12.2. Assay selection and validation

Full validation of molecular techniques, as per guidelines issued by the World Organization for Animal Health (OIE) is a relatively new concept in the field of honey bee virus diagnosis. In general terms, a diagnostic protocol is designed in response to a particular diagnostic need and this is then developed into an optimized, documented and fixed procedure, using a series of intra-laboratory validation steps that demonstrate the reliability of results and the performance of the method. Recently, a new standard (XP U47-600) was developed by the French Standards Institute (AFNOR) concerning the minimum requirements for the development, validation and implementation of veterinary PCR-based diagnostic methods in animal health, based on the recommendations by the OIE and following the ISO/IEC 17025 criteria (NF, 2005; OIE, 2010). The validation procedure establishes the performance characteristics for each test method, such as sensitivity, specificity, detection and/or quantification limits.

The initial validation of a RT-qPCR assay involves two steps. The first concerns the validation of the qPCR assay itself, in terms of:

1. Analytical specificity.
2. The PCR detection limit (DLPCR).
3. The PCR quantification limit (QLPCR).  
4. The linearity and efficiency of the qPCR assay.

The second step concerns the evaluation of the entire diagnostic protocol in terms of:

1. The method’s detection limit (DLmethod).
2. The diagnostic specificity and sensitivity on samples of known status.
3. The method’s quantitation limit (QLmethod) based on a validation range and accuracy profile. 

In each of the two steps, various performance parameters are calculated, including measurement uncertainty (MU), deviations of repeatability and intermediate reliability.

The following is a step-by-step outline of how to develop an accredited RT-qPCR assay for the detection and quantitation of honey bee viruses, based on the successful development of such an assay for CBPV (Blanchard et al., 2012).

12.2.3. qPCR dynamic range and quantitation limit