Detection of PepMV and ringtest results

(1)

Detection of PepMV

and ringtest results

HJ (Joe) Vetten

(2)

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)

(3)

Ringtesting of methods

–

Assessment of available

methods

–

Selection of methods

•

ELISA, RT-PCR, TaqMan

–

Important for EPPO diagnostic

protocol

PEPEIRA:

(4)

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]

(5)

Detection methods

Which detection methods were considered for

the EPPO Diagnostic Protocol?

Bioassay on test plants

ringtest (all)

Lateral-flow immunoassay

Immunoelectron microscopy

DAS-ELISA

ringtest (all)

(conventional) RT-PCR

ringtest (14)

(6)

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.

(7)

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

(8)

Ringtest samples

(9)

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.

(10)

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:

Need for validated seed-testing methods

•

proof of PepMV absence in the area

(survey) or

•

in the seed crop

(inspection) or

(11)

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

(12)

Methods to be

ringtested

With reference to the ISF method:

ELISA

§

+ bioassay* followed by ELISA

RT-PCR

§

qRT-PCR

§

* each sub-sample to be inoculated on 2

N. benth./occid.

(13)

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

(14)

Genetic variability of

PepMV

Ch2

US1

Peru

EU (tomato)

recombinant isolate

Phylogenetic tree of complete genome (nt) sequences of

(15)

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)

(16)

Ringtest Samples

Spiking ratio*

Seeds contaminated

with PepMV strains

Clean

seeds

PepMV-infected

leaves

buffer

EU

Ch2 US1 Peru

1: 50 [5 + 245]

+

(4x)

+

1 : 250

+

1 : 1,250

+

1 : 6,250

+

•

8 x 250 x 2 (sets, replicates) = 4000 seeds [x ~20 labs]

•

Each lab: 20 x 2 (replicates) = 40 samples (+ controls)

Freshly harvested seeds contaminated with PepMV strains were

used for spiking of clean seeds

(17)

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

(18)

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

(19)

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 0.177 ? US1 0.488 Pos CH2 1.848 Pos

EU 0.199 ? US1 1.110 Pos CH2 2.490 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

(20)

Ringtest participants

majority of PEPEIRA consortium labs

(combinations of methods, e.g., ELISA

and qRT-PCR)

Seed industry (< 5 labs) invited to

participate

(21)

Ringtest results

Bioassay

ELISA

End-point (EP, conv.) RT-PCR

(22)

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

(23)

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

(24)

Three of the 20 labs had some clearly positive

bioassay results

Bioassay: Results

1

PepMV strain PO4 buffer clean seeds EU Ch2 US1 Peru 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

2

3

4

(25)

1 lab indicated to have 6 and 3 ’bioassay

positives’ for seed sets A and B, respectively

Bioassay: Results

Set A PepMV strain PO4 buffer clean seeds EU Ch2 US1 Peru 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 buffer clean seeds EU Ch2 US1 Peru 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

’

Bioassay positives‘

were clearly

ELISA-negative

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 pCtrl: 0.59 [2 h], Th.V.: 0.181 [2 h]

(26)

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

(27)

DAS-ELISA

René van der Vlugt

(28)

ELISA ringtest overview

•

18 labs participated

– 15 partners

– 3 additional labs (ISHI)

•

– 17 full sets (2 sets repeated)

(29)

ELISA: Comparison table

‘True’

+

-ELISA

+

939 (83.8%)

1 (0.4%)

-

181 (16.2%)

283 (99.6%)

1120

284

(30)

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

(31)

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?

(32)

End-point (EP) RT-PCR

(33)

Ringtest overview: EP RT-PCR

• 14 laboratories participated

13 PEPEIRA partners

1 seed company

•

13 full (2 sets – repeated a few days apart)

1 lab did not test the 1:50 dilutions of set B

(34)

• 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 )

(35)

EP RT-PCR: Comparison table

‘True’

+

-EP RT-PCR

+

283 (63.2%)

1 (0.9%)

-

165 (36.8%)

111 (99.1%)

448

112

(36)

PEPEIRA ring test

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

CH2 28 0 27 1 24 4 13 17

US1 27 1 26 2 12 16 6 22

Maximum number of samples per PepMV strain and dilution: 28

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

(37)

EP RT-PCR:

Two contrasting examples

H H US1 Peru B M

Seed batch A

PepMV strain clean seeds pos ctrl buffer EU Ch2 US1 Peru 0 +? 0 1:50 ++ +++ +++ ++ 1:250 0 0 ++ + 1:1250 +? + + 0 1:6250 0 0 0 0 Seed batch B

PepMV strain clean seeds pos ctrl buffer EU Ch2 US1 Peru 0 0 0 1:50 + +++ + + 1:250 0 +++ 0 +++ 1:1250 ++ ++ 0 0 1:6250 0 +? 0 0 Seed batch A

PepMV strain clean seeds pos ctrl buffer EU Ch2 US1 Peru 0 +++ 0 1:50 +++ +++ ++ ++ 1:250 +++ +++ ++ ++ 1:1250 +++ +++ ++ ++ 1:6250 ++ +++ ++ + Seed batch B

PepMV strain clean seeds pos ctrl buffer EU Ch2 US1 Peru 0 ++ 0 1:50 +++ +++ + + 1:250 +++ +++ + + 1:1250 ++ +++ + + 1:6250 + +++ + +?

Lab X

Lab Y

(38)

RT-PCR:

Two contrasting examples

M H H US1 Peru B M

(39)

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?])

•

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).

(40)

Real-time PCR

Rick Mumford

PEPEIRA ring test

(41)

Ringtest overview: TaqMan

•

11 laboratories participated

•

8 partners

•

3 additional seed companies

•

8 full (2 sets – repeated 4 days apart)

•

3 partial (1 set only)

(42)

• 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)

(43)

TaqMan: Comparison table

‘True’

+

-TaqMan

+

265 (87.2%)

2 (2.6%)

-

39 (12.8%)

74 (97.4%)

304

76

(44)

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)

•

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

(45)

Ringtest data

(46)

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)

(47)

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

(48)

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

recommended

•

Conventional RT-PCR