Study on detection of Salmonella in poultry samples by real-time PCR with Taqman probes / 中华预防医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To develop a RT-PCR method for a rapid and sensitive detection of Salmonella in poultry samples.</p><p><b>METHODS</b>The RT-PCR method was established and its specificity was testified on the basis of invA gene. Serial 10-fold diluted pure suspension culture of CMCC 50041 was detected by RT-PCR, the standard curve was constructed and the amplification efficiency was calculated. Artificially contaminated experiment was done, six artificially-inoculated samples containing final concentration of Salmonella CMCC 50041 (1, 10, 10(2), 10(3), 10(4), 10(5) and 10(6) CFU per 25 g poultry samples) were prepared respectively. All the samples were incubated for 0, 4, 8, 12 and 18 h and the DNA was extracted for RT-PCR detection, meanwhile by PCR detection and the traditional method. The sensitiveness and specificity were compared among the three methods. At the same time, 16 samples of retail whole poultry were collected from markets and detected by the above three methods as well, and thereby to further compare the positive detection among the three methods.</p><p><b>RESULTS</b>The established RT-PCR method was specific for the detection of Salmonella. The sensitivity was 5.2×10(3) CFU/ml for pure Salmonella culture without enrichment. Correlation coefficients of standard curves constructed using the Ct versus log value of concentration of Salmonella showed good linearity over a 8-log dynamic range (5.2×10(3)-5.2×10(10) CFU/ml), with the R(2) at 0.999. RT-PCR detection limit for artificially contaminated samples after enriching for 12 h was 1 CFU/25 g sample, which was the same with the limit of PCR and 10 times more sensitive than the limit of traditional method. Standard curve of sample after enrichment for 12 h was established. Seven of 16 samples were detected positive by RT-PCR, which were also tested positive by PCR, while only five samples were positive by traditional method. The positive ones were quantitatively analyzed using standard curve of sample and determined the initial Salmonella numbers of CFU/25 g.</p><p><b>CONCLUSION</b>The established RT-PCR technology was simple, rapid, sensitive and specific, which was suitable to quickly detect Salmonella in poultry samples.</p>