Establishment And Application Of Reverse Linear Probe Hybridization Detecting Method For Salmonella

Salmonella (Salmonella SPP.), all over the world, is a commonpathogen found in human and animals. Food poisoning, caused bysalmonella, often account for the first or the second in the wordwide.Salmonella infections are also one of the main factors, which affect thequality of laboratory animals and process of animal experiments. Asproducing of multiple drug-resistant strains in succession, the timelydetection of salmonella is effective in preventing salmonella spreading,ensuring the quality of laboratory animals and animal experiments carryout smoothly. Therefore, developed a simple and rapid detectiontechnology has a great application value. Reverse linear probehybridization method (RLPH) is able to highly specific and sensitiveamplify the objective gene. As a simple, rapid, reliable and low costmethod, it can meet the requirement.This study determined the reverse linear probe hybridization methodfor detection of salmonella, and prepared the salmonella test article. Forsalmonella’s argT genes, one pair of specific primers were designed, whoseupstream primer was labeled with biotin. Using Salmonella DNA astemplate, amplified a fragment of496bp.Two specific probes weredesigned according to the conservative region of PCR products sequence.Hybridize the PCR products and the probe.Then we optimized the reactionconditions. By optimizing,we set conditions as follows, annealing temperature was55℃,adding15c on both ends of the probe, hybridizationtime were30min, elution temperature was50℃, the graduate of antibodywas0.4u l. In this study, we have used the RLPH method primers ofSalmonella to amplify the bacteria such as, salmonella, staphylococcusaureus and epidermis staphylococcus aureus, escherichia coli, waxybacillus coli, pseudomonas aeruginosa, proteus, which were separated andpreserved by Department of inspection Chong Qing medical university.Amplification results of agarose gel electrophoresis results showed thatonly salmonella are positive. Further proved that, argT gene can be used asa specific and reliable target sequence for the detection of salmonella. Morethan3ng/μl of PCR products of Salmonella could be effectively detectedby RLPH. And no cross reaction with other bacteria was found. Reactionresults can be directly observed with macroscopic by adding NBT/BCIPalkaline phosphatase chromogenic. Salmonella RLPH method,with theadvantage of rapid, sensitive, specific, simple in operating and efficient inworking, not only can be used in the detection of salmonella inexperimental animals, also applies to food, clinical detection and diagnosisof infection with salmonella. This method can satisfy the needs of largequantities of samples and rapid detection.