Combination Of Direct-ELISA And PCR For Rapid Detection Of Salmonella

Salmonella is a common, important pathogen of zoonosis, the number of food poisoning cases caused by Salmonella ranks first among all food poisoning cases, which caused great losses to national economy. Salmonella is of great importance in public health, food hygiene, animal science and veterinary service, and import-export inspection and quarantine.A PCR technique for the detection of Salmonella was established using a pair of primers designed according to the sequence of histine trans-operon gene. It was shown that a specific 495 bp fragment was amplified by this PCR procedure from all Salmonella genomic DNA samples of 15 reference strains and 27 isolates, while control strains, including Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Streptococcus faecalis, Pseudomonas aeruginosa, Clostridium welchii, Listeria grayi, Shigella, Citrobacter, could not be demonstrated the 495 bp band. The sequence of the PCR products accorded with the known sequence of histine trans-operon gene. These results suggested that this PCR method was specific for Salmonella genus. It was further confirmed that PCR had high specificity by dot hybridization using sequenced PCR product as a probe. The detection threshold of PCR was determined as 14 pg of Salmonella DNA sample. When Salmonella and E. coli were mixed at a ratio of 1:100, both PCR and direct-ELISA can detect the positive signals. When the ratio was 1:1000, PCR but not direct-ELISA gives positive results. Through comparison among 5 different methods of DNA template extraction, heat-lyse treatment was selected for its easy-operation, low-cost and celerity.An Mab-based direct-ELISA and its kit for the detection of Salmonella was developed previously. To compare the accuracy of both direct-ELISA and PCR for therapid detection of Salmonella, they were used to detect Salmonella in fresh milk, lobster, shelled shrimp, cooked food samples depending on the National Standard (GB) method to verify the results. The result coincidence of these two methods reached 97.6%. Furthermore, it was also compared the efficacy of two methods to detect Salmonella in the different enrichment broth of shelled shrimp and stool samples. In the pre-enrichment broth, PCR can amplify the specific band, which was verified by GB method, while direct-ELISA gives less than 0.5 of the OD(490) value. In selective enrichment and post-enrichment broth, Salmonella can be detected by both methods. These results suggest that the sensitivity of PCR is higher than that of direct-ELISA.We use direct-ELISA to detect water samples from downtown area of Yangzhou city, and use PCR to test the ELISA positive samples and randomly selected negative samples, the coincidence of them is also 97.2%. In the 2002 spring physical examination of Yangzhou Guangling district for employees in the restaurant industry, 1426 human feces samples were detected by the combination strategy of direct-ELISA and PCR method. Compared with the results of National Standard method, the sensitivity and specificity of direct ELISA was 100% and 97%, respectively, while those of PCR method reached both 100%. It also indicated that combination use of two methods could give positive report within 40 hrs, and also achieve high sensitivity and specificity .Based on all the results, an opitimized rapid protocol for the detection of Salmonella was developed, that is, use direct-ELISA to screen the large number of samples first, and then use PCR to test the ELISA positive samples, the final step is, if needed, to use National Standard method to determine the serotypes of Salmonellae. This combined method was highly specific, sensitive and very quick. It provides an efficient tool to handle many samples in diseases prevention, animal quarantine, food and feed hygiene. This procedure ensures the accuracy and reliability of detecting results and saves lots of material and manpower, which would create great social and economic interest.