INTERLABORATORY COMPARISON EU-XF-IC-2020-03 Evaluation …

EU-XF-IC-2020-03 v2

REPORT

INTERLABORATORY COMPARISON EU-XF-IC-2020-03

Evaluation of molecular methods for the detection of Xylella fastidiosa

September – November 2020

This report was prepared in January 2021 analyzing the qPCR results received by 23 Participant

Laboratories: https://www.xfactorsproject.eu/research-results-xylella-fastidiosa/

Authors:

Maria Saponari, IPSP-CNR

Giuliana Loconsole, IPSP-CNR

https://www.xfactorsproject.eu/research-results-xylella-fastidiosa/

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Contents LIST OF ABBREVIATIONS AND TERMS USED IN THIS REPORT .......................................................................... 4

1. GENERAL INFORMATION .............................................................................................................................. 5

1.1 BACKGROUND ......................................................................................................................................... 5

1.2 ORGANIZERS ............................................................................................................................................ 6

1.3 PARTICIPATING LABORATORIES .............................................................................................................. 6

1.4 DOCUMENTS AND INSTRUCTIONS .......................................................................................................... 7

1.5 TIMELINE OF THE TEST PERFORMANCE STUDY ....................................................................................... 7

2. PANEL OF EXPERIMENTAL SAMPLES ............................................................................................................ 7

3. DIAGNOSTIC PROCEDURES ...................................................................................................................... 9

3.1 DIAGNOSTIC PROCEDURES EVALUATED IN FRAMEWORK OF THE TPS ................................................... 9

3.2 DIAGNOSTIC PROCEDURES EVALUATED IN FRAMEWORK OF THE PT ................................................... 10

4. ANALYSIS OF THE RESULTS ......................................................................................................................... 13

4.1 TEST PERFORMANCE STUDY .................................................................................................................. 13

4.2. PROFICIENCY TEST ................................................................................................................................ 15

4.2.1 CATEGORIZATION OF THE LABORATORIES BASED ON THEIR PERFORMANCE IN THE PT .................. 15

5. RESULTS ................................................................................................................................................... 16

5.1 HOMOGENEITY AND STABILITY ASSESSMENT ....................................................................................... 16

5.2 TEST PERFORMANCE STUDY ON PLANT SAMPLES: QUALITATIVE RESULTS .......................................... 22

5.2.1 DIAGNOSTIC AND ANALYTICAL SENSITIVITY ...................................................................................... 22

5.2.2 DIAGNOSTIC SPECIFICITY .................................................................................................................... 23

5.2.3 ACCURACY .......................................................................................................................................... 25

5.2.4 REPEATABILITY AND REPRODUCIBILITY .............................................................................................. 26

5.2.5 TEST PERFORMANCE STUDY ON PLANT SAMPLES: QUANTITATIVE RESULTS .................................... 27

5.3 TEST PERFORMANCE STUDY ON INSECT SAMPLES: QUALITATIVE RESULTS ......................................... 29

5.3.1 DIAGNOSTIC AND ANALYTICAL SENSITIVITY ...................................................................................... 29

5.3.2 DIAGNOSTIC SPECIFICITY .................................................................................................................... 30

5.3.3 ACCURACY .......................................................................................................................................... 31

5.3.4 REPEATABILITY AND REPRODUCIBILITY .............................................................................................. 32

5.3.5 TPS ON INSECT SAMPLES: QUANTITATIVE RESULTS........................................................................... 33

5.4 RESULTS OF THE PROFICIENCY TEST ...................................................................................................... 35

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6. CONCLUSIONS ......................................................................................................................................... 38

6.1 TEST PERFORMANCE STUDY .................................................................................................................. 39

6.2 PROFICIENCY TEST ................................................................................................................................. 41

7. REFERENCES ................................................................................................................................................ 42

8. DIAGOSTIC PROTOCOLS .............................................................................................................................. 43

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LIST OF ABBREVIATIONS AND TERMS USED IN THIS REPORT

Term Meaning

CTAB CTAB DNA extraction protocol [EPPO PM7/24 (4 )

Mericon Modified DNeasy MericonTM Food Standard Protocol (Qiagen) [EPPO PM7/24(4 )

Maxwell RSC Maxwell® RSC PureFood GMO and Authentication Kit

DSP Diagnostic specificity

DSN Diagnostic sensitivity

PT Proficiency test

TPS Test performance study

Cq Quantification cycle

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1. GENERAL INFORMATION

1.1 BACKGROUND

This interlaboratory comparison (EU-XF-IC-2020-03) has been organized in the framework of the

activities related to the experimental plan foreseen in WP4/WP9 of the Horizon 2020 project “XF-

ACTORS – 727987”, and follows the previous European proficiency testing EU-XF-PT-2017-02 carried

out in 2017.

This interlaboratory comparison was developed for the identification of X. fastidiosa in plants and

insects by comparing different procedures of DNA extractions followed by qPCR using the

primers/TaqMan probe described by Harper et al. (2010), and had two main scopes:

- A test performance study (TPS) to assess the performance of a fully automatized DNA extraction

protocol by Promega (Maxwell® RSC PureFood GMO and Authentication Kit AS1600 - based on

magnetic beads and without chloroform treatment) in comparison with the procedures previously

validated (CTAB and Modified DNeasy MericonTM Food Standard Protocol - Qiagen) and described

in the EPPO diagnostic standard 7/24 (4). The TPS was performed on plant and insect samples.

- A laboratory proficiency test (PT) to assess the efficiency of different laboratories performing

molecular detection of X. fastidiosa.

The study was organized in accordance with the EPPO 7/122 guidelines and the following

performance criteria were analyzed:

Diagnostic sensitivity: proportion of positive samples giving a positive result.

Diagnostic specificity: proportion of negative samples giving a negative result.

Accuracy: the accuracy is the closeness of agreement between a test result and the accepted reference value.

Repeatability (or accordance): level of agreement between replicates of a sample tested under the same conditions.

Reproducibility (only for TPS): ability of a test to provide consistent results when applied to aliquots of the same sample tested under different conditions (time, persons, equipment, location, etc.).

Limit of detection (analytical sensitivity): Smallest amount of target that can be detected reliably in matrix.

The diagnostic procedures tested in the different laboratories are reported in the Annex I-IV. The samples

were processed either manually or using automatized dedicated platforms.

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1.2 ORGANIZERS

This study has been conceived and organized by the Institute for Sustainable Plant Protection, CNR,

in the framework of the project XF-ACTORS.

1.3 PARTICIPATING LABORATORIES Participant laboratories included national reference laboratories and expert laboratories, most of which

were proficient during the previous intervalidation test PT EU-XF-PT-2017-02. Laboratories are listed below

with an alphanumeric code assigned, to ensure results confidentiality.

INSTITUTION COUNTRY INSTITUTION COUNTRY

AGES - Austrian Agency for Health and Food Safety

AUSTRIA Unisalento - Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento

ITALY

HCPHS - Croatian Centre for Agriculture, Food and Rural Affairs

CROATIA Fondazione Minoprio- Laboratorio Servizio Fitosanitario Regione Lombardia

ITALY

CISTA - Central Institute for Supervising and Testing in Agriculture

CZECH REPUBLIC

Servizio Fitosanitario Regione Toscana

ITALY

Anses - Laboratoire de la santé des végétaux

FRANCE Regione Veneto - Unità Organizzativa Fitosanitaria

ITALY

JKI - Julius Kuehn Institute GERMANY IPSP-CNR – Institute for Sustainable

Plant Protection

ITALY

BPI - Benaki Phytopathological Institute

GREECE IVIA - Instituto Valenciano de Investigaciones Agrarias

SPAIN

DAFM- Plant Health Laboratory, Department of Agriculture, Food and the Marine

IRELAND CSIC - Institute for Sustainable Agriculture

SPAIN

CIHEAM-IAMB Istituto Agronomico Mediterraneo -Bari

ITALY LOSVIB - Laboratorio Oficial de Sanidad Vegetal de las Islas Baleares

SPAIN

CREA-PAV - Consiglio per la ricerca e la sperimentazione in Agricoltura, Centro di ricerca per la Patologia Vegetale

ITALY LAPHC- Laboratory for Agriculture and Plant Health of Catalonia

SPAIN

CRSFA - Centro di Ricerca, Sperimentazione e Formazione in Agricoltura “Basile Caramia”

ITALY AGAPA - Agencia de Gestión Agraria y Pesquera de Andalucía Laboratorio de Produccion y Sanidad Vegetal de Almeria

SPAIN

SAFE - Dipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, Università di Foggia

ITALY AGROSCOPE

SWIZTERLAND

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AGRIS- Sardegna Servizio Ricerca studi ambientali, difesa delle colture e qualità delle produzioni -LABORATORIO FITOPATOLOGICO REGIONALE Azienda sperimentale San Michele - Ussana (CA)

ITALY

1.4 DOCUMENTS AND INSTRUCTIONS Participants received the following documents containing the information for the contract and the

instructions describing the protocols and the panel of samples to be used for each diagnostic procedures

tested:

- Announcement of the EU-XF-IC-2020-03;

- Participant contract;

- Instruction and protocols;

- Excel files with spreadsheet to register and report the results.

1.5 TIMELINE The panels of samples distributed to the expert laboratories and used for homogeneity and stability tests

were prepared during the last week of August and early September 2020 (24 August-4 September).

The homogeneity tests for all diagnostic protocols tested in this interlaboratory comparison were performed

(on september 2-4) immediately after preparing the different batches of spiked samples and prior to ship the

samples on 7-8 September, 2020.

Participants were requested to perform the diagnostic tests and send the results to the organizers by

October 9, 2020. This was then extended to the end of October. Consequently, the stability tests were

performed on November 2-3, 2020.

Each laboratory registered the results in the excel template provided by the organizers. For each sample, the

DNA yield (concentration ng/µl) and the values of the quantification cycle (Cq) were registered along with

the status assigned to each samples (positive/negative/undetermined).

This final report has been completed and submitted in January 2021.

2. PANEL OF EXPERIMENTAL SAMPLES The panel of samples consisted of crude sap prepared from leaf petioles of Polygala mirtifolia and from

specimens of Philaenus spumarius macerated using the extraction buffers selected based on the different

extraction protocols evaluated. The panel of samples included: randomized Xylella-free preparations and

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samples spiked with heat-inactivated (incubation at 70°C for 15min) bacterial suspensions at different

concentrations (CFU/ml), prepared by scraping 10-days old colonies of X. fastidiosa subsp. pauca strain De

Donno (CFBP 8402). The stock bacterial suspension consisted of a PBS-suspension with an OD600 value of 0.16,

corresponding based on previous experiments (with this strain and using BCYE agar medium) to 10^8 CFU/ml.

From this suspension, 10-fold serial dilutions were prepared by diluting it in plant sap or insect homogenate.

The samples did not contain living bacteria and did not pose any danger to humans, animals, plants or

environment.

The samples provided to each laboratory included:

1) LABORATORY PARTICIPATING TO THE TPS:

Set (A): 22 plant samples (from ID 1 to ID 22) for each DNA extraction procedure selected, consisting of: - 3 replicates for each 10-fold serial dilutions from 10^6 to 10 CFU/ml of bacterial suspension spiked in plant sap from healthy Poligala myrtifolia; - 3 replicates of plant sap from healthy P. myrtifolia; - 1 lure sample - 1 PAC Set (B) 21 insect samples (from ID 23 to ID 43) to be processed using CTAB and ‘Maxwell (PROMEGA)’ DNA extraction procedures: - 3 replicates for each 10-fold serial dilutions from 10^6 to 10 CFU/ml of bacterial suspension spiked in Xylella-free P. spumarius homogenate; - 3 replicates of Xylella-free P. spumarius. 2) LABORATORY PARTICIPATING TO THE PT: A total of 13 samples (from ID 44 to ID 56) for each method of DNA extraction selected, consisting of: - 3 replicates for each 10-fold serial dilution from 10^6 to 10^4 CFU/ml of bacterial suspension spiked in plant sap from healthy Poligala myrtifolia; - 3 replicates of plant sap from healthy P. myrtifolia; - 1 lure sample; - 1 tube of Positive Amplification Control (PAC) for qPCR assay. Each sample was identified by an alphanumeric code: - a number associated randomly to the different samples.

- a letter: the letter associated to each number indicates the protocol, i.e. C stands for “CTAB”,

M stands for “Mericon food kit Qiagen”, P stands for “Maxwell Promega”, Q stands for Quick pick, R stands for Roche

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3. DIAGNOSTIC PROCEDURES

3.1 DIAGNOSTIC PROCEDURES EVALUATED IN FRAMEWORK OF THE TPS

The 16 expert laboratories participating to the TPS (L01 - L16) tested the following DNA extraction

procedures:

DNA extraction from plant sap:

- CTAB DNA extraction protocol [EPPO PM7/24 (4 ) Appendix 3]

- Modified DNeasy MericonTM Food Standard Protocol (Qiagen) [EPPO PM7/24(4 ) Appendix 3],

manual or using the dedicated Qiacube platform.

- Maxwell® RSC PureFood GMO and Authentication Kit AS1600 – (Promega) using the dedicated

Maxwell® RSC Instruments.

DNA extraction from insect preparations:

- CTAB DNA extraction protocol [EPPO PM7/24(4 ) Appendix 3]

- Maxwell® RSC PureFood GMO and Authentication Kit AS1600 – (Promega) by the Maxwell® RSC

Instruments.

The recovered total DNA extracts were then subjected to real time qPCR (Harper et al., 2010 - erratum 2013)

using different thermocycler instruments (Table 1) and following the amplification conditions (reagents and

cycles) recommended in EPPO, 2019, Diagnostic protocol PM 7/24 (4) Xylella fastidiosa Appendix 5 – Real-

time PCR (Harper et al., 2010 - erratum 2013).

Fourteen out of 16 laboratories compared all three extraction protocols proposed for this TPS, whereas the

remaining laboratories compared only two protocols: laboratory L06 evaluated CTAB and Maxwell RSC;

laboratory L07 used Mericon and Maxwell RSC.

L02, L07 and L15 perfomed the extraction with Mericon using the Qiacube platform. The other laboratories

processed the samples manually.

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Table 1. Thermocycler instruments used by the expert laboratories participating to the TPS.

Lab. Code

Thermocycler Lab. Code Thermocycler

L01 StepOnePlus Real-Time PCR System

(Applied Biosystems) L09 CFX96 (BIORAD)

L02 CFX96 (BIORAD) L10 CFX96 Touch™ Real-Time PCR (BIORAD)

L03 LightCycler480

(Roche) L11 CFX96 (BIORAD)

L04 LightCycler480

(Roche) L12 CFX96 (BIORAD)

L05 Brand: Analytik Jena Model: Qtower 3G

L13 QuantStudio3

(Applied Biosystems)

L06 QuantStudio5

(Applied Biosystems) L14

StepOne Plus (Applied Biosystems)

L07 StepOne Plus

(Applied Biosystems) L15 CFX96 (BORAD)

L08 CFX96 (BIORAD) L16 CFX96 Touch

Detection system (BIORAD)

3.2 DIAGNOSTIC PROCEDURES EVALUATED IN FRAMEWORK OF THE PT

A total of 14 laboratories participated to the PT (L04, L06, L12 and from L17 to L23). Participant

laboratories were asked to use the diagnostic method(s) of their choice and no further requirements

were imposed regarding the protocols.

The DNA extraction protocols used by each laboratory and the information on the thermocycler and qPCR

master mix routinely used are reported in Table 2. L01, L02, L04, L06, L12, L14 and L16 participated to both

TPS and PT. L01, L02, L14 and L16 used the same protocols and conditions for PT and TPS.

Regarding the Mericon DNA extraction, all lab performed it manually, only L02 and L22 used the Qiacube

platform (Qiagen).

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Table 2. Laboratories participating to the PT with the specifications of the DNA extraction procedure

performed, the type of thermocycler and qPCR master mix used.

Lab. Code

CTAB MERICON (QIAGEN)

MAXWELL RSC

AS1600 (PROMEGA)

QUICK PICK (BIONOBILE) + Kingfisher Flex

(Thermo fisher)

ROCHE (High pure

PCR template

kit

innuPREP Plant

(Analytik Jena AG)

Thermocycler qPCR master mix

L01 X X CFX96 (BIORAD) TaqMan™ Fast Universal PCR

Master Mix (AB) (Cod.)

4352042

L02 X X X CFX96 (BIORAD) TaqMan™ Fast Universal PCR


4352042

L04 X X X LightCycler480 (Roche)

GoTaq(R) Probe qPCR (Promega)

L06 X* QuantStudio5 (Applied

Biosystems)

TaqMan™ Fast Universal PCR

Master Mix (Applied

Biosystems)

L12 X X X CFX96 (BIORAD) BIORAD

L14 X StepOne Plus (Applied

Biosystem)



4352042

L16 X X CFX96 Touch Detection system

(BIORAD)



4352042

L17 X X X Realplex 4S Mastercycler

Gradient (Eppendorf)

Quanta Tough Mix

L18 X X Mx3000P Real-time PCR, Agilent;

MxPro ET qPCR Software

Taqman Universal PCR

Master Mix (Applied

Biosystems, 4304437)

L19 X X StepOnePlus (Applied

Biosystems)

TaqMan Universal PCR

Master Mix (Applied

Biosystems)

L20 X** X C1000 Touch thermal cycler / Bloc CFX96 Real

TaqMan™ Fast universal PCR Master Mix

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optics module, BIORAD

(Applied Biosystems)

L21 X BioRad CFX96 Touch

GoTaq Probe qPCR (Promega)

L22 X X Biorad CFX-96



4352042

L23 X X GoTaq Probe qPCR (Promega)

QuantStudio3 (Applied

biosystems)

* L06 used for DNA extraction Maxwell® RSC Cultured Cells DNA Kit by Promega, cat. number AS1620.

** L20 performed Maxwell® RSC PureFood GMO and Authentication Kit – (Promega) AS1600 on Kingfisher Flex (Thermo fisher).

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4. ANALYSIS OF THE RESULTS

4.1 TEST PERFORMANCE STUDY Results were analyzed based on the qualitative (negative, positive, undertermined) and quantitative

data (Cq values). Even if the panel of Xylella-contaminated samples tested contained from 10^6 to 10 CFU/ml.

Because of inconsistent results generated during the homogenicity and stability tests for the replicates

containing 10 CFU/ml, the analyses performed for all performance criteria were limited to the results

recorded for the samples containing from 10^6 to 10^2 CFU/ml.

In each laboratory, samples were categorized as negative, positive or undetermined, according to the yielded

Cq values. For each laboratory, the Organizers determined the number of positive agreements (PA), negative

agreements (NA), positives deviations (PD) and negatives deviations (ND) according to the parameters

described in Table 3. The recorded values were then used to calculate the performance criteria (Table 4) for

each protocol.

It is worth noting that because each laboratory to interpret the status of the samples used different cut-off

values, in some cases (mostly for the most diluted samples yielding high Cq values) the Organizers, based on

the Cq values, revised the assessment made by each laboratory in order to harmonize the interpretation of

the results following the criteria summarized in Table 5).

The performance was expressed as percentage, with 100% being the highest performance level (see for more

information Chabirand et al., 2014; OEPP/EPPO PM 7/122 (1), 2014).

Table 3. Definition of the parameters adapted from ISO 16140

Laboratory Results Assigned value

Positive Negative

Positive PA= positive agreement PD= positive deviation

Negative ND= negative deviation NA= negative agreement

Undetermined ND= negative deviation PD=positive deviation

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Table 4. Details on the performance criteria (Chabirand et al., 2014; OEPP/EPPO PM 7/122 (1), 2014)

Performance criteria Definition Calculation

Accuracy (AC) Closeness of agreement between the

laboratory result and the assigned value

AC= (NPA+NNA)/N

Sensitivity (SE) Closeness of agreement between the


for samples for which the assigned value

is positive

SE= NPA/N+

Specificity (SP) Closeness of agreement between the


for samples for which the assigned value

is negative

SP=NNA/N-

Repeatibility (DA) Closeness of agreement between

independent test results obtained under

conditions of repeatability, i.e.

conditions under which independent

test results are obtained by the same

method, on identical test samples in the

same laboratory, by the same operator,

using the same equipment, within a

short period of time

DA denotes the percentage

chance of obtaining the same

result (positive, negative or

indeterminate) from two

identical samples analyzed in

the same laboratory

Reproducibility as the ability of a test to provide

consistent results when applied to

aliquots of the same sample tested

under different conditions (time,

persons, equipment, location, etc)

based on the number of

interlaboratory pairs of same

results/total number of

Interlaboratory pairs

Analytical sensitivity Smallest amount of target that can be detected reliably in plant and insect matrix

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Table 5. Rules followed to harmonize the interpretation of the results related to the samples yielding high

values of quantification cycle (Cq)

Results gathered for each sample Final assessment

Well 1 Well 2

Cq > 35 Cq > 35 Sample was assigned as positive as long as: - the three replicates tested were within 3 cycles from the previous serial dilution, and - no amplification was observed in the no template control (NTC) and Xylella-free replicates. Sample was assigned as undetermined if the conditions

described above were not met.

Cq > 35 N/A Sample was assigned as negative

4.2. PROFICIENCY TEST Results were analyzed based on the qualitative diagnostic value assigned to each sample (negative, positive

or undetermined) by the participant laboratories. Using these data, the Organizers determined for each

method the number of positive agreements (PA), negative agreements (NA), positives deviations (PD) and

negatives deviations (ND) according to the parameters described in Table 3. The recorded values were then

used to calculate the performance criteria (Table 4), except that the reproducibility.

The proficiency of a given laboratory was then expressed as percentage, with 100% being the highest

performance level (see Chabirand et al., 2014 for more detailed information).

4.2.1 CATEGORIZATION OF THE LABORATORIES BASED ON THEIR

PERFORMANCE IN THE PT Based on the values of accuracy generated for each protocols tested, the laboratories were categorized as:

(i) “highly proficient” when the level of accuracy corresponded to the highest value (i.e. 100%); this was

the case of the laboratories in which all the samples produced the expected positive and negative

results, without any positive deviation (false positive) or negative deviation (false negative). In addition,

the three replicates of each sample produced identical results.

(ii) “proficient” when the level of accuracy was in the range of 90-100%. This category included

the laboratories that obtained either one positive deviation or one negative deviation.

(iii) “non-proficient” when the level of accuracy was lower than 90%. This category included the

laboratories that obtained more than one positive or negative deviation.

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5. RESULTS The analytical results collected from each laboratory are available at the following link

https://www.xfactorsproject.eu/research-results-xylella-fastidiosa/

5.1 HOMOGENEITY AND STABILITY ASSESSMENT The homogeneity and the stability of the panel of samples used for this interlaboratory validation

was assessed for all diagnostic procedures using the whole set of sample distributed (i.e. the panel of plant

and insect samples containing from 10^6 CFU/ml to 10 CFU/ml).

Homogeneity tests were performed before shipping the samples, while stability tests were

performed once all laboratories had completed their tests.

Values of the Cq yielded during the homogeneity and stability tests are reported in Table 6, along

with the average Cq values yielded by the participant laboratories for each given sample.

As expected, the Cq values showed a direct correlation with the bacterial concentrations. The values

of the standard deviation (SD) and ∆Cq between the homogeneity and stability tests were in the acceptable

range of variations for the samples containing from 10^6 CFU/ml to 10^2 CFU/ml of bacterial suspension,

regardless the DNA extraction procedure used. These replicates were considered to be sufficiently

homogenous and stable (Table 6-11) and thus suitable to be used for the validation purposes. On the

contrary, qPCR reactions on the samples containing 10 CFU/ml of X. fastidiosa cells, yielded inconsistent

results between the replicates and/or Cq values very close to those recorded for the previous dilution (10^2

CFU/ml), i.e. between the two dilutions the ∆Cq was much less than 3 as expected. Due to these results,

these samples were considered not sufficiently homogenous (not conform), and thus excluded from all the

evaluations made in this interlaboratory validation.

The conformity (yes/no) of the samples for the homogeneity and stability is reported in Table (7-11).

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Table 6. Quantitation cycle (Cq) yielded for each extraction procedure in qPCR assays. Values include the

average of the overall Cq obtained by the participating laboratories to the TPS and the Cq values recovered

for the homogeneity and stability tests. SD indicates the standard deviation among the replicates of the same

sample.

Concentration of Xylella fastidiosa in the samples

10^6 CFU/ml 10^5 CFU/ml 10^4 CFU/ml 10^3 CFU/ml 10^2 CFU/ml 10 CFU/ml

CTAB - plant samples

Average from laboratories

20.27 23.98 27.71 30.90 34.35 -

SD 1.53 1.73 1.49 1.21 1.41 -

Homogeneity 20.36 25.42 28.47 31.72 35.03 -

SD 1.18 0.37 0.73 0.67 0.92 -

Stability 21.54 24.92 29.08 31.09 34.82 -

SD 0.90 1.46 1.48 1.66 0.80 -

Mericon - plant samples


21.85 25.20 28.72 32.02 35.23 -

SD 1.26 1.35 1.28 1.17 1.72 -

Homogeneity 22.02 25.47 29.11 32.27 34.86 -

SD 0.38 0.15 0.37 0.41 0.36

Stability 22.56 26.08 29.10 32.62 35.53 -

SD 0.50 0.37 0.27 0.48 1.30 -

Maxwell RSC - plant samples


21.47 24.96 28.29 31.68 35.09 -

SD 1.27 1.23 1.29 1.36 1.50 -

Homogeneity 22.24 25.25 28.33 31.99 35.79 -

SD 0.21 0.16 0.16 0.25 0.96 -

Stability 22.12 25.54 28.95 31.83 35.37 -

SD 0.34 0.34 0.13 0.31 0.47 -

CTAB – insects


19.65 22.89 26.27 29.94 32.83 -

SD 1.38 1.73 1.68 1.96 1.75 -

Homogeneity 21.31 24.56 27.76 31.12 34.36 -

SD 0.68 0.31 0.59 0.45 0.63 -

Stability 20.15 24.78 29.19 30.49 33.19 -

SD 2.22 1.31 0.68 0.74 1.06 -

Maxwell RSC – insects


20.93 24.29 27.59 31.00 34.52 -

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SD 1.20 1.24 1.39 1.36 1.17 -

Homogeneity 19.51 22.73 26.25 30.29 33.34 -

SD 0.33 0.25 0.45 1.03 0.80 -

Stability 20.77 24.23 27.36 30.34 33.77 -

SD 0.06 0.23 0.16 0.15 0.51 -

Table 7. Results of the real time PCR assays (Cq values) performed on the plant samples for the homogeneity

tests. The conformity (yes/no) of each sample is defined based on the quantitative and qualitative results of

the homogeneity tests.


CTAB-plant


Replicate 1

repetition 1

18.21 25.02 27.60 31.91 35.81 38.1

Replicate 2 19.89 25.10 27.66 32.40 34.38 37.72

Replicate 3 20.73 25.80 28.31 30.91 33.97 n/a

Replicate 1

repetition 2

20.67 25.33 29.34 30.86 35.55 n/a

Replicate 2 21.35 25.94 28.96 32.12 36.16 n/a

Replicate 3 21.31 25.34 28.95 32.12 34.30 38.15

Quantitative homogeneity Yes Yes Yes yes yes no

Qualitative homogeneity Yes Yes Yes yes yes no

Mericon -plant


Replicate 1

repetition 1

21.97 25.25 29.35 31.48 34.79 n/a

Replicate 2 22.00 25.53 29.11 32.69 34.69 37.15

Replicate 3 21.51 25.40 29.29 32.36 34.29 n/a

Replicate 1

repetition 2

22.35 25.70 28.95 32.44 34.89 n/a

Replicate 2 21.74 25.41 28.46 32.27 35.34 n/a

Replicate 3 22.54 25.51 29.48 32.36 35.13 n/a



Maxwell RSC-plant


Replicate 1

repetition 1

22.39 25.17 28.53 31.76 36.73 36.72

Replicate 2 22.45 25.56 28.22 32.01 36.43 35.56

Replicate 3 21.94 25.12 28.12 32.48 34.17 n/a

Replicate 1 repetition 2

22.29 25.18 28.23 31.95 35.55 36.14

Replicate 2 22.35 25.27 28.35 31.90 35.35 n/a

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Replicate 3 22.03 25.17 28.50 31.85 36.48 36.57



Table 8. Results of the real time PCR assays (Cq values) performed on the insect samples for the homogeneity

tests. The conformity (yes/no) of each sample is defined based on the quantitative and qualitative results of

the homogeneity tests.


CTAB-insect


Replicate 1

repetition 1

20.46 22.49 26.71 30.01 33.02 35.74

Replicate 2 19.62 22.65 26.16 31.80 32.59 34.85

Replicate 3 20.11 22.55 25.57 29.37 32.37 n/a

Replicate 1

repetition 2

20.52 22.72 26.73 30.06 33.53 35.40

Replicate 2 19.29 22.76 26.40 31.26 33.06 35.38

Replicate 3 19.79 23.20 25.95 29.25 32.66 n/a

Quantitative homogeneity yes Yes Yes yes yes no

Qualitative homogeneity yes Yes Yes yes yes no

Maxwell RSC-insect


Replicate 1

repetition 1

20.54 24.45 28.55 31.48 33.86 35.68

Replicate 2 21.31 24.84 27.12 30.62 35.30 37.12

Replicate 3 22.55 24.05 27.18 30.80 34.57 36.82

Replicate 1

repetition 2

21.42 24.80 28.32 31.82 33.49 n/a

Replicate 2 20.96 24.81 27.57 30.94 34.59 36.92

Replicate 3 21.08 24.43 27.80 31.03 34.34 n/a

Quantitative homogeneity yes yes Yes yes yes no

Qualitative homogeneity yes yes Yes yes yes no

20 EU-XF-IC-2020-03 v2

Table 9. Results of the real time PCR assays (Cq values) performed on the plant samples for the stability tests.

The conformity (yes/no) of each sample is defined based on the quantitative and qualitative results of the

stability tests.


CTAB-plant


Replicate 1

repetition 1

21.87 26.25 30.52 33.76 35.20 n/a

Replicate 2 20.72 23.43 26.41 28.77 33.71 36.19

Replicate 3 21.56 25.02 29.49 31.29 34.00 36.14

Replicate 1

repetition 2

20.34 23.12 28.46 30.19 35.54 35.74

Replicate 2 22.83 26.77 30.13 31.64 35.60 37.31

Replicate 3 21.90 24.93 29.46 30.88 34.85 n/a

Quantitative stability yes yes Yes yes yes no

Qualitative stability yes yes Yes yes yes no

Mericon -plant


Replicate 1

repetition 1

22.47 26.08 29.21 33.06 37.32 n/a

Replicate 2 22.96 26.07 29.13 32.22 34.57 n/a

Replicate 3 22.58 26.44 29.53 33.23 36.90 36.77

Replicate 1

repetition 2

22.55 26.03 28.84 32.82 35.46 n/a

Replicate 2 21.67 26.44 28.81 32.32 34.79 36.65

Replicate 3 23.12 25.44 29.06 32.09 34.13 n/a

Quantitative stability yes yes yes yes yes no

Qualitative stability yes yes yes yes yes no

Maxwell RSC -plant


Replicate 1

repetition 1

21.47 26.15 29.08 32.01 36.06 37.15

Replicate 2 22.44 25.55 28.86 31.77 35.14 37.12

Replicate 3 22.18 25.27 29.11 32.23 35.53 37.27

Replicate 1

repetition 2

22.10 25.67 28.87 31.41 35.64 36.35

Replicate 2 22.33 25.27 28.80 31.99 35.09 37.16

Replicate 3 22.21 25.30 28.97 31.56 34.75 37.22

Quantitative stability yes yes yes yes yes no

Qualitative stability yes yes yes yes yes no

21 EU-XF-IC-2020-03 v2

Table 11. Results of the real time PCR assays (Cq values) performed on insect samples for the stability tests.

The conformity (yes/no) of each sample is defined based on the quantitative and qualitative results of the

stability tests.


CTAB-insect


Replicate 1

repetition 1

18.69 24.58 28.95 30.30 33.33 n/a

Replicate 2 18.47 22.78 28.61 30.20 32.89 36.04

Replicate 3 21.91 25.69 29.12 29.36 31.62 33.78

Replicate 1

repetition 2

18.96 25.29 29.33 30.87 32.67 n/a

Replicate 2 19.01 23.91 28.69 30.63 33.88 35.05

Replicate 3 23.88 26.43 30.46 31.57 34.72 34.35

Quantitative stability yes yes yes Yes yes no

Qualitative stability yes yes yes Yes yes no

Maxwell RSC-insect


Replicate 1

repetition 1

20.85 24.26 27.47 30.58 34.25 36.30

Replicate 2 20.77 24.02 27.55 30.66 34.22 30.46

Replicate 3 20.81 24.63 27.14 30.16 33.16 37.05

Replicate 1

repetition 2

20.78 24.25 27.42 30.31 33.22 37.30

Replicate 2 20.77 23.98 27.35 30.20 34.17 37.04

Replicate 3 20.66 24.24 27.20 30.36 33.57 36.00

Quantitative stability yes yes yes Yes yes no

Qualitative stability yes yes yes Yes yes no

22 EU-XF-IC-2020-03 v2

5.2 TEST PERFORMANCE STUDY ON PLANT SAMPLES: QUALITATIVE

RESULTS

The results of the TPS showed that the bacterium was correctly identified in the total DNA extracts

recovered from the plant samples containing from 10^6 CFU/ml to 10^2 CFU/ml, regardless the extraction

procedure. Conversely, the detection failed or produced inconsistent results for the plant samples containing

a bacterial concentration of 10 CFU/ml. This is in agreement with the results of the homogeneity and stability

tests (table 6). Thus, for the evaluation of the performance criteria, 15 expected positive samples (N+) were

considered, while the results of the replicates containing 10 CFU/ml were not used for the analyses.

5.2.1 DIAGNOSTIC AND ANALYTICAL SENSITIVITY

The diagnostic sensitivity calculated based on the panel of 15 N+ samples for the three DNA

extraction procedures (Table 12) was higher than 98%, reaching values of 98,67% for CTAB, of 99,17 for the

Maxwell RSC, and 99,56% when the DNA was extracted using the Mericon kit. These percentages represent

the average values calculated from the results reported by 16 laboratories participating to the TPS and

comparing the three extractions procedures (with the exception of L06 and L07 that compared only 2

methods). However, it is worth noting that the diagnostic sensitivity for all three DNA extraction methods

was 100% in almost all laboratory: 13 out 15 laboratories for CTAB; 14 out 15 for Mericon; 15 out 16 for

Maxwell RSC. The diagnostic sensitivity recorded in the remaining laboratories was affected by the

occurrence of false negatives among the replicates tested (negative deviation – ND), more precisely:

(i) Diagnostic sensitivity was 93,33% for CTAB in laboratory L06. In this laboratory, the bacterium was

not correctly detected in one of the replicates containing 10^4 CFU/ml (i.e. 1 ND). This sample yielded

a very low value of DNA concentration (ng/µl) indicating that most likely a technical problem

occurred during the extraction steps.

(ii) Diagnostic sensitivity was 86,67% for CTAB in laboratory L14. In this laboratory, the bacterium was

not correctly detected in two replicates containing 10^2 CFU/ml (i.e. 2 ND); both replicates generated

Cq values higher than 37,50 and thus categorized as negative.

(iii) Diagnostic sensitivity was 93,33% for Mericon in laboratory L09. In this laboratory, the bacterium

was not correctly detected in one of the replicates containing 10^3 CFU/ml (i.e. 1 ND).

23 EU-XF-IC-2020-03 v2

(iv) Diagnostic sensitivity was 86,67% for the Maxwell RSC in laboratory L15. In this laboratory, the

bacterium was not correctly detected in two replicates containing 10^2 CFU/ml (i.e. 2 ND).

Given that the samples containing the lowest bacterial concentration (10 CFU/ml) were excluded from the

analysis, the analytical sensitivity (the lowest detectable concentration of X. fastidiosa) was assessed to be

10^2 CFU/ml for all three extraction procedures.

Table 12. Diagnostic sensitivity (%) of the real time PCR (Harper et al., 2020) performed on the DNA extracted using

CTAB, Mericon and Maxwell RSC protocols, and calculated based on the positive agreement (PA) refered to 15 expected

positive samples (N+).

Laboratory Code

CTAB MERICON Maxwell RSC

DSN(%) N. PA/N+ DSN(%) N. PA/N+ DSN(%) N. PA/N+

01 100.00 15/15 100.00 15/15 100.00 15/15

02 100.00 15/15 100.00 15/15 100.00 15/15

03 100.00 15/15 100.00 15/15 100.00 15/15

04 100.00 15/15 100.00 15/15 100.00 15/15 05 100.00 15/15 100.00 15/15 100.00 15/15 06 93.33 14/15 - - 100.00 15/15 07 - - 100.00 14/14 100.00 15/15 08 100.00 15/15 100.00 15/15 100.00 15/15 09 100.00 15/15 93.33 14/15 100.00 15/15 10 100.00 15/15 100.00 15/15 100.00 15/15 11 100.00 15/15 100.00 15/15 100.00 15/15 12 100.00 15/15 100.00 15/15 100.00 15/15 13 100.00 15/15 100.00 15/15 100.00 15/15 14 86.67 13/15 100.00 15/15 100.00 15/15 15 100 15/15 100.00 15/15 86.67 13/15 16 100 15/15 100.00 15/15 100.00 15/15

Average values 98.67% 99.56% 99.17%

5.2.2 DIAGNOSTIC SPECIFICITY The diagnostic specificity of the qPCR assays performed on the DNA extracted using the three

extraction procedures was 100% in almost all laboratories. For all three methods, only in one

laboratory the values were lower than 100% (Table 13). More specifically, exceptions were

encountered in the laboratories L04, L13 and L16. In the first two laboratories, one positive

deviation (false positive replicate) occurred for the samples tested using the Mericon kit and CTAB

24 EU-XF-IC-2020-03 v2

protocol, respectively. Whereas, two positive deviations were recorded in L16 within the panel of

samples processed using the Maxwell RSC.

Overall, the average diagnostic specificity recorded among the 16 participating laboratories was

97,78 for CTAB and Mericon and 95,83 for Maxwell RSC.

Table 13. Diagnostic specificity (%) generated by CTAB, Mericon and Maxwell RSC followed by qPCR (Harper

et al., 2020) and based on number of negative agreement (NA) calculated on rules listed in table 6,

considering 3 expected positive samples.

Laboratory Code

CTAB MERICON Maxwell RSC DSP(%) DSP(%) DSP(%)

01 100.00 100.00 100.00

02 100.00 100.00 100.00

03 100.00 100.00 100.00

04 100.00 66.77 100.00 05 100.00 100.00 100.00 06 100.00 - 100.00 07 - 100.00 100.00 08 100.00 100.00 100.00 09 100.00 100.00 100.00 10 100.00 100.00 100.00 11 100.00 100.00 100.00 12 100.00 100.00 100.00 13 66.67 100.00 100.00 14 100.00 100.00 100.00 15 100.00 100.00 100.00 16 100.00 100.00 33.33

Total 97.78% 97.78% 95.83%

25 EU-XF-IC-2020-03 v2

5.2.3 ACCURACY

The accuracy calculated based on the diagnostic sensitivity and specificity for each extraction protocol

corresponded to the maximum value - 100% - in most of the laboratories (Table 14), with few exceptions (as

detailed before in 5.2.1 and 5.2.2). Overall, all three extraction procedures showed an accuracy ranging from

98,52% for CTAB, to 98,61% for Maxwell RSC and 99,26% for Mericon.

Table 14. Accuracy (%) generated by CTAB, Mericon and Maxwell RSC followed by qPCR (Harper et al., 2020)

and based on diagnostic sensitivity and specificity values.

Laboratory Code


Accuracy (%) Accuracy (%) Accuracy (%)

01 100.00 100.00 100.00

02 100.00 100.00 100.00

03 100.00 100.00 100.00

04 100.00 94.44 100.00

05 100.00 100.00 100.00

06 94.44 - 100.00

07 - 100.00 100.00

08 100.00 100.00 100.00

09 100.00 94.44 100.00

10 100.00 100.00 100.00

11 100.00 100.00 100.00

12 100.00 100.00 100.00

13 94.44 100.00 100.00

14 88.89 100.00 100.00

15 100.00 100.00 88.89

16 100.00 100.00 88.89

Total 98.52% 99.26% 98.61%

26 EU-XF-IC-2020-03 v2

5.2.4 REPEATABILITY AND REPRODUCIBILITY

The repeatability (intra-laboratory variability) of the qPCR reactions performed using the DNA extracted with

the three methods was 100% in almost all laboratories (Table 15). The average value of repeatability assessed

for CTAB was 97.78%, 98.52% for Mericon and 98.61% for Maxwell RSC.

Similarly, the values of reproducibility (number of interlaboratory pairs gathering the same results on the

total number of interlaboratory pairs) corresponded to 98,52% for CTAB, 99,30 for Mericon and 98,68%

for Maxwell RSC.

Table 15. Repeatability (%) generated by CTAB, Mericon and Maxwell RSC followed by qPCR (Harper et al.,

2020) and based on obtaining the same result (positive, negative/undetermined) from two identical samples

analysed in the same laboratory.

Laboratory Code


Repeatability (%) Repeatability (%) Repeatability (%)

01 100.00 100.00 100.00

02 100.00 100.00 100.00

03 100.00 100.00 100.00

04 100.00 88.89 100.00

05 100.00 100.00 100.00

06 88.89 - 100.00

07 - 100.00 100.00

08 100.00 100.00 100.00

09 100.00 88.89 100.00

10 100.00 100.00 100.00

11 100.00 100.00 100.00

12 100.00 100.00 100.00

13 88.89 100.00 100.00

14 88.89 100.00 100.00

15 100.00 100.00 88.89

16 100.00 100.00 88.89

Total 97.78% 98.52% 98.61%

27 EU-XF-IC-2020-03 v2

5.2.5 TEST PERFORMANCE STUDY ON PLANT SAMPLES: QUANTITATIVE

RESULTS

This section includes the comparative analysis of the values of the quantitation cycles (Cq) generated in the

qPCR assays performed on the DNA extracted using the three tested procedures. The average Cq values

obtained from the series of samples processed by each DNA extraction procedure are graphically shown in

Figure 1.

The qPCR efficiencies were determined using the slope of the standard curve generated for each DNA

extraction procedure by the panel of samples containing from 10^6 CFU/ml to 10^2 CFU/ml.

The slopes of the linear regression, that measure the assay’s efficiency, were -3.508 for CTAB

protocol, -3.353 for Mericon and -3.396 for Maxwell RSC, corresponding to the optimal qPCR efficiency values

respectively of 93%, 98% and 97% (Table 16). The standard curves had correlation coefficients (R2 value)

comprised between 0.9991 and 1, regardless the procedures used to prepare the DNA templates.

Regardless the method used for the purification of the DNA, similar Cq values were generated for the

samples with the same bacterial content. However, CTAB extraction procedure generated Cq values generally

slightly lower (∆Cq= ~ 1) than Mericon and Maxwell RSC kits for each dilution, with a maximum ΔCq of 1.58

for the samples containing 10^6 CFU/ml. Similar Cq values were recovered from the qPCR assays carried out

on the DNA extracted with Maxwell RSC and Mericon kits.

Table 16. Efficiency of the qPCR assays in relation to the procedure used to recover the DNA

qPCR (Harper et al., 2010)

DNA extraction procedures Linear regression and R2 values Efficiency (%)

CTAB y = 3.508x + 16.918 R² = 0.9991

93%

Mericon y = 3.358x + 18.53 R² = 0,9997

98%

Maxwell RSC y = y = 3,396x + 18,11 R² = 1

97%

28 EU-XF-IC-2020-03 v2

Figure 1. Standard curves represented as linear regression of the quantitation cycle (Cq) values (Y axis) versus the concentration of the spiked samples (X axis). Different colors indicate the Cq generated using CTAB, Mericon and the Maxwell RSC.

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

10^6 cfu/ml 10^5 cfu/ml 10^4 cfu/ml 10^3 cfu/ml 10^2 cfu/ml

Cq

Val

ue

s

CFU/ml

qPCR assays using plant samples

Promega

Mericon

CTAB

Maxwell RSC Mericon CTAB

29 EU-XF-IC-2020-03 v2

5.3 TEST PERFORMANCE STUDY ON INSECT SAMPLES: QUALITATIVE

RESULTS Similarly, to the results gathered for the plant samples, none of the replicates containing 10 CFU/ml gave

consistent, homogeneous and stable results. Thus, the replicates containing the lowest amount of bacterial

cells were all excluded from the analyses of the performance criteria, with 15 expected positive samples (N+)

used as reference number.

5.3.1 DIAGNOSTIC AND ANALYTICAL SENSITIVITY

The diagnostic sensitivity reached, for both DNA extraction procedures, values of 100% in most of the

participating laboratories (Table 17), i.e. all Xylella-positive samples were correctly identified. In few cases

for both extraction procedures, the diagnostic sensitivity was lower, more specifically:

- qPCR assays on the DNA extracted with CTAB generated values of 60% in L16 (due to 4 negative deviations

produced for all replicates containing 10^5 CFU/ml and one replicate containing 10^2 CFU/ml), 80% in L05

(the bacterium was not detected in the 3 replicates containing 10^2 CFU/ml), 86.67% in L09 (the bacterium

was not detected in one of the replicates containing 10^3 CFU/ml and 10^2 CFU/ml);

- qPCR assays using the DNA extracted with the Maxwell RSC kit generated values of 93,33% in L07, L09 and

L15 due to the occurrence of one negative deviation in the replicates containing 10^2 CFU/ml (L07 and L15)

and 10^3 CFU/ml (L09).

Overall, based on the values generated by all laboratories, the average value of the diagnostic sensitivity of

the qPCR assays performed on the DNA extracted with the Maxwell RSC kit was 98,75%, while it was 95,11%

for the qPCR assays performed on the DNA extracted using CTAB.

As for the plant samples, since all laboratories failed to detect the bacterium in the replicates containing 10

CFU/ml, the analytical sensitivity of the qPCR assays set up with the insect DNA extracted using both

procedures was assessed to be 10^2 CFU/ml.

30 EU-XF-IC-2020-03 v2

Table 17. Diagnostic sensitivity (%) generated by CTAB and Maxwell RSC followed by qPCR (Harper et al.,

2020) and based on number of positive agreement (PA) calculated on rules listed in table 6, considering 15

expected positive samples (N+).

Laboratory Code

CTAB Maxwell RSC

DSN(%) N. PA/N+ DSN(%) N. PA/N+

01 100.00 15/15 100.00 15/15

02 100.00 15/15 100.00 15/15

03 100.00 15/15 100.00 15/15

04 100.00 15/15 100.00 15/15 05 80.00 12/15 100.00 15/15 06 100.00 15/15 100.00 15/15

07 - - 93.33 14/15 08 100.00 15/15 100.00 15/15 09 86.67 13/15 93.33 14/15 10 100.00 15/15 100.00 15/15

11 100.00 15/15 100.00 15/15 12 100.00 15/15 100.00 15/15

13 100.00 15/15 100.00 15/15 14 100.00 13/15 100.00 15/15 15 100.00 15/15 93.33 14/15

16 60.00 9/15 100.00 15/15

Total 95.11% - 98.75%

5.3.2 DIAGNOSTIC SPECIFICITY The diagnostic specificity of the qPCR assays set up with the DNA extracted using the two tested

procedures yielded values of 100% in most laboratories (Table 18). However, in 3 laboratories (L01,

L12 and L13) false positive reactions were recorded for one or two Xylella-free replicates using the

CTAB-extracted DNA. As such the diagnostic specificity of the qPCR assays performed in these

laboratories was as low as 33,33% (in the case of two negative deviations) and 66.67% (in the case

of one negative deviation).

When the qPCR assays were performed using the DNA recovered from Maxwell RSC kit, only one

negative deviation occurred in one laboratory (L01), lowering the diagnostic specificity reached in

this lab to 66,67%.

The average values (from all laboratories) gathered for the two DNA extraction procedures were

31 EU-XF-IC-2020-03 v2

88.89% and 97.92% for CTAB and Maxwell RSC, respectively.

Table 18. Diagnostic specificity (%) generated by CTAB and Maxwell RSC followed by qPCR (Harper et al.,

2020) and based on number of negative agreement (PA) calculated on rules listed in table 6, considering 3

expected positive samples.

Laboratory Code

CTAB Maxwell RSC DSP(%) DSP(%)

01 33.33 66.67

02 100.00 100.00

03 100.00 100.00

04 100.00 100.00 05 100.00 100.00 06 100.00 100.00 07 - 100.00 08 100.00 100.00 09 100.00 100.00 10 100.00 100.00 11 100.00 100.00 12 66.67 100.00 13 33.33 100.00 14 100.00 100.00 15 100.00 100.00 16 100.00 100.00

Total 88.89% 97.92%

5.3.3 ACCURACY Based on the values of diagnostic sensitivity and specificity, the accuracy of the qPCR assays set up using the

DNA extracted using the two methods compared in this validation was equal to 100% in most laboratories

(Table 19). However, the occurrence of positive and negative deviations in some laboratories determined an

average accuracy value of 94.07% for CTAB and 98.61% for Maxwell RSC.

32 EU-XF-IC-2020-03 v2

Table 19. Accuracy (%) generated by CTAB, and Maxwell RSC followed by qPCR (Harper et al., 2020) and

based on diagnostic sensitivity and specificity values.

Laboratory Code

CTAB Maxwell AS100 (Promega)

Accuracy (%) Accuracy (%)

01 88.89 94.44

02 100.00 100.00

03 100.00 100.00

04 100.00 100.00

05 83.33 100.00

06 100.00 100.00

07 - 94.44

08 100.00 100.00

09 88.89 94.44

10 100.00 100.00

11 100.00 100.00

12 94.44 100.00

13 88.89 100.00

14 100.00 100.00

15 100.00 94.44

16 66.67 100.00

Total 94.07% 98.61%

5.3.4 REPEATABILITY AND REPRODUCIBILITY

The repeatability of the qPCR assays reached values of 100% using the DNA extracted with both procedures

in most of the laboratories (Table 20), with few exceptions. The repeatability average values were 96.30 %

and 97.22% for CTAB and for Maxwell RSC, respectively.

With regard to the reproducibility, based on the number of interlaboratory pairs with the same results on

total number of interlaboratory pairs, a value of 94.07 % and 98.61% were generated respectively for CTAB

and Maxwell RSC.

33 EU-XF-IC-2020-03 v2

Table 20. Repeatability (%) generated by CTAB and Maxwell RSC followed by qPCR (Harper et al., 2020) and

based on obtaining the same result (positive, negative/undetermined) from two identical samples analysed

in the same laboratory.

Laboratory Code

CTAB Maxwell RSC

Repeatability (%) Repeatability (%)

01 88.89 88.89

02 100.00 100.00

03 100.00 100.00

04 100.00 100.00

05 100.00 100.00

06 100.00 100.00

07 100.00 88.89

08 100.00 100.00

09 77.78 88.89

10 100.00 100.00

11 100.00 100.00

12 88.89 100.00

13 88.89 100.00

14 100.00 100.00

15 100.00 88.89

16 100.00 100.00

Total 96.30% 97.22%

5.3.5 TPS ON INSECT SAMPLES: QUANTITATIVE RESULTS This section includes the comparative analysis of the Cq values generated in the qPCR assays performed on

the DNA extracted from the insects samples using the two procedures evaluated. The Cq values obtained

from the series of samples processed by each one of the two DNA extraction procedures are graphically

shown in Figure 2.

The qPCR efficiencies were determined using the slope of the standard curve generated for each

method of DNA extraction by panel of samples containing from 10^6 CFU/ml to 10^2 CFU/ml.

The slopes of the linear regression, that measure the assay’s efficiency, were -3.3422 for CTAB

protocol and -3.3895 for Maxwell RSC extraction, corresponding to the optimal qPCR efficiency values of 99%

and 97%, respectively (Table 21). The standard curves had an R2 correlation coefficient of 0.9989 (CTAB) and

0.9999 (Maxwell RSC).

34 EU-XF-IC-2020-03 v2

The Cq values generated in the qPCR assays performed using the DNA extracted using CTAB were

slightly lower than those generated using the DNA recovered from the Maxwell RSC kit, with ΔCq in the range

of 1.06-1.69.

Table 21. Efficiency of the qPCR assays in relation to the procedure used to recover the DNA

qPCR by Harper et al., 2010

DNA extraction procedures Linear regression and R2 values Efficiency (%)

CTAB y = 3.3422x + 16.291 R² = 0.9989

99.16 %

Maxwell RSC y = 3.3895x + 17.499 R² = 0.9999

97.26 %

Figure 2. Standard curves represented as linear regression of the quantitation cycle (Cq) values (Y axis) versus the concentration of the spiked samples (X axis). Different colors indicate the Cq generated using CTAB and Maxwell RSC extraction procedures.

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

10^6 cfu/ml 10^5 cfu/ml 10^4 cfu/ml 10^3 cfu/ml 10^2 cfu/ml

Cq

Val

ue

s

CFU/ml

qPCR assays using insect samples

Promega

CTAB

Maxwell RSC CTAB

35 EU-XF-IC-2020-03 v2

5.4 RESULTS OF THE PROFICIENCY TEST The laboratories participating to the PT performed their qPCR assays on plant samples containing the

bacterium from 10^6 to 10^4 CFU/ml and on Xylella-free replicates. In all laboratories, at these

concentrations, all samples were correctly identified as positive samples, regardless the procedure used to

recover the DNA and regardless the qPCR master mixes used to set up the qPCR reactions (Table 2). For

laboratories L01, L02, L14 and L16 the results on plant samples from 10^6 to 10^4 CFU/ml and on Xylella-

free replicates were extrapolated from the TPS and used to calculate the performance criteria for the PT.

None of the laboratory produced negative or positive deviations, reaching values of 100% for all performance

criteria (diagnostic sensitivity, diagnostic specificity, accuracy and repeatability) (Table 22), thus all resulting

“highly proficient” for the identification of X. fastidiosa in plant samples.

However, as expected some differences were recorded for the Cq values generated by the different qPCR

assays. In Figure 3 are graphically shown the Cq values obtained in each laboratory for the different DNA

extracts. As shown, the range of Cq values recovered for the Xylella-contaminated samples, varied in the

different laboratories, as a consequence of the (i) different quantity/quality of the DNA extracts recovered

using the different extraction protocols, and/or (ii) reagents and master mixes used.

For example, L18 consistently yielded Cq values higher than the average Cq values recorded in the other

laboratories, regardless the DNA extraction procedure, indicating that most likely the qPCR master mix and

amplification conditions used in this laboratory had a lower efficiency.

Indeed, at a given concentration of the bacterium in the samples, differences were detected in Cq values

generated in the same laboratory (i.e. L17, L19) using the different extraction procedures tested [Roche,

Innuprep, AS120 (Promega) and AS100 (Promega) processed on kingfisher], suggesting that the method of

extraction affected the quantity and quality of the recovered DNA.

Another example is L04, in this laboratory the Cq values generated were consistently lower than those

obtained in other laboratories using the same methods of extraction, indicating that the qPCR master mix

and the amplification conditions used in this laboratory were highly efficient.

36 EU-XF-IC-2020-03 v2

Table 22. Values of the performance criteria generated by the laboratories participating to the PT for the

different DNA extraction procedures performed.

Lab. Code Extraction Procedure Diagnostic sensitivity

Diagnostic specificity

Accuracy Repeatability

L01

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%

L02

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%

Maxwell RSC AS1600 (PROMEGA)

100% 100% 100% 100%

L04

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%


100% 100% 100% 100%

L06 Maxwell® RSC Cultured Cells DNA (PROMEGA)

(AS1620) 100% 100% 100% 100%

L12

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%


100% 100% 100% 100%

L14 Maxwell RSC AS1600

(PROMEGA) 100% 100% 100% 100%

L16

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%

L17

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%

innuPREP Plant (Analytik Jena AG)

100% 100% 100% 100%

L18

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%

L19 MERICON (QIAGEN)

100% 100% 100% 100%

37 EU-XF-IC-2020-03 v2

ROCHE (High pure PCR template kit)

100% 100% 100% 100%

L20

Quick pick (BIONOBILE)

100% 100% 100% 100%

Maxwell RSC AS1600 (Promega) + kingfisher

(Thermofisher) 100% 100% 100% 100%

L21 CTAB 100% 100% 100% 100%

L22

CTAB 100% 100% 100% 100%

MERICON (QIAGEN)

100% 100% 100% 100%

L23

CTAB 100% 100% 100% 100%


100% 100% 100% 100%

38 EU-XF-IC-2020-03 v2

Figure 3. Range of the values of the quantitation cycle (Cq) recovered in each laboratory on the DNA extracts

prepared from the samples containing 10^6, 10^5 and 10^4 CFU/ml, using different extraction procedures.

6. CONCLUSIONS This TPS allowed to extend the panel of procedures available and validated for obtaining DNA from

host plants and insect vectors for the identification of X. fastidiosa by qPCR. The commercial kit “Maxwell

RSC AS1600” from Promega included in the TPS showed performance values similar to the currently used

protocols, representing an alternative procedure for those laboratories that are equipped with the dedicated

Promega automatized platform.

It should be pointed out that this validation included one plant matrice, and more validations are

necessary to extend the panel of host plants for which the protocols have been validated, as well as the

bacterial strains used as target of the diagnosis.

The data generated from the PT showed that the participating laboratory were all proficient for

performing the diagnosis of X. fastidiosa. Differently from other validations, this PT included in the validation

process the extraction step and not only the qPCR assay. Thus, it was an important opportunity to test the

proficiency of the laboratories in performing one of the critical step of the diagnostic protocol, the extraction

and purification of DNA of high quality.

39 EU-XF-IC-2020-03 v2

6.1 TEST PERFORMANCE STUDY In the framework of this interlaboratory validation, different methods of DNA extractions were

compared for the identification of X. fastidiosa in plant samples and insect samples. The efficacy of the

extraction procedures was measured through qPCR assays (Harper et al., 2010) by using the qualitative and

the quantitative (Cq values) results. The data presented for each tested procedure represent the average

values of the performance criteria recorded in the 16 different laboratories participating to this TPS.

The values of accuracy, repeatability and reproducibility (Table 23) recorded for the tested extraction

procedures ranged from 98.52% to 99.30% when testing plant samples, and from 94.07% to 98.75% when

testing insect samples. It is worth noting that the validations included samples containing the target pathogen

at concentrations close or equal to detection limit of the qPCR assay. Analyzing separately the results of the

panel of samples containing the bacterium at concentrations above this threshold (i.e. from 10^6 CFU/ml to

10^4CFU/ml), the above mentioned performance criteria reach values of 100% for all tested procedures, as

also shown by the data collected for the PT.

The data gathered analyzing the plant samples confirmed the accuracy and reliability of the procedures

previously validated and revealed that the novel protocol tested yielded similar performance values, thus

indicating its suitability for the identification of this pathogen in plant and insect samples.

As expected, higher total DNA yields (and consequently lower Cq values) were obtained using CTAB

compared to both commercial kits, however the latter showed performance criteria always slightly higher

than CTAB. This was more apparent for the insect samples, where manipulation of small volume of insect sap

probably reduced the efficiency of the CTAB protocol, with the automatized protocol being more efficient.

On the other hand, commercial kits combined with the use of automatized platforms yielded better

performance than the manual CTAB procedure, reducing the risks of false positives, as shown for the insects.

Overall, all DNA extraction procedures tested, showed robustness (EPPO PM 7/76) and proved to be suitable

for the identification of X. fastidiosa in plant and insect samples.

40 EU-XF-IC-2020-03 v2

Table 23. Summary of the values of the performance criteria obtained for the different DNA extraction

procedures used to process plant and insect samples.

DNA extracts Accuracy %

Sensitivity %

Specificity %

Repeatability %

Reproducibility %

PLANT SAMPLES

CTAB 98.52 98.67 97.78 97.78 98.52

Mericon 99.26 99.56 97.78 98.52 99.30

Maxwell RSC 98.61 99.17 95.83 98.61 98.68

Analytical sensitivity 10^2 CFU/ml

INSECT SAMPLES

CTAB 94.07 95.11 88.89 96.30 94.07

Maxwell RSC 98.61 98.75 97.92 97.22 98.61

Analytical sensitivity 10^2 CFU/ml

41 EU-XF-IC-2020-03 v2

6.2 PROFICIENCY TEST Using the panel of plant samples containing from 10^6 to 10^4 CFU/ml of X. fastidiosa, all

laboratories participating to this interlaboratory validation proved to be highly proficient. All

laboratories produced values of 100% for all performance criteria evaluated, regardless the

procedure of extraction, the qPCR master mix and the amplification conditions used.

In conclusion, this PT provided a good overview on the laboratory performance for the diagnostics

currently used in the EU/Mediterranean countries for the detection of X. fastidiosa in the plant

samples.

42 EU-XF-IC-2020-03 v2

7. REFERENCES

- Chabirand A, Anthoine G, Pierson O, Hostachy B, 2014. The organization of proficiency testing in

plant pathology (qualitative methods of analysis) according to the ISO/IEC 17043: example of the

French national reference laboratory. Accred Qual Assur (2014) 19: 111–125 DOI 10.1007/s00769-

014-1034-y.

- International Organization for Standardization (2010). Conformity assessment—general

requirements for proficiency testing. ISO/IEC 17043:2010. ISO, Geneva

- Harper SJ, Ward LI, Clover GRG, 2010. Development of LAMP and real-time PCR methods for the

rapid detection of Xylella fastidiosa for quarantine and field applications. Phytopathology 100,

1282–1288.

-PM 7/122 (1), Guidelines for the organization of interlaboratory comparison by plant pest

diagnostic laboratories. EPPO Bull, 44 (3), 390-399.

- European and Mediterranean Plant Protection Organization (EPPO). PM 7/24 (4) Xylella fastidiosa.

EPPO Bull. 2019, 49, 175–227.

- PM 7/76 (4) (2017) Use of EPPO diagnostic protocols. EPPO Bull, 47: 1365-2338. 10.

doi:1111/epp.12365

43 EU-XF-IC-2020-03 v2

8. DIAGOSTIC PROTOCOLS

ANNEXES_1-4

ANNEX 1* CTAB

ANNEX 2* DNeasy Mericon food kit – QIAGEN

ANNEX 3 Maxwell® RSC PureFood GMO and Authentication Kit – Promega

ANNEX 4* Real time PCR Harper et al., 2010 erratum 2013

*The protocols herein reported refer to those recommended in the EPPO, 2019, Diagnostic protocol PM 7/24 (4) Xylella fastidiosa

IMPORTANT NOTES BEFORE STARTING

Before starting the DNA extractions, it is necessary to thaw and vortex the 2.0 ml

microcentrifuge tubes with the plant sap and insect homogenate. Samples can be directly processed as indicated in Annex 1-3.

ANNEX 1

CTAB-based extraction from plant sap FOR PT: The plastic bag labelled as “CTAB PLANT” contains 13 tubes with 1.2 ml of CTAB-plant sap numbered from 44 to 56. This panel of sample has the letter “C” in the ID

FOR TPS: The plastic bag labelled as “CTAB PLANT” contains 22 tubes with 1.2 ml of CTAB-plant sap numbered from 1 to 22. This panel of sample has the letter “C” in the ID.

1. Incubate the samples (2 ml microcentrifuge tubes) at 65°C for 30 minutes. 2. Centrifuge samples at 12,000 g for 5 minutes and transfer 1ml of supernatant to a new 2ml micro-centrifuge tube, being careful not to transfer any of the plant debris. Add 1 ml of Chloroform:Isoamyl Alcohol 24:1 and mix well by shaking. 3. Centrifuge sample at 16,000 g for 10 minutes. Transfer 700 µl to a 1.5ml microcentrifuge tube and add 450 µl (approximately 0.6V) of cold 2-Propanol. Mix by inverting 2 times. Incubate at -20°C for 20 minutes. 4. Centrifuge the samples at 16,000 g for 20 minutes and decant the supernatant. 5. Wash pellet with 1ml of 70% ethanol. 6. Centrifuge sample at 16,000 g for 10 minutes and decant 70% ethanol. 7. Air dry the samples or use the vacuum. 8. Re-suspend the pellet in 120 µl of RNAse- and DNase-free water. 9. Extracts of total nucleic acid can be stored at 4º C for immediate use or at -20ºC for use in the future. 10. Determine the concentration at the spectrophotometer (Nanodrop 1000 or similar). Read the absorption (A) at 260nm and at 280 nm. Optimal A260/280 ratio should be close to 2 for high quality nucleic acid.

CTAB-BASED TOTAL NUCLEIC ACID EXTRACTION FROM INSECT HOMOGENATE

FOR TPS: The plastic bag labelled as “CTAB INSECT” contains 21 tubes with 500 l of CTAB-homogenate of insects, numbered from 23 to 43. This panel of sample has the letter “C” in the ID.

1. Heat the samples (2 ml microcentrifuge tubes) at 65°C for 30 minutes.

2. Add 500 l of Chloroform:Isoamyl Alcohol 24:1 and mix well by shaking or vortexing.

3. Centrifuge sample at 13,000 rpm for 10 minutes. Transfer 400 l to a 1.5 ml microcentrifuge tube and

add 240 l (approximately 0.6 V) of cold 2-Propanol. Mix by inverting 2 times. Incubate at -20°C for 20 minutes.

4. Centrifuge the samples at 13.000 rpm for 20 minutes and decant the supernatant. 5. Wash pellet with 1ml of 70% ethanol. 6. Centrifuge sample at 13,000 rpm for 10 minutes and decant 70% ethanol. 7. Air dry the samples or use the vacuum. 8. Re-suspend the pellet in 30 µl of TE or RNAse- and DNase-free water. 9. Determine the concentration at the spectrophotometer (Nanodrop 1000 or similar). Read the

absorption (A) at 260nm and at 280 nm. Optimal A260/280 ratio should be close to 2 for high quality nucleic acid.

ANNEX 2

DNeasy® mericon™ Food Standard Protocol – Qiagen (Modified) for manual and automated extraction from plant sap

FOR PT: The plastic bag labelled as “MERICON KIT” contains 13 tubes with 1 ml of plant

sap prepared in “Food lysis buffer”, numbered from 44 to 56 . This panel of sample has the

letter “M” in the ID.

FOR TPS: The plastic bag labelled as “MERICON KIT” contains 22 tubes with 1 ml of

plant sap prepared in “Food lysis buffer”, numbered form 1 to 22. This panel of sample has

the letter “M” in the ID.

1. Incubated for 30 min at 60°C. To enhance inhibitor precipitation, cool the sample to room temperature (15–25°C) on ice after incubation.

2. Centrifuge for 5 min at 2500 x g.

3. Pipet 500 μl chloroform into a 2 ml microcentrifuge tube.

4. Carefully transfer 700 μl of the clear supernatant from step 2 to the microcentrifuge tube

containing the chloroform. Be sure not to carry over material from the bottom phase, which

contains precipitated food debris.

5. Vortex the microcentrifuge tube from step 4 vigorously for 15 s and centrifuge at 14,000 x

g for 15 min.

6. Pipet 350 μl of Buffer PB into a fresh 2 ml microcentrifuge tube. From this step, it is

alternatively possible to use the automated platform QIACUBE (see below).

7. Add 350 μl of the upper, aqueous phase from step 5 and mix thoroughly by vortexing.

8. Pipet 600 μl of the mixture from step 7 into the QIAquick spin column placed in a 2 ml

collection tube. Centrifuge at 17,900 x g for 1 min and discard the flow-through. Reuse the

collection tube in step 9.

9. Add 500 μl Buffer AW2 to the QIAquick spin column, centrifuge at 17,900 x g for 1 min and

the discard flow-through. Reuse the collection tube and centrifuge again at 17,900 x g for 1

min to dry the membrane.

10. Transfer the QIAquick spin column to a 1.5 ml or 2 ml microcentrifuge tube (not supplied),

and pipet 100 μl Buffer EB directly onto the QIAquick membrane. Incubate for 1 min at room

temperature (15–25°C), and then centrifuge at 17,900 x g for 1 min to elute.

11. Determine the concentration at the spectrophotometer (Nanodrop 1000 or similar). Read

the absorption (A) at 260nm and at 280 nm. Optimal A260/280 ratio should be close to 2 for

high quality nucleic acid.

Method with Qiacube

Transfer 350 μl of the aqueous phase from step 7 to a 2 ml safe-lock tube, and place it onto the QIAcube shaker

Set up 100 µl

ANNEX 3

Maxwell RSC PureFood GMO and Authentication Kit performed on MaxwellR RSC automated

platform (Promega) for DNA extraction from plant sap

FOR PT: The plastic bag labelled as “PROMEGA” contains 13 tubes, numbered from 44 to 56, with

1 ml of plant sap prepared in CTAB buffer provided by Promega. This panel of sample has the

letter “P” in the ID.

FOR TPS: The plastic bag labelled as “PROMEGA” contains 22 tubes, numbered form 1 to 22,

with 1 ml of plant sap prepared in in CTAB buffer provided by Promega. This panel of sample

has the letter “P” in the ID.

1. Add 20μl of RNase A Solution to each microcentrifuge tubes (to eliminate RNA) and 40μl of

Proteinase K (PK) Solution.

2. Tap, invert and vigorously vortex tubes until the sample is resuspended.

3. Place in a heat block at 65°C for 30 minutes.

4. Prepare cartridges as instructed in Section 5.A during the incubation (see next pages).

5. After incubation, invert or vortex tubes with lysate to mix thoroughly.

6. Place tubes with lysate into a microcentrifuge and spin at room temperature for 10 minutes at

≥16,000 × g to separate any oils and solids.

7. Transfer only 300μl of clear lysate sample into well #1 of the reagent cartridge. Avoid pipetting

any solid material from the bottom of the tube or on the surface of the liquid. Also avoid oil on the

surface. Transferring these materials may inhibit downstream assays. If necessary, transfer the

cleared lysate to a new tube and centrifuge again to avoid oils and solids.

Some lysate will remain in the tube after transferring the 300μl aliquot to the cartridge.

8. Proceed to Section 5.A for purification on the Maxwell® Instrument according to the

manufacturer's instructions. IMPORTANT NOTE: Add 100 μl of Elution Buffer according to step 3

of Section 5.A (next pages) for plant sap; Maxwell® Instrument Setup and Run (according to

manufacturer's instructions 5.B).

9. Determine the concentration at the spectrophotometer (Nanodrop 1000 or similar). Read the

absorption (A) at 260nm and at 280 nm. Optimal A260/280 ratio should be close to 2 for high

quality nucleic acid.

100 μl EB for plant

50 μl EB for insects

Maxwell RSC PureFood GMO and Authentication Kit performed on MaxwellR RSC automated

platform (Promega) for DNA extraction from insect homogenate

FOR TPS: The plastic bag labelled as “PROMEGA” contains 21 tubes, numbered from 23 to 43,

with 0.5 ml of insect homogenate prepared in in CTAB buffer provided by Promega. This panel

of sample has the letter “P” in the ID.

1. Add 10μl of RNase A Solution to each microcentrifuge tubes (to eliminate RNA) and 20μl of

Proteinase K (PK) Solution.

2. Tap, invert and vigorously vortex tubes until the sample is resuspended.

3. Place in a heat block at 65°C for 30 minutes.

4. Prepare cartridges as instructed in Section 5.A during the incubation (see next pages).

5. After incubation, invert or vortex tubes with lysate to mix thoroughly.

6. Place tubes with lysate into a microcentrifuge and spin at room temperature for 10 minutes at

≥16,000 × g to separate any oils and solids.

7. Transfer only 300μl of clear lysate sample into well #1 of the reagent cartridge. Avoid pipetting

any solid material from the bottom of the tube or on the surface of the liquid. Also avoid oil on the

surface. Transferring these materials may inhibit downstream assays. If necessary, transfer the

cleared lysate to a new tube and centrifuge again to avoid oils and solids.

Some lysate will remain in the tube after transferring the 300μl aliquot to the cartridge.

8. Proceed to Section 5.A for purification on the Maxwell® Instrument according to the

manufacturer's instructions. IMPORTANT NOTE: add 50 μl of Elution Buffer for insect homogenate

according to step 3 of Section 5.A .

9. Maxwell® Instrument Setup and Run (according to manufacturer's instructions 5.B).

10. Determine the concentration at the spectrophotometer (Nanodrop 1000 or similar). Read the

absorption (A) at 260nm and at 280 nm. Optimal A260/280 ratio should be close to 2 for high

quality nucleic acid.

ANNEX 4

Real-time PCR (qPCR)

The qPCR must be performed using the primers and the TaqMan probe designed by Harper et al.

(2010; erratum 2013), and reagents reported in Appendix 5 – Real-time PCR (Harper et

al., 2010; erratum 2013) simplex [EPPO 2019, PM 7/24(4)].

In each test, each sample must be run in double wells and negative amplification control (NAC)

(reaction mix added with molecular-grade water) and the Positive Amplification Control (PAC)

(supplied by the Organizer) must be included.

UltraPure™ BSA (50 mg/mL) Invitrogen, catalogue number: AM2616 Sequence of the primers/probe: XF-F (forward) 5’-CAC GGC TGG TAA CGG AAG A-3’ XF-R (reverse) 5’-GGG TTG CGT GGT GAA ATC AAG-3’ XF-P (probe) 5’ 6FAM -TCG CAT CCC GTG GCT CAG TCC-BHQ-1- 3’

- PCR conditions: 95°C for 10 minutes, followed by 40 cycles of 94°C for 10 seconds and 62°C for 40 seconds.

RESULTS INTERPRETATION:

Verification of the controls: • PAC amplification curves should be exponential. • NAC should give no amplification. When these conditions are met: • A test will be considered positive if it produces an exponential amplification curve. • A test will be considered negative if it does not produce an amplification curve or if it produces a curve which is not exponential. • Tests should be repeated if any contradictory or unclear results are obtained.

Reagents [Concentrated

Sol.]

[Final Sol.]

Vol. for one tube

Molecular-grade water 6,48 µL TaqMan™ Fast Universal PCR Master

Mix (AB) (Cod.) 4352042

2

x

1

x

10

µL

XF-F 10 µM 0.30 µM 0.6 µL XF-R 10 µM 0.30 µM 0.6 µL XF-P 10 µM 0.10 µM 0.2 µL

Molecular grade BSA (non acetylated) * 50 µg/µL 0.30 µg/µL 0.12 µL

PCR Mix Volume 18 µL DNA Sample/PAC Volume 2 µL

Total Volume total per reaction 20 µL

INTERLABORATORY COMPARISON EU-XF-IC-2020-03 Evaluation …

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