Study on the Reverse Linear Probe Hybridization for Detecting Salmonella in Laboratory Animals
Author of the article:WEI Liwen1, WEI Li1, ZHANG Wenlu2, PAN Yongquan1, TAN Yi1, LAI Guoqi1*, XU Yongzhu3*
Author's Workplace：(1. Laboratory Animal Center, Chongqing Medical University, Chongqing 400016, China; 2. Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, China; 3. Chongqing Health Services Center, Chongqing 400020, China)
Key Words：salmonella; laboratory animals; polymerase chain reaction; RLPH
Abstract：Objective To establish a simple, rapid, accurate, sensitive and specific detection method of salmonella in laboratory animals. Methods Based on sequence of argT, universal primers and specific probes were designed. 5' of upstream primer was labeled by biotin. Specific probes, 3′ and 5′ of which were added poly C respectively, were fixed on the nitrocellulose membrane linearly, so as to make the amplification products of salmonella to hybridize with probes. By optimizing the hybridization conditions, a reverse linear probe hybridization method (RLPH) was established. 74 laboratory animals’ serum samples from Chongqing were detected by this method, compared with traditional culture method. Results More than 3 ng/µL of PCR products of salmonella was effectively detected by RLPH. The whole process, containing bacterium's culturing, DNA extraction, PCR and RLPH, was finished within 27 hours. The specificity is 100% (6/6) to test six other non-salmonella samples. The consistency is 100%. Conclusion The reverse linear probe hybridization method to salmonella is rapid, reliable, sensitive and specific.