PEPEIRA partners 3 seed companies 22 data sets in total 18 from labs summarizing the results for the 2 sets supplied 4 (2 labs detailing the results for each set)

Bioassay: Overall results 15 of the 20 labs had clearly negative bioassay results (but 1 lab did not do ELISA on test plants inoculated with seed extracts diluted higher than 1:50) Only 3 labs had clearly positive bioassay results 1 lab apparently had a contamination problem (or mixed up samples) 1 lab had bioassay positives based on questionable ELISA reactions

Bioassay: Results Three of the 20 labs had some clearly positive bioassay results 1 PepMV strain PO4 clean EU Ch2 US1 Peru buffer seeds 0 0 1: 50 2 0 0 0 1: 250 2 0 0 0 1:1250 2 0 0 0 1:6250 1 0 0 0 3 PepMV strain PO4 clean EU Ch2 US1 Peru buffer seeds 0 0 1: 50 0 0 0 1 1: 250 0 0 0 0 1:1250 0 0 0 0 1:6250 0 0 0 0 2 PepMV strain PO4 clean EU Ch2 US1 Peru buffer seeds 0 0 1: 50 0 2 0 0 1: 250 0 2 0 0 1:1250 0 0 0 0 1:6250 0 0 0 0 4 PepMV strain PO4 clean EU Ch2 US1 Peru buffer seeds 0 1 1: 50 0 0 0 0 1: 250 0 0 0 0 1:1250 0 0 0 1 1:6250 0 0 0 0

Bioassay: Results 1 lab indicated to have 6 and 3 bioassay positives for seed sets A and B, respectively Set A PepMV strain PO4 clean EU Ch2 US1 Peru buffer seeds 0 0 1: 50 0 1 1 0 1: 250 0 1 1 0 1:1250 0 0 0 0 1:6250 0 1 1 0 Set B PepMV strain PO4 clean EU Ch2 US1 Peru buffer seeds 0 0 1: 50 1 0 0 0 1: 250 1 0 0 0 1:1250 0 0 0 1 1:6250 0 0 0 0 0.100 + 0.205 0.142 + 0.189 0.110 + 0.132 0.120 + 0.206 0.216 + 0.153 0.178 + 0.164 0.130 + 0.128 0.198 + 0.121 Bioassay positives were clearly ELISAnegative pctrl: 0.59 [2 h], Th.V.: 0.181 [2 h]

Bioassay Results A total of ~1680 test plants (N. occidentalis 37B [or N. benthamiana]) were inoculated: only 12 plants (0.7%) were clearly bioassay-positive i.e., 3 of 160 PepMV-contaminated seed samples were shown to contain infectious PepMV False positives Only 1 sample in 1 of 20 labs (contamination?) False negatives Real false negatives not possible? Or Are the majority of samples false negatives?

PEPEIRA ring test DAS-ELISA René van der Vlugt

ELISA ringtest overview 18 labs participated 15 partners 3 additional labs (ISHI) 35 data sets in total 17 full sets (2 sets repeated) 1 partial (not tested in duplicate)

ELISA: Comparison table True + - + 939 (83.8%) 1 (0.4%) ELISA - 181 (16.2%) 283 (99.6%) 1120 284

ELISA: Overall results All 4 PepMV strains readily detected All four concentrations detected % pos 1: 50 1: 250 1:1,250 1:6,250 EU 97 94 77 56 Ch2 100 97 96 86 US1 97 94 80 57 Peru 94 87 84 44

ELISA: Unmatched results False positives One out of 18 labs Healthy control values were higher than those of the positive control supplied False negatives Real false negatives not possible? Some labs had problems with overall sensitivity

PEPEIRA ring test End-point (EP) RT-PCR

Ringtest overview: EP RT-PCR 14 laboratories participated 13 PEPEIRA partners 1 seed company 28 data sets in total 13 full (2 sets repeated a few days apart) 1 lab did not test the 1:50 dilutions of set B

EP RT-PCR: Overall results All 4 strains were readily detected (by most labs) All 4 concentrations were detected (by only few labs) The two highest dilutions were often not detected No internal control was used Did extraction procedure perform well? (in COX assay (TaqMan): OK )

EP RT-PCR: Comparison table True + - EP RT-PCR + 283 (63.2%) 1 (0.9%) - 165 (36.8%) 111 (99.1%) 448 112

PEPEIRA ring test EU Ch2 US1 Peru 1: 50 55,0 84,0 71,0 55,5 1: 250 47,0 73,0 49,0 39,0 1:1250 27,0 54,5 24,0 19,0 1:6250 14,0 34,5 12,0 7,5 143,0 246,0 156,0 121,0 1: 50 1:250 1:1250 1:6250 pos neg pos neg pos neg pos neg EU 25 3 17 11 14 14 7 21 Maximum number of samples per PepMV strain and dilution: 28 CH2 28 0 27 1 24 4 13 17 US1 27 1 26 2 12 16 6 22 Peru 25 3 17 11 11 17 5 23

EP RT-PCR: Two contrasting examples Lab X Seed batch A PepMV strain EU Ch2 US1 Peru 1:50 ++ +++ +++ ++ 1:250 0 0 ++ + 1:1250 +? + + 0 1:6250 0 0 0 0 clean pos buffer seeds ctrl 0 +? 0 Seed batch B PepMV strain clean pos buffer EU Ch2 US1 Peru seeds ctrl 0 0 0 1:50 + +++ + + 1:250 0 +++ 0 +++ 1:1250 ++ ++ 0 0 1:6250 0 +? 0 0 Lab Y Seed batch A PepMV strain clean EU Ch2 US1 Peru seeds pos ctrl buffer 0 +++ 0 1:50 +++ +++ ++ ++ 1:250 +++ +++ ++ ++ 1:1250 +++ +++ ++ ++ 1:6250 ++ +++ ++ + H H US1 Peru B M Seed batch B PepMV strain EU Ch2 US1 Peru 1:50 +++ +++ + + 1:250 +++ +++ + + 1:1250 ++ +++ + + 1:6250 + +++ + +? clean seeds pos ctrl buffer 0 ++ 0

RT-PCR: Two contrasting examples M H H EU Ch2 PC M M H H US1 Peru B M

EP RT-PCR: Unmatched Results False positives 1 lab in total: Only 1 positive (out of 112 healthy samples; 0.9%) (suspect this was accidental sample switch [or contamination?]) False negatives 7 of 14 labs in total: 7 labs had issue with overall sensitivity (failed to detect most strains at higher dilutions, and positive control provided was not [or only weakly] detected). 1 lab failed to detect any US1 positives in set B (but 2 in set A)

PEPEIRA ring test Real-time PCR Rick Mumford

Ringtest overview: TaqMan 11 laboratories participated 8 partners 3 additional seed companies 19 data sets in total 8 full (2 sets repeated 4 days apart) 3 partial (1 set only)

TaqMan: Overall results All 4 strains were readily detected All 4 concentrations were readily detected Extraction procedure performed extremely well No failures seen (when assessed using COX assay)

TaqMan: Comparison table True + - TaqMan + 265 (87.2%) 2 (2.6%) - 39 (12.8%) 74 (97.4%) 304 76

TaqMan: Unmatched Results False positives 2 labs in total: Only 2 positives (out of 76 healthy samples; 2.6%) Strongly suspect 1 was accidental sample switch (adjacent sample number was false negative: all other results were correct) False negatives 4 labs in total: 1 lab had issue with overall sensitivity (detection of all strains but not at higher dilutions). Peruvian was worst (1:50 only) 1 lab failed to detect any Ch2 or Peruvian positives 1 lab failed to detect any Peruvian positives 1 lab only detected one Peruvian 1:50 positive (out of 8)

Ringtest data Conclusion

Comparison of ELISA and RT-PCR data True (in %) + ELISA EP RT- PCR TaqMan ELISA EP RT- PCR TaqMan + 83.8 63.2 87.2 0.4 0.9 2.6 16.2 36.8 12.8 99.6 99.1 97.4 (1120) (448) (304) (284) (112) (76)

Conclusions ELISA and qrt-pcr are very sensitive and robust tests and can be recommended for PepMV detection on seeds (and in plants) Also EP RT-PCR (using the primers of Ling et al. 2008) is potentially sensitive and robust [but why did 7 of the 14 labs encounter problems with EP RT-PCR???]; currently not recommended Bioassay appears to be an insensitive method for detection of [infectious] PepMV on seeds, Because of the seed transmission risk and EU decision 2004/200/EC the PEPEIRA consortium feels the bioassay cannot be recommended for seed testing

EPPO Diagnostic Protocol (DP) A first draft of a DP for PepMV is available and will soon be submitted to EPPO Tests included in the EPPO DP: Lateral-flow immunoassay Immunoelectron microscopy DAS-ELISA Conventional RT-PCR Real-time (q) RT-PCR recommended recommended