In Thailand, several PCR-based methods are used by private and public service laboratories for the detection of white spot
syndrome virus (WSSV) infection in penaeid shrimp post larvae (PL) before they are stocked in rearing ponds. Conflicting test
results for similar samples sent to two service laboratories has decreased confidence in PCR testing. Thus, we compared the
sensitivity of several PCR methods commonly employed in Thailand using Taqman real-time PCR as the gold standard with a
purified WSSV template stock. Using this stock for assays, we found no significant inhibitory effect by WSSV-free host shrimp
DNA over the range 0 to 300 ng per reaction or by added DNA from WSSV-infected shrimp. Real-time PCR could detect WSSV
with certainty at dilutions of approximately 5 copies per reaction while 1000 copies were needed for a common one-step PCR
method and 50 for a common single-tube nested PCR (1N-PCR) method. Of 2 two-tube nested PCR protocols tested, one required
100 and the other 1000 copies. In addition to these sensitivity tests, a triple-blind ring test was carried out employing sets of 10
WSSV-infected DNA extracts sent to 12 commercial and public laboratories in Thailand, without specifying the PCR method to be
used. Returned results included no false positives and two false negatives, the latter both from light infection vials. This translated
into a test sensitivity of 97.3% and a specificity of 100%. Overall, the results confirmed the validity of PCR-based methods in
Thailand for detection of WSSV in shrimp DNA extracts.
© 2005 Elsevier B.V. All rights reserved.
Keywords: PCR-based methods; White spot syndrome virus (WSSV) infection; Penaeus monodo
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