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Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

Specific scope

This guideline includes specific quality management requirements for laboratories preparing for accreditation according to the ISO/IEC Standard 17025 General requirements for the competence of testing and calibration laboratories (references to relevant parts of ISO/IEC Standard 17025 are included). It should be noted that in EPPO standards the verb ‘should’ carries the highest level of obligation.

Specific approval and amendment

First approved in 2009–09.


1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

Development of quality management systems (also referred to as management systems or quality systems) and accreditation have become a concern for many laboratories in the EPPO region. A Standard PM 7/84 Basic requirements for quality management in plant pest diagnosis laboratories was adopted in 2007. PM 7/84 describes basic requirements to assist laboratories conducting plant pest diagnosis in designing their management system for quality. This new Standard includes additional requirements for laboratories applying for accreditation. It is based on the ISO/IEC Standard 17025 General requirements for the competence of testing and calibration laboratories (ISO/IEC, 2005) and should be used together with PM 7/84. Laboratories usually apply for accreditation only for the most relevant pests, and not for all pests on which they are likely to perform a diagnosis.

Accreditation against the ISO/IEC Standard 17025 is granted by national accreditation bodies, it is important that laboratories develop good communication procedures and establish regular contact with their national accreditation body throughout the process.

This document concerns the quality of a diagnosis and does not specifically deal with health and safety matters. However, laboratory practices should conform to national health and safety regulations.

2. Scope of accreditation: fixed scope and flexible scope

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

Historically, the accreditation of laboratories has been usually based on a fixed scope which should define clearly and unambiguously the range of tests covered by the laboratory’s accreditation (e.g. immunofluorescence test for the detection of Ralstonia solanacearum on potato tubers). However, this does not readily allow new or modified tests to be added to a laboratory’s scope, even when the competence of the laboratory in performing and validating related tests has already been evaluated by an accreditation body. Although applications for an extension to scope can be made at any time, the timescales involved may actually prevent quick reactions to client’s demands. Consequently the concept of flexible scope has been developed.

A flexible scope of accreditation allows a laboratory to undertake certain tests, and to report the results as accredited, even though these tests are not explicitly stated in the laboratory’s scope (‘Requirements for the Accreditation of Flexible ScopesEA-2/15, 2008). Examples of situations where the need for flexible scope may arise are:

  • • 
    Optimisation of a given test.
  • • 
    Modification of an existing test to broaden its applicability (e.g. to deal with new matrices).
  • • 
    Inclusion of a test equivalent to the one that is already covered by accreditation.

The concept of flexible scope encompasses a degree of flexibility which is usually agreed in consultation with the accreditation body. Nevertheless, it should be noted that this degree of flexibility has a varying interpretation at the national level. The experience in plant pest laboratories so far is that flexible scope places more responsibility on the laboratory for demonstrating that tests are valid, suitable for circumstances of use and are performed competently and consistently. If the laboratory decides to report a test as accredited and an audit later identifies problems with the procedures used, results may not be valid and all diagnosis reports issued may have to be withdrawn. Therefore it is highly recommended that experience with a fixed scope accreditation is obtained before a laboratory applies for flexible scope as all requirements of the ISO/IEC Standard 17025 have to be fulfilled in both cases. Nevertheless, a laboratory may already be accredited for activities other than plant pest diagnostics. Experience with a fixed scope accreditation in another activity may be sufficient for the direct application for a flexible scope for plant pest diagnostic activities.

3. Terms and definitions

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

Definitions of terms used in this standard are included in PM 7/76 Use of EPPO diagnostic protocols (revision in preparation). Definitions of the performance criteria are included in the Introduction to EPPO PM 7 Standards.

In this Standard ‘test’ refers to the application of a method to a specific pest and a specific matrix. Methods concerned include the following: bioassay methods, biochemical methods, fingerprint methods, isolation/extraction methods, molecular methods, morphological and morphometrical methods, pathogenicity assessment, and serological methods.

4. Management requirements (ISO 17025 point 4)

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

The laboratory should establish, implement and maintain a quality management system covering all facilities and activities in the scope of the accredited plant pest diagnosis.

The management system should describe the facilities and activities covered (including details of the customers and tested pests). The quality system should be documented and the quality documents should be archived (see also below).

As stated in PM 7/84, the management system of the laboratory should ensure that:

  • • 
    Appropriate resources are available to conduct the diagnosis, for example personnel, facilities, and consumables (see also the section ‘Technical requirements’).
  • • 
    Purchased supplies, reagents and consumable material are appropriate for the intended use.
  • • 
    Responsibilities and tasks of personnel are clearly defined (e.g. by organisational flow charts) and appropriately assigned.
  • • 
    Possible conflicts of interest between personnel and activities performed are identified and prevented.
  • • 
    Training is documented and assessed (see also the section ‘Technical requirements’).
  • • 
    Procedures and instructions are available to and implemented by the personnel. This includes Standard Operating Procedures (SOPs).
  • • 
    The client is informed upon request of the relevant data regarding the diagnosis of its sample.
  • • 
    Any subcontracted work is performed by a laboratory that is following the requirements of this standard, which should be checked by the laboratory (the minimum being a written declaration of compliance).
  • • 
    Confidentiality of diagnosis results to customer is guaranteed2
  • • 
    A mechanism is in place to deal with complaints.
  • • 
    Mechanisms are in place to record, analyse and correct any deviation from procedures or requirements of the customer.
  • • 
    All documentation mentioned above is maintained and archived. The quality management system should be reviewed periodically by the top management. This implies a periodical assessment of all the components of the system and routine recording of deviations in the system and subsequent corrective actions that have been taken.

4.1 Commitment of top management (ISO 17025 point 4.2.3)

The laboratory top management should commit itself to bringing into effect the goals of quality management and to continually improve the effectiveness of its management system. It should provide evidence of this commitment.

4.2 Continuous improvement (ISO 17025 point 4.10)

The laboratory’s management system, including testing, should be reviewed periodically by the top management to ensure its continuing suitability and effectiveness, and to introduce necessary changes or improvements. A continuous improvement programme should be implemented by the laboratory. In-house mechanisms and external evaluation can provide the necessary information. Internal mechanisms include:

  • • 
    The definition of quality objectives and adequate quality indicators (e.g. reduction of number of complaints in the forthcoming year with xx %). These should be reviewed by the management.
  • • 
    The organisation of staff meetings to:
    • o
      Plan preventive actions and assess corrective actions for their effectiveness.
    • o
      Analyse thoroughly internal audits results.
    • o
      Evaluate complaints and their corrective actions, etc.
    • o
      Identify training needs.
    • o
      Collect propositions for improvement by the staff.
  • • 
    Procedures for seeking feedback from clients.

Assessments by external audits, results of proficiency tests or other inter-laboratory tests are also important elements for continuous improvement.

5. Technical requirements (ISO 17025 point 5)

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

5.1 General (ISO 17025 point 5.1)

See same section of PM 7/84.

5.2 Personnel (ISO 17025 point 5.2)

See same section of PM 7/84.

5.3 Accommodation and environmental conditions (ISO 17025 point 5.3)

See same section of PM 7/84.

5.4 Diagnostic methods (ISO 17025 point 5.4)

5.4.1 General (ISO 17025 point 5.4.1)

The laboratory should use appropriate methods and procedures for all tests within its scope. These include sampling where relevant, handling, transport, storage, preparation and testing of samples. It is expected that diagnostic laboratories will have an understanding of the biology of organisms and take this into account when sub-sampling and/or when preparing the sample for analysis. Purchased supplies, reagents and consumable material should be appropriate for the intended use.

All instructions, standards, technical manuals and reference data relevant to the work of the laboratory should be kept up-to-date and made readily available to personnel. Deviation from SOP when applying tests should occur only if documented, technically justified and authorised by an appropriate person.

5.4.2 Selection of tests (ISO 17025 point 5.4.2)

The laboratory should use diagnostic tests that are suitable according to the circumstances of use (see EPPO Standard PM 7/76 Use of EPPO diagnostic protocols). Tests described in the legislation (e.g. European Union or national legislation) are mandatory for the countries concerned. If no test is mandatory, tests published as international, regional or national standards should, preferably, be used. Whenever such tests are not available or whenever performance could be improved laboratory-developed or adapted tests could be considered.

The laboratory should ensure that it uses the latest valid edition of a test unless it is not appropriate or possible to do so. When necessary, the test should be supplemented with additional details to ensure consistent application.

A laboratory preparing for accreditation should only use validated tests. Tests may have been validated with validation data available. If this is not the case, tests should undergo a validation process within the laboratory (see 5.4.3). When a validated test is used, the laboratory should provide objective evidence that it can operate the test according to the established performance characteristics (see 5.4.4). A test is considered as a fully validated test when it provides data for the following performance criteria: analytical sensitivity, analytical specificity, reproducibility and repeatability. Depending on the scope of the test selectivity may also need to be determined. If a change to a validated test is made by the laboratory, a judgement should be made and documented, as to whether such a change requires validation or verification.

Tests included in EPPO Diagnostics protocols do not all include performance criteria. A survey carried out in 2008 on the use of tests included in EPPO Diagnostic Protocols (Petter et al., 2009) showed that those presented in Appendix 1 (in preparation) are widely used. Consequently they can be considered as giving appropriate confidence regarding repeatability, reproducibility and selectivity. A laboratory implementing these tests should at least produce validation data regarding analytical sensitivity. It may also need to produce additional data on analytical specificity depending on local circumstances (e.g. likelihood of false reactions with closely-related organisms). Once these data are provided, these tests are considered validated. The regular revision programme of EPPO Diagnostic Protocols includes the addition of validation data.

Validated tests providing performance criteria are considered in this document as ‘standard tests’ and are referred to as ‘standard methods’ in ISO 17025.

5.4.3 Validation of tests (ISO 17025 point 5.4.5)

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

Validation is carried out to provide objective evidence that the test is suitable for the circumstances of use (see EPPO Standard PM 7/76 where the different intended uses are described). In plant pest diagnosis the test should be fit for routine diagnosis. As already stated, the minimum test performance criteria to be defined are: analytical sensitivity, analytical specificity, repeatability, reproducibility and if appropriate selectivity. If values for these criteria are not available or accessible (e.g. published sources), the laboratory should produce the missing data or justify why they could not be produced. When values for performance criteria are partly available, the laboratory should verify that it can perform the test according to them (see 5.4.4). Validation is to be performed with reference material (see definition in PM 7/84), including artificially infected/infested samples (spiked samples). When using cultures or isolates for biological tests, care should be taken that they have a proven virulence.

Validation of a test by comparison with a ‘standard test’ is also possible and guidance for such validation is given in Appendix 2. Nevertheless, such test cannot be regarded as a ‘standard test’ as it does not have full performance criteria. This process can only demonstrate that the test is as good as the standard test.

Collected data and results of laboratory-performed validations (in particular related to reproducibility), as well as results of inter-laboratory comparison (evaluation of a test performance), can also provide an indication of the robustness of the test, e.g. to what extent different reagents or altered test conditions affect the established test performance values. These may also provide data on diagnostic sensitivity and diagnostic specificity by comparison to (an) alternative test(s).

The validation procedures described here (and in particular the explanations given in Tables 4–9 in Appendix 5) should be regarded as general guidance according to which a test can be validated. Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented. It is not possible to provide a detailed description for each combination in this document.

Tests in plant pest diagnostics can be qualitative or quantitative. A qualitative test establishes the presence or absence of a plant pest in a sample. A quantitative test establishes a certain quantity of a plant pest in a sample. Whenever possible the limit of detection of a test should be determined. Nevertheless this limit cannot always be established absolutely while detecting plant pests. There are organisms that cannot be cultured (obligate pathogens), which can not be quantified (fungi), which are only present in the plant and which cannot be purified (e.g. phytoplasmas). For this reason, exact concentration of these organisms cannot or can hardly ever be established accurately and so estimates have to be used. Even with those which can be purified (many bacteria and viruses), the concentration can only be estimated (e.g. CFUs or mg mL−1). This estimation is often based on an indirect measurement.

Validation process

As mentioned in ISO 17025 ‘the laboratory shall record the results obtained and the procedure used for the validation’.

The general process for validation is described below (see also Fig. 1). More detailed guidance is given in Tables 4–9 in Appendix 5.

image

Figure 1.  Validation process.

Download figure to PowerPoint

  • 1
    Identify the scope of the test e.g. detection and/or identification of organism x in matrix y by method z considering any specific requirement related to the circumstances of use of the test (for examples, see PM 7/76).
  • 2
    Consider the technical requirements to determine analytical specificity, analytical sensitivity, selectivity, reproducibility and repeatability performance characteristics, by consulting the guidelines in Tables 1–6 as required. Then define the type and constitution of samples needed for the validation.
  • 3
    Plan and perform the validation for individual performance characteristics or in a combined test setup. It is advised to follow the steps described in Fig. 1.
  • 4
    Present the results in a validation report with a conclusion on whether the validated test meets the requirement identified in point 1.
  • 5
    Use of a ‘Statement on test validation’ form can be valuable, and may be presented as in Appendix 3.
Table 1.   Guidance on the verification of performance criteria
Analytical sensitivityAnalytical specificityRepeatabilityReproducibility
  1. *Artificial subsamples created from 1 sample can be used.

  2. †At the limit of detection.

Analyse at least 5–10 samples at the established limit of detection. This can be combined with repeatability/reproducibility.Select a few of the most relevant targets (e.g. different strains) and non-targets. Tests should be performed at medium levels of organisms.Perform at least 2 experiments with 3 levels of target organism* (low/medium/high) with the same operator and equipment at the same day.Perform at least 3 experiments with 3 levels of target organism* (low/medium/high) but at different moments, when possible with different operators, and when relevant with different equipment.

5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

The laboratory should confirm that it can properly carry out the selected standard test, for the intended use. This verification should be repeated if the standard test is changed.

The process required to provide objective evidence that the laboratory is competent to perform a standard test according to its established performance characteristics is described below.

Testing outside the original scope needs revalidation according to 5.4.3 (e.g. detection in a new host). If the laboratory makes a significant change to a standard test, the test should be validated again. Verification can also be done by participating in a proficiency test or in an inter-laboratory comparison (ring test/collaborative study), provided that these allow you to fulfil the requirements in Table 1.

Choice of reagent can be critical for the performance of a test. A change of reagent (or lots/batches of reagent) or reagent supplier may influence the performance of a test. In such case a verification of the performance of the reagent should be done by comparison with the reagent previously used or according to Table 1 columns 1 and 2.

Verification process (Fig. 2)

image

Figure 2.  Verification process.

Download figure to PowerPoint

  • 1
    Perform the validated test as described or with minor changes to take account of local conditions (e.g. suppliers of reagents or equipments, unless it is specifically required in the validated test) to evaluate whether the laboratory meets the performance criteria values from the validation data (see guidelines in Table 1). Selectivity does not need to be verified.
  • 2
    Results of verification test:
    •   Performance values are not met: go to point 3.
    •   Performance values are met: the laboratory can apply the test for routine use – go to point 4.
  • 3
    If deviation from conditions described in the validated test affect test results, investigate the reasons for this deviation. Correct, verify the test again or revalidate if required following the procedure described in section ‘Validation of tests’. If performance values are not met, investigate whether the minor changes that have been introduced in the test are the cause. If it is not the case, seek external guidance (e.g. contact the author of the test).
  • 4
    Present the results in a verification report with a conclusion as to whether the verified test meets the requirements identified in point 1.
  • 5
    Use of a ‘Statement on test verification’ form can be valuable, and may be presented as in Appendix 4.
Uncertainty of measurement (ISO 17025 5.4.6)

Laboratories should attempt to identify the factors influencing the uncertainty of a test such as staff, equipment and biological properties (i.e. serotypes, pathotypes). Whenever possible, appropriate measures should be taken to control this uncertainty. If no measures are taken the reasons for this should be recorded and the client should be made fully aware of uncertainty surrounding the test.

Although in most cases tests used for plant pest diagnosis provide qualitative results, these qualitative results may be based on measurement (morphometrical data, counting of cells). This measurement may be just one part of the diagnostic process but if this is critical for a diagnosis its uncertainty should be estimated.

5.5 Equipment (ISO point 5.5)

See same section of PM 7/84.

5.6 Reference materials (ISO point 5.5.3)

See same section of PM 7/84.

5.7 Sampling (ISO Point 5.7)

See same section of PM 7/84.

5.8 Sample handling (ISO point 5.8)

See same section of PM 7/84.

5.9 Ensuring the quality of diagnosis (ISO point 5.9)

See same section of PM 7/84.

6 Reporting the results (ISO point 5.10.3)

See EPPO Standard PM 7/77 Documentation and reporting on a diagnosis.

Footnotes
  • 2

    Nevertheless, laboratories are encouraged to develop a procedure to report findings of regulated pests to the NPPO when the NPPO is not the customer.

References

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices
  • AFNOR (1995). XP V03-111 Agricultural and Food Products Analysis. Protocol for the intra-laboratory evaluation of an alternative method of qualitative analysis against a reference method. Association Française de Normalisation, La Plaine Saint-Denis (FR).
  • EA (2008) EA-2/15 Requirements for the Accreditation of Flexible Scopes. http://www.eurolab.org/docs/EA/EA-2_15.pdf . European Association for Accreditation.[accessed on 16 September 2009.]
  • Hughes KJD, Griffin RL, Tomlinson JA, Boonham N, Inman AJ & Lane C (2006) Development of a one step real-time PCR assay for diagnosis of Phytophthora ramorum. Phytopathology 96, 975981.
  • ISO/IEC (2005) Standard 17025 General requirements for the competence of testing and calibration laboratories (available on http://www.iso.org).
  • ISO (2003) ISO 16140 Microbiology of food and animal feeding stuffs Protocol for the validation of alternative methods (available on http://www.iso.org).
  • Petter F & Suffert M (2010) Survey on the use of tests mentioned in EPPO diagnostic protocols. Bulletin OEPP/EPPO Bulletin 40, 121126.

Appendices

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Scope of accreditation: fixed scope and flexible scope
  5. 3. Terms and definitions
  6. 4. Management requirements (ISO 17025 point 4)
  7. 5. Technical requirements (ISO 17025 point 5)
  8. 5.4.3 Validation of tests (ISO 17025 point 5.4.5)
  9. 5.4.4 Verification of the performance of the laboratory to undertake a specified test (ISO point 5.4.2 second paragraph last sentence)
  10. References
  11. Appendices

Appendix 1 – List of tests included in EPPO Diagnostic protocols that are widely used (in preparation)

This list will be prepared in consultation with the relevant EPPO Panels on diagnostics.

Appendix 2 – Procedure for validation of a test by comparison with a ‘standard test’

The comparison of the test with the ‘standard test’ should be performed as follows:

Perform 3 repetitions with the target organism and 3 with each of the non-target organisms as indicated in Table 2. Samples are processed with the two tests in parallel. It should be noted that this procedure may not be applicable to all disciplines for example morphological identification of insects.

The number of samples indicated in this table has been determined by comparison with published standards e.g. ISO 16140 Microbiology of food and animal feeding stuffs Protocol for the validation of alternative methods (ISO, 2003) and AFNOR XP V03-111 Agricultural and food products analysis. Protocol for the intra-laboratory evaluation of an alternative method of qualitative analysis against a reference method (AFNOR, 1995).

Correlation between results obtained with the standard test and the new test should be evaluated for the different pest levels. Results can be presented as shown in Table 3 and relative performance characteristics calculated.

Table 2.   Minimum number of samples required when comparing a test to a ‘standard test’
Type of materialLevel of organism
LowMediumHigh
  1. *The total number of samples of non-target(s) should not exceed twice the total number of samples of target.

Isolates of pure cultures of target or samples spiked with target1075
Isolates of pure cultures of non-target(s) or samples spiked with non-target(s)22–44*
Naturally contaminated sample with target organismAdequate dilution series are prepared with 15 positive samples previously identified with the standard tests to reach the limit of detection of the standard test.

Appendix 3 – Statement on test validation

Test name:

Scope of test:

Additional comments:

Documentation for the validity of the test and the requirements that the test should meet are available in the laboratory. Documentation includes laboratory books and other information as indicated below which shows how procedures have been validated in this study.

Performance criteriaABCDWhere to find documentation
Analytical sensitivity     
Analytical specificity     
Selectivity (when relevant)     
Repeatability     
Reproducibility     

A = Data from own laboratory experiments.

B = Data from proficiency tests or ring tests.

C = Information from manufacturers.

D = External information (literature, etc.).

Other information (optional).

Table 3.   Example of results from a correlation between a standard test and a new test Thumbnail image of
 Where to find documentation
Diagnostic sensitivity 
Diagnostic specificity 

On the basis of the above statement the validity of the test is judged suitable for the scope of the test.

Name in capital letters:Laboratory position:
Signature:Date:
Name in capital letters:Laboratory position:
Signature:Date:

Appendix 4 – Statement on test verification

Test name:

Standard test:

Scope of the standard test:

Minor changes:

Documentation for the validity of the test and the requirements that the test should meet are available in the laboratory. Documentation includes laboratory books and other information which shows how the verification test has been performed in this study.

Performance criteriaValues from the standard testMeet requirements of standard test Yes/noWhere to find documentation
Analytical sensitivity   
Analytical specificity   
Repeatability   
Reproducibility   

On the basis of the above statement the verification of the test is judged suitable for the scope of the test.

Name in capital letters:Laboratory position:
Signature:Date:
Name in capital letters:Laboratory position:
Signature:Date:

Appendix 5 – Tables giving detailed guidance for the validation process by field (Bacteriology, Botany, Entomology, Nematology & Virology)

Table 4. Table 4 Bacteriology Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented
Method for extraction of target organism from matrix Remark. Extraction is always validated by a test.
 Analytical sensitivityThe method should be able to extract viable cells of the target bacterium from the sample material to allow sensitive detection. Perform extractions from at least 3 samples with high/medium/low levels of contamination. Samples may consist of explicit infected material (diagnosis) or samples may be produced by adding infected material with known cell density of the target bacterium to the sample material (detection of latent infection or contamination).
 Analytical specificityThis parameter is not relevant. Extraction of the target organism from a sample is per definition non-specific.
 SelectivityThis parameter is not relevant. Extraction of the target organism from a sample is per definition non-selective.
 RepeatabilityPerform extraction on 8 replicate samples. For detection of latent infection, repeatability should be validated at the analytical sensitivity limit.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Molecular methods, e.g. hybridization, PCR and real time PCR Remark. This step also includes methods for isolation of DNA from the sample material
 Analytical sensitivityAnalyse at least 3 series of spiked sample extracts with a range of 10–106 cells of the target organism per mL. Preferentially this is done by making decimal diluted cell suspensions of the target bacterium in the sample extracts. Determine the lowest cell density giving a positive test result. If consistent results are not obtained after 3 series, then additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. brand of PCR reagents (in particular DNA polymerase) and PCR cycle conditions.
 Analytical specificityAnalyse (i) strains of the target bacterium covering genetic diversity, different geographic origin and hosts and (ii) a set of non-target bacteria, in particular those associated with the sample material. Use cell suspensions of pure cultures at approximately 106 cells mL−1. In addition, the test results can be supported by ‘in silico’ comparison of probe/primer sequences to sequences in genomic libraries.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityAnalyse 8 replicates of spiked sample extracts with medium/low level of contamination. For detection of latent infection, repeatability should be validated at the analytical sensitivity limit. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Serological methods: IF and ELISA
 Analytical sensitivityAnalyse at least 3 series of spiked sample extracts with a range of 102–106 cells of the target organism per mL. Preferentially, this is done by making decimal diluted cell suspensions of the target bacterium in the sample extracts. Determine the lowest cell density giving a positive test result at the working dilution of the antiserum/antibodies. If consistent results are not obtained after 3 series, additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. the number of microscope fields to read in the IF test and the OD threshold in the ELISA test.
 Analytical specificityDefine specificity of antibodies on (i) strains of the target bacterium covering genetic diversity, different geographic origin and hosts and (ii) on a set of non-target bacteria, in particular those associated with the sample material. Use cell suspensions of pure cultures at approximately 106 cells mL−1 and use antiserum/antibodies at their working dilution.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityAnalyse 8 replicates of spiked sample extracts with medium/low level of contamination. For detection of latent infection, repeatability should be validated at the analytical sensitivity limit. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible and on different days and with different equipment when relevant.
Plating methods: selective isolation
 Analytical sensitivityAnalyse at least 3 series of spiked sample extracts with a range of 10–106 cells of the target organism per mL. Preferentially, this is done by making decimal diluted cell suspensions of the target bacterium in the sample extracts. Determine the lowest cell density giving a positive test result. If consistent results are not obtained after 3 series, additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. brand of ingredients for the medium (in particular antibiotics and preparation of stock solutions) and incubation conditions.
 Analytical specificityDefine specificity of the culture medium on (i) strains of the target bacterium covering genetic diversity, different geographic origin and hosts and (ii) for a set of non-target bacteria, in particular those associated with the sample material. Use a cell suspension at approximately 106 cells mL−1 and analyse by dilution plating.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of host plants.
 RepeatabilityAnalyse 8 replicates of spiked sample extracts with medium/low level of contamination. For detection of latent infection, repeatability should be validated at the analytical sensitivity limit. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible and on different days and with different equipment when relevant.
Bioassay methods: selective enrichment in host plants
 Analytical sensitivityAnalyse at least 3 series of spiked sample extracts with a range of 102–106 cells of the target organism per mL. Preferentially this is done by making decimal diluted cell suspensions of the target bacterium in the sample extracts. Determine the lowest cell density giving a positive test result. This implies isolation from test plants with or without symptoms of infection. If consistent results are not obtained after 3 series, additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. stage of test plants, inoculation method and incubation conditions.
 Analytical specificityDefine specificity of the bioassay on (i) strains of the target bacterium covering genetic diversity, different geographic origin and hosts and (ii) for a set of non-target bacteria, in particular those associated with the sample material. Use a cell suspension at approximately 106 cells mL−1.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars including the most susceptible cultivar(s).
 RepeatabilityAnalyse 8 replicates of spiked sample extracts with medium/low level of contamination. For detection of latent infection, repeatability should be validated at the analytical sensitivity limit. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s), if possible, and on different days and with different equipment when relevant.
Pathogenicity test
 Analytical sensitivityThis parameter is not relevant for the pathogenicity test which is generally performed with cell suspensions of approximately 106 cells mL−1. However, analytical sensitivity may be considered when inoculating in different growth stages of the host plant.
 Analytical specificityDefine specificity of the pathogenicity test on a set of strains of the target bacterium covering genetic diversity, different geographic origin and hosts and on a set of non-target bacteria which, in particular those associated with the sample material. Use cell suspensions of approximately 106 cells mL−1. A positive result implies expression of symptoms and re-isolation of the target bacterium (Koch’s postulates).
 SelectivityDetermine the relative insensitivity of the pathogenicity test by using different cultivars of the host plant.
 RepeatabilityAnalyse 3 replicates of a set of strains of the target bacterium covering variability in identification tests and virulence. Use cell suspensions of approximately 106 cells mL−1. A positive result implies expression of symptoms and re-isolation of the target bacterium (Koch’s postulates).
 ReproducibilityAs for repeatability but with different operator(s) if possible and on different days and with different equipment when relevant.
Fingerprint methods: protein profiling, fatty acid profiling & DNA profiling
 Analytical sensitivityDetermine the minimum quantity of harvested bacteria from selected culture media to perform a reliable analysis. Test parameters should be stringently defined and standardised, e.g. culture medium, stage of culture for harvesting of cells.
 Analytical specificityDefine specificity of the fingerprint method on (i) strains of the target bacterium covering genetic diversity, different geographic origin and hosts and (ii) for a set of non-target bacteria, in particular those associated with the sample material. Provide markers for differentiation at subspecies or pathovar level.
 SelectivityNot applicable.
 RepeatabilityAnalyse 3 replicates of the protein/fatty acid/DNA extract.
 ReproducibilityAs for repeatability but with different operator(s) if possible and on different days and with different equipment when relevant.
Table 5.   Botany Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented
Method for extraction of target organism from matrix Extraction is always validated by a test.
 Analytical sensitivityDetermine the percentage of weed seeds that is recovered by the extraction method.
 Analytical specificityNot applicable.
 SelectivityNot applicable.
 RepeatabilityAt least 8 samples of seed or grain lots, plant products for extraction of the same target organisms.
 ReproducibilityAs for repeatability, but with different operator(s) if possible, on different days and with different equipment when relevant.
Morphological and morphometrical methods intended for identification
 Analytical sensitivityEstablish the minimum number of target organism (seed or plant) needed to perform a reliable analysis. The lowest number of target organism depends on the condition of the sample (good to very poor), the development stage, the known interspecies variability, the difficulty to interpret features, etc.
 Analytical specificityScreen against a range of morphologically similar target and non target organisms.
 SelectivityNot applicable.
 RepeatabilityAt least 8 identifications of the same target organism (seed or plant) as a blind test within a range of morphologically similar target and non target organisms. The blind test could be conducted as follows: the same target organism is presented among several non target organisms. This is repeated 8 times, and non target organism may vary between repetitions.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Table 6Entomology.    Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented
Method for extraction of target organism from matrix Remark. Extraction is always validated by a test.
 Analytical sensitivityExtraction of insects: when applicable, determine the percentage of insects that is recovered by the extraction method. DNA extraction: validation is included in molecular methods validation.
 Analytical specificityNot applicable.
 SelectivityNot applicable.
 RepeatabilityNot applicable.
 ReproducibilityNot applicable.
Morphological and morphometrical methods intended for identification
 Analytical sensitivityWhenever possible, establish the minimum number of individuals or features to perform a reliable analysis with a minimum of three repetitions. The lowest number of target individuals depends on the condition of the sample (good to very poor), the development stage, the known intraspecies variability, the difficulty to interpret features, etc.
 Analytical specificityWhenever possible, screen against a range of morphologically similar target and non target organisms.
 SelectivityNot applicable.
 RepeatabilityAt least 8 identifications of the same target preparation as a blind test within a range of morphologically similar non-target organisms. The blind test could be conducted as follows: the target specimen is presented among several non target specimens. This is repeated 8 times, and non target specimen(s) may vary between repetitions.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Molecular methods, e.g. hybridisation, PCR Remark. This step also includes methods for isolation of DNA from the sample material.
 Analytical sensitivityPrepare a relative number of individuals. This number varies depending on the genus, species and stage. Determine the minimum number of individuals or part of individuals to be detected. Perform at least 3 experiments. If consistent results are not obtained after 3 series then additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. brand of PCR reagents (in particular DNA polymerase) and PCR cycle conditions.
 Analytical specificityScreen at least once against a range of target organism(s), covering genetic diversity, different geographic origin and hosts, and relevant non-target organism(s), in particular those associated with the sample material. In addition, the test results can be supported by ‘in silico’ comparison of probe/primer sequences to sequences in genomic libraries.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityAnalyse 1 sample for each of the 2 levels modalities (at and above the limit of detection of target organism) with a minimum of 8 replicates of the sample extract. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Table 7Mycology.    Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented
Method for extraction/isolation/baiting of target organism from matrix Remark. Extraction is always validated by a test.
 Analytical sensitivityThe method should be able to extract/isolate/bait a sufficient quantity of the target organism to allow it to be cultured or analysed further. Whenever possible, determine by blending healthy and infected tissue the lowest amount of diseased tissues or features required to be plated or identified in order to perform a reliable analysis. Extract/isolate/bait the target from at least 3 samples (naturally infected or artificially infected samples). This may include washing procedure and membranes to trap spores.
 Analytical specificityThis parameter is not relevant. Extraction of the target organism from a sample is per definition non-specific.
 SelectivityThis parameter is not relevant. Extraction of the target organism from a sample is per definition non-selective.
 RepeatabilityUse one sample with low concentration of target and make 8 subsamples (extractions). Assess extraction efficiency by the relevant test method.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Morphological and morphometrical methods intended for identification:
 Analytical sensitivityEstablish the minimum number of features to perform a reliable identification with a minimum of three repetitions whenever possible. The lowest number of features depends on the condition of the sample (good to very poor), the development stage, the known intraspecies variability, the difficulty to interpret features, etc.
 Analytical specificityScreen against a range of morphologically similar target and non target organisms.
 SelectivityNot applicable.
 RepeatabilityAt least 8 identifications of the target preparation as a blind test within a range of morphologically similar target and non target organisms. The blind test could be conducted as follows: the same target preparation is presented among several non target preparations. This is repeated 8 times, and non target preparations may vary between repetitions.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Molecular methods, e.g. hybridization, PCR Remark. This step also includes methods for isolation of DNA from the sample material.
 Analytical sensitivityDetermine the minimum quantity of target (e.g. number of conidia or weight of infected material in healthy material) from which a detectable amount of target DNA can be obtained. Perform at least 3 experiments with 8 serial dilutions, preferably in host plant DNA. If consistent results are not obtained after 3 series then additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. brand of PCR reagents (in particular DNA polymerase) and PCR cycle conditions.
 Analytical specificityScreen at least once against a range of target and relevant non-target organisms (e.g. phylogenetically close fungi) that might be present in the sample and sample extract. In addition the test results can be supported by ‘in silico’ comparison of probe/primer sequences to sequences in genomic libraries.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityPerform 3 experiments at above and at the limit of detection with a minimum of 8 replicates. This can give indications on the level of certainty of measurement.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Serological methods: ELISA
 Analytical sensitivityDetermine, using at least 3 experiments the limit of detection e.g. number of conidia or weight of infected material in healthy material. Determine threshold level whereby consistent correct diagnosis is achieved (usually this is between 2 or 3 times the negative control). When consistent results are not obtained after 3 experiments additional series should be conducted.
 Analytical specificityScreen once against a range of related target organisms and relevant non-related target organisms (e.g. phylogenetically close fungi) that might be present in the sample and sample extract.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityPerform 3 experiments at above and at the limit of detection with a minimum of 8 replicates on the same day/same operator/same media. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Bioassay methods: baiting
 Analytical sensitivityDetermine the necessary quantity of matrix or matrix extract (e.g. grams of leaves, soil) to produce symptoms. Perform 3 experiments with 8 dilutions series with 10 plants. If consistent results are not obtained after 3 series then additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. stage of test plants, inoculation method and incubation conditions.
 Analytical specificityDefine specificity of the bioassay on (i) strains of the target fungi covering genetic diversity, different geographic origin and (ii) for a set of non-target fungi, in particular those associated with the sample material.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityPerform 3 experiments at above and at the minimal amount of inoculum to produce symptoms with a minimum of 8 replicates. For detection of latent infection, repeatability should be validated at the analytical sensitivity limit. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days when relevant.
Table 8.   Nematology Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented
Method for extraction of target organism from matrix Remark. Extraction is always validated by a test.
 Analytical sensitivityDetermine the percentage of nematodes that is recovered by the extraction method with a minimum of three samples.
 Analytical specificityNot applicable.
 SelectivityEvaluate the selectivity of the test regarding the diversity of the matrix (e.g. type of soils for cysts extractors).
 RepeatabilityUse one sample of target and make 8 subsamples (extractions). By the relevant test method, assess that the recovery percentage among the subsamples is in the same range.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Morphological and morphometrical methods intended for identification
 Analytical sensitivityEstablish the minimum number of individuals to perform a reliable identification with a minimum of three repetitions whenever possible. The lowest number of target individuals depends on the condition of the sample (good to very poor), the development stage, the known intraspecies variability, the difficulty to interpret features, etc.
 Analytical specificityScreen against a range of morphologically similar target and non target organisms.
 SelectivityNot applicable.
 RepeatabilityAt least 8 identifications of the same slide as a blind test within a range of morphologically similar target and non target organisms. The blind test could be conducted as follows: the same target preparation is presented among several non target preparations. This is repeated 8 times, and non target preparations may vary between repetitions.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Molecular methods, e.g. hybridisation, PCR Remark. This step also includes methods for isolation of DNA from the sample material.
 Analytical sensitivityPrepare a relative number of individuals. This number varies depending on the genus, species and stage. Determine the minimum number of individuals or part of individuals to be detected or identified. Perform at least 3 experiments. If consistent results are not obtained after 3 series then additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. brand of PCR reagents (in particular DNA polymerase) and PCR cycle conditions.
 Analytical specificityScreen at least once against a range of target organism(s), covering genetic diversity, different geographic origin and hosts, and relevant non-target organism(s), in particular those associated with the sample material. In addition the test results can be supported by ‘in silico’ comparison of probe/primer sequences to sequences in genomic libraries.
 SelectivityNot relevant for nematodes identification as they are previously isolated from the matrix. If the PCR test is used as a detection test, insensitivity of the test to variation of the matrix (e.g. soil, plant) should be determined.
 RepeatabilityAnalyse 1 sample for each of the 2 levels modalities (at and above the limit of detection of target organism) with a minimum of 8 replicates of the sample extract. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Biochemical methods: e.g. enzyme electrophoresis, protein profiling Remark. This step also includes sample preparation
 Analytical sensitivityDetermine the minimum number of individuals to be detected to perform a reliable analysis with a minimum of three samples whenever possible. The lowest number of target individuals depends on the condition of the sample (good to very poor), the known intraspecies variability, the difficulty to interpret features, etc. Test parameters should be stringently defined and standardised.
 Analytical specificityScreen against a range of target organism(s) and non target genus and/or species.
 SelectivityNot applicable.
 Repeatability1 sample for each of the 2 levels above and at the lowest number of individuals. At least 8 replicates of the same sample extract.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Bioassay methods: including baiting, pathogenicity tests
 Analytical sensitivityDetermine the minimum number of individuals to produce symptoms or multiply in plant material with a minimum of three repetitions whenever possible. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. stage of test plants, inoculation method.
 Analytical specificityScreen against a range of target organism(s).
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars.
 RepeatabilityUse at least 3 replicates with the minimum number of individuals to produce symptoms. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Table 9.   Virology This table covers viruses, viroids and phytoplasmas. Figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnostics. Deviations from this guidance may be necessary depending on pest/plant combination. In such case reasons for deviation should be documented
Method for extraction of target from matrix (genomic material/organism) Remark. Extraction is always validated by a test.
 Analytical sensitivityThe method should be able to extract a sufficient quantity of target organism or its DNA/RNA/protein from a relevant test sample. Isolate the target from at least 3 samples.
 Analytical specificityThis parameter is not relevant. Extraction of the target organism from a sample is per definition non-specific.
 SelectivityTest selectivity of the extraction, as described in sensitivity, on different cultivars of the host plant.
 RepeatabilityUse one sample with low concentration of target and make 8 subsamples (extractions). Assess extraction efficiency by the relevant test method.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Molecular methods, e.g. hybridization, PCR and real-time PCR Remark. This step also includes methods for isolation of RNA/DNA from the sample material.
 Analytical sensitivityBecause the concentration of viruses, viroids and phytoplasmas is never known, determine the maximum dilution of RNA/DNA detected. The sensitivity determined here is not an absolute sensitivity but a relative sensitivity. Perform at least 3 experiments with eight serial dilutions. If consistent results are not obtained after 3 series then additional series should be prepared and tested. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. brand of PCR reagents (in particular DNA polymerase) and PCR cycle conditions.
 Analytical specificityScreen once against a range of target and relevant non-target organisms that might be present in the sample. In addition the test results can be supported by ‘in silico’ comparison of probe/primer sequences to sequences in genomic libraries.
 SelectivityPossible influences on the analytical sensitivity have to be considered. Determine the influence of the matrix by adding positive sample to sap from different cultivars of the crop.
 RepeatabilityAt least one sample for each of the 2 modalities (regarding analytical sensitivity results) low/medium levels of target organism, min. 2 subsamples, at least 3 but preferably 8 replicates.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Serological methods: ELISA and Direct Tissue Blot Immuno Assay
 Analytical sensitivityBecause the concentration of viruses is never known: use one healthy sample (same matrix as to be validated), prepare at least 8 subsamples to determine the standard deviation in the optical density (OD) to determine the background reaction. Perform at least 3 experiments of eight serial dilutions of positive sample in the healthy sample selected. In each experiment, perform two repetitions for each serial dilution. Determine the highest dilution of sample extracts which could be detected. If consistent results are not obtained after 3 series then additional series should be prepared and tested. The sensitivity determined here is not an absolute sensitivity but a relative sensitivity. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. the OD threshold in the ELISA test.
 Analytical specificityScreen antibodies against a range of related target organisms and relevant non-related target organisms that might be present in the sample.
 SelectivityPossible influences on the analytical sensitivity have to be considered. Determine the influence of the matrix by adding positive sample to sap from different cultivars of the crop. Use at least 2 subsamples for each target organism.
 RepeatabilityUse at least 3 samples at a low target level (regarding analytical sensitivity results), 2 subsamples for each and 8 different plates (tests). This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Bioassay methods: plant test (mainly used as verification test for viruses or viroids but not for phytoplasmas) and grafting.
 Analytical sensitivityDetermine the maximum dilution of inoculum in the buffer to produce symptoms or multiply in plants. This is only an estimation of dilutions that can be used. Use 1 representative sample and 3 subsamples for each dilution. The sensitivity determined here is not an absolute sensitivity but a relative sensitivity. Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardised, e.g. stage of test plants, inoculation method and incubation conditions. Not relevant for grafting.
 Analytical specificityDefine specificity of the bioassay on (i) strains of the target organism covering genetic diversity, different geographic origin and hosts and (ii) for a set of non-target organisms, in particular those associated with the sample material.
 SelectivityDetermine the relative insensitivity of the test to variations of the sample material, e.g. by using different cultivars of the host plant.
 RepeatabilityUse one representative sample (with eight plants for each host) of appropriate dilution determined in sensitivity assay and use the host plants determined in specificity assay. This assessment will provide information on the level of uncertainty of the test result.
 ReproducibilityAs for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Biochemical methods: e.g. electrophoresis, R-PAGE
 Analytical sensitivityDetermine the maximum dilution of target individuals/nucleic acid/protein to perform a reliable analysis. The sensitivity determined here is not an absolute sensitivity but a relative sensitivity.Specify positive and negative reactions. Test parameters should be stringently defined and standardised.
 Analytical specificityCompare with relevant organisms/proteins/contaminants and show differentiation can be made. Investigate also intraspecific variability. Define the characteristics to be identified.
 SelectivityNot applicable.
 RepeatabilityAt least 8 replicates of same sample extract with low levels of target organism. These levels should be defined as proportional to the analytical sensitivity.
 ReproducibilityAs for repeatability but vary relevant conditions, e.g. different operator, time (delay and storing conditions after sample preparation), to assess flexibility of the test. These require at least 8 replicates. Also equipment and reagents may be varied as part of robustness. These conditions require at least 3 replicates with comparable results.