[Study on detection of Salmonella in poultry samples by real-time PCR with Taqman probes].
OBJECTIVE
To develop a RT-PCR method for a rapid and sensitive detection of Salmonella in poultry samples.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The established RT-PCR technology was simple, rapid, sensitive and specific, which was suitable to quickly detect Salmonella in poultry samples.
Division of Risk Surveillance and Alert, China National Center for Food Safety Risk Assessment, Beijing 100022, China.
Lin
L
Yan
Email: xywang6401@126.com.
Xiaoying
X
Wang
Yunchang
Y
Guo
Xiaoyan
X
Pei
Dongmin
D
Yu
Dajin
D
Yang
D000818:Animals; D001426:Bacterial Proteins; D005516:Food Microbiology; D011200:Poultry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; C075518:invA protein, Bacteria