Inter-Laboratory Comparison of qPCR Assays for Piscirickettsia salmonis in Atlantic Salmon (Salmo salar L.) in 11 Chilean Laboratories.

Abstract

Real-time PCR (qPCR) testing is an essential component of early detection surveillance systems for Piscirickettsia salmonis infection in Atlantic salmon farms in Chile. Currently, all 11 laboratories in the authorised diagnostic laboratory network use assays based on published protocols. Compared with other P. salmonis qPCR assays, these assays have the advantage of targeting two different genes, that is, the 16S ribosomal gene and the internal transcribed spacer (ITS), potentially allowing for higher diagnostic accuracy. However, variation and lack of harmonisation of qPCR testing systems (e.g., primers/probe, RNA/DNA as target, extraction methods, etc.) may contribute to among-laboratory variation in qPCR results and an increased frequency of false-negative and false-positive results. The purpose of the ring trial reported herein was to compare qPCR results from 11 laboratories in Chile routinely testing Atlantic salmon for P. salmonis as part of a national control program. The panel of 14 samples included duplicates of three concentrations of P. salmonis in a homogenised head kidney, LF89 and EM90 bacteria and two negative controls (blank and a suspension of Flavobacterium psychrophilum). The sample order was randomised across labs, samples were tested blinded and analysed without knowledge of the source lab. Of the laboratories, 8 (72.7%) had at least one incorrect result out of 14 tested samples. Low-concentration samples (C_t of about 30) were more often incorrectly classified by reverse transcription-qPCR (RT-qPCR) (3/6 labs) than by qPCR (0/5). Six (54.5%) labs had at least one false-positive result indicating that cross-contamination was likely during sample processing. Affected laboratories are advised to conduct internal investigations to confirm the causes of false-positive results and recommendations for design and implementation of future ring trials are discussed.