Research And Development Of Multiplex-pcr Kit Detecting Four Foodborne Pathogenic Bacteria In Aquatic Products

China is a big producer and consumer of aquatic products and has became one of the most important countries in world fishery industry. However, the aquatic products are vulnerable to bacterial contamination during the processes of aquaculture, fishing, storage, processing, transportation and sale. Therefore detection of pathogenic bacteria is an significant work in hygienic inspection of aquatic and processing products to ensure health and life safety of consumers. Among these pathogenic bacteria, Salmonella, Shigella, Slaphylococcus aureus and Vibrio parahaemolylicus were considered as the most typical pathogens in aquatic and processing products which has been paid close attention by each country's inspection department and center of disease control. The item that non-pathogens should be detected in aquatic products has been clearly stipulated in Chinese National Standard DB11/519-2008. However, traditional detecting method exists some disadvantages like detecting period as long as5-10days, complicated detecting procedures and heavy workload. It is necessary and urgent to establish a modern detecting method which is rapid, accurate and easy to operate as well as with high sensitivity and speciality to satisfy the need of modern food safety rapid detection.This study was aimed to establish a rapid method to detect four foodborne pathogenic bacteria including Salmonella spp., Siaphylococcus aureus, Vibrio parahaemolylicus and Shigella spp. in aquatic products based on multiplex-PCR technique. This method was optimized and evaluated from several aspects and finally was intergrated as a rapid detecting kit named SSSV m-PCR kit.'The detailed study contents and results as follows:1. A selective enrichment broth (SSSV) was formulated to allow simultaneous growth of Salmonella spp., Shigella spp., Staphylococcus anreiis, and Vibrio parahaemolyticus in order to achieve rapid enrichment of these four foodborne pathogenic bacteria in aquatic products. Appropriate additives including accelerants, inhibitors and nutrient substance based on Buffered Peptone Water (BPW) were selected. The optimized ingredients and proportions of additives were determined by the single factor experiment and orthogonal test. The selectivity of SSSV and growth properties of four target pathogens in SSSV were also evaluated. The growth models of these four pathogens were built by the Gompertz equation, and characteristic parameters including exponential growth rate (EGR), lag-phase duration (LPD), generation time (GT) and maximum population density (MPD) were obtained. The resuscitative ability of SSSV to cold-stressed target pathogens in aquatic products at cold and freezing storage conditions was assessed by artificial contamination. And finally the detection effect of SSSV applying to practical aquatic samples was compared with Nutrient Broth (NB) and selective enrichment broths recommended by National Standards. The results showed that this enrichment broth, SSSV, was obtained to enrich simultaneously the growth of four pathogens and inhibited some non-target competitive microflora. The number of pathogens mixing with different concentrations grew form104to107CFU/mL in the SSSV broth at37℃for8h. The population of pathogens in the peeled shrimp, cuttlefish rolls and fish balls inoculated artificially and stored at4℃and-18C increased form105to106CFU/mL using SSSV enrichment at37℃for8h and12h, respectively. SSSV broth could recover and resuscitate these four pathogens suffered cold stress. The enrichment effect of SSSV for four target bacteria in practical aquatic samples was superior than Nutrient Broth, but was slightly inferior to the selective enrichment broths recommended by National Standards. 2. Gene code invasion protein A (invA) of Salmonella spp., heat stable nuclease (nuc)of Staphylococcus aureus, toxin regulatory protein (loxR) of Vibrio parahaemolylicus and invasion plasmid antigen H (ipaH) of Shigella spp. were selected as target genes to design their corresponding pairs of oligonucleotide primers. Then a rapid multiplex-PCR method was established and optimized to detect these four common foodborne pathogens. As showed by the results, the amplified fragment sizes of Salmonella, Slaphylococcus aiuvns, Shigella and Vibrio parahaemolylicus were549bp,426bp,348bp,243bp respectively and the accuracy of amplification was demonstrated by sequencing and BLAST alignments. This method has satifying specificity and high sensitivity, the limits of detection were as low as101CFU/mL for Salmonell, Vibiro parahaemolylicus and102CFU/mL for Shigella, Slaphylococcus aureus. The multiplex-PCR method combined with SSSV enrichement broth was applied to detect50aquatic product samples compared with national standard methods, the coincidence rate of two methods was more than97%, which was no significant difference was observed between these two methods (P>0.05). This combined method could shorten detection time and was able to achieve the detection task within24hours.3. The reagents was assemblied as a detection kit based on the method above. This SSSV m-PCR kit was developed to rapid and convenient detect Salmonella, Staphylococcus aureus, Vibrio parahaemolylicus and Shigella in aquatic products. The preservation method, stability and reproducibility were determined and evaluated. The results indicated that the detection effect of this kit was stable and reproducible. The shelf life of SSSV m-PCR kit preserved at4℃was as long as30-60days, while it approached or reached1year at the preservation temperatures of-20"C and-80"C respectively. However repeated freezing and thawing had great influence on the quality of kit, it is recommended that users should choose approriate preservation method according to different detecting needs and conditions.