Validation of a Nested PCR assay for detection of pv. in anthurium tissues in a European multicenter collaborative trial

(1)

Validation of a Nested PCR assay for detection of

Xanthomonas

axonopodis

pv.

dieffenbachiae

in anthurium tissues in

a European multicenter collaborative trial

X. a. pv. dieffenbachiae (Xad): a quarantine organism in Europe (EPPO A2 list)

Comparison of methods

1

•

st

step of the NF

EN ISO 16140:2003 european standard

(3)

N-PCR assay and EPPO reference method 2004

•

(4)

were more efficient (relative

ac-curacy>95%, detection threshold ≈ 10

3

CFU.mL

-1

) than DAS-ELISA and IF

 N-PCR

(1)

included in the EPPO diagnostic protocol revised in 2009

(2)

Collaborative trial

2

•

nd

step of

the

ISO 16140:2003 standard

project presented at the European Standing Committee on Plant Health

• objective: determine the variability of the results obtained by several

• labs and compare results with those obtained during the methods

comparative study

Two methods are particularly remarkable:N-PCR and reference methods

Protocol

Conclusions on the results of the collaborative trial

N-PCR and EPPO reference method 2004: excellent results for AC, SP and SE (≥95%)

• and no significant differences from the expected theoretical results and between labs

DAS-ELISA results explained by the lack of performance on the sample 10

•

4

CFU.mL

-1

IF results: an important variation between laboratories: only labs with IF experience for

• detection of Xad in anthurium obtained correct results

Conclusions on the whole validation study

N-PCR equivalent to the reference method. Its high specificity makes the

pathogeni-•

city tests to anthurium optional, drastically reducing the amount of time for result

deli-very (2 days vs. 10 days). ET medium replacable by NCTM4

(5)

for isolation step

I-ELISA not recommended for detection purpose

• DAS-ELISA not recommended for detection on asymptomatic samples and for

identifi-•

cation purposes within the framework of the EPPO scheme

IF requires appropriation by laboratories before routine analyses

•

Jouen E.1, Chabirand A.2, Robène-Soustrade I.1, Gagnevin L.1, Chiroleu F.1,

Saison A.2, Boyer C.1, Cassam N.2, Laurent A.1, Hostachy B.2,

Bergsma-Vla-mi M.3, Bianchi G.4, Cozzolino L.5, Cudejkova M.6, Elphinstone J.7,

Hole-va M.8, Manole F.9, Martini P.10, Minatchy J.11, Op de Beeck G.12, Sigillo L.13,

Siverio de la Rosa F.14, Soubelet H.15, Van Vaerenbergh J.16 and Pruvost O.1

Ref.

method N-PCR DAS-ELISA IF significant

differen-ce from the expected theoretical results§

NA _(p=0.15)no* _(p<0.001)yes** yes**_(p<0.05)

significant variation

between laboratories§§ _(p=1.00)no _(p>0.05)no*

yes** for sample

104 CFU.mL-1 (p<0.001)

yes** for all samples

(p-values <0.05 to <0.001)

AC : Relative accuracy

• - degree of correspondence between results obtained with a method and expected theoretical results

SE : Relative sensitivity

• - probability that a method gives a posi-tive result when the expected theoretical result is posiposi-tive

SP : Relative specificity

• - probability that a method gives a negative result when the expected theoretical result is nega-tive

CO : Concordance

• - probability of finding the same result for 2 identical samples analysed in 2 different labs (≈ reproducibility)

DA : Accordance

• - probability of finding the same result for 2 identical samples analysed in the same lab (≈ repeatability)

COR : Concordance odds ratio

• - ratio DAx(100-CO)/COx(100-DA) 60% 80% 100% AC SP SE DA CO 70% 95%

Revised EPPO flow diagram for the detection and identification of Xad in sympto-matic or asymptosympto-matic samples (2)_._{Main additions are highlighted in pink.}

plant sample

Rapid screening test molecular test (N-PCR) or serological test (DAS-ELISA or IF) pathogen extraction positive

Xad not detected

Isolation

Dilution plating on YPGA medium and on semi-selective medium:

NCTM4 ,CS or modified ET

colonies with typical or suspected morphology

after isolation

negative negative

Isolation

Identification tests on pure culture (perform two tests

with different biological principle): catabolic

biochemical tests/ELISA using monoclonal antibodies/IF/

N-PCR

positive

Xad identified

Isolation

Confirm pathogenicity by host test on Anthurium optional

no

yes

Statistical significance tests. NA: no test available, too few discordant results (<6) between the reference method and

the expected theoretical results. *: results obtained when laboratory N was excluded from the statistical analysis. This laboratory obtained half of the false positive results from the sample contaminated with the saprophytic strain. When this laboratory is kept in the analysis, there is a significant difference from the expected theoretical results (p=0.020) and a significant variation between laboratories for the sample contaminated with non-target organism (p=0.011) **: results obtained with ambiguous results analysed as theoretical expected results. §_{: results obtained using McNemar’s}

test (alpha=5%) when the total number of discordant results was ≥ 25 (DAS-ELISA and IF). When this number was < 25, results were obtained using a binomial distribution (alpha=5%) (PCR). §§_{: results obtained using the Fisher’s exact test}

(alpha=5%) on the Concordance Odds Ratio (COR).

References : (1) Robène-Soustrade I., et al. 2006. Appl. Environ. Microbiol. 72(2):1072-1078 ; (2) Anonymous. 2009. EPPO Bull. 39(3):246-253 ; (3) Anonymous. 2003. ISO 16140. AFNOR ; (4) Anonymous. 2004. EPPO Bull. 34:183-186 ; (5) Laurent P., et al. 2009. Letters Appl. Microbiol. 49(2):210-216

Evaluation of 5 statistical criteria on the results obtained with the 4 evaluated methods

EPPO reference method 2004

1. Isolation

on non selective (YPGA) and semi-selective (ET) media

2. identification Indirect ELISA (monoclonal serum Xcd108) optional methods DAS-ELISA (polyclonal serum) AND/OR ImmunoFluorescence (polyclonal serum) Nested PCR assay 1. DNA extraction

(Qiagen DNeasy Plant kit)

2. two PCR rounds 32 blinded subsamples

non-target

organism

target organism at 3

contamination levels

8 replicates 8 replicates 8 replicates 8 replicates

0 CFU.mL-1 104 CFU.mL-1 105 CFU.mL-1 107 CFU.mL-1

15 European participating laboratories

Organizing lab : 1 CIRAD UMR PVBMT and 2 LNPV, La Réunion, France 3 Plant Protection Service, Wageningen, The Netherlands

4 ERSA, Pozzuolo del Friuli, Italy

5 ArBoPaVe, University of Naples, Portici, Italy 6 SPA, Olomouc, Czech Republic

7 FERA, York, United Kingdom

8 Benaki Phytopathological Institute, Kifissia, Greece 9 CLPQ, Voluntari-City, Romania

10 IRF, Sanremo, Italy

11 FDGDON Réunion, La Réunion, France 12 LDA972, Martinique, France

13 ENSE, Battipaglia, Italy

14 Laboratorio de Sanidad Vegetal, Canary Islands, Spain 15 LNPV, Angers, France

Validation of a Nested PCR assay for detection of pv. in anthurium tissues in a European multicenter collaborative trial