Application Of Multiplex PCR And Recombinant DNA Technologies In The Control Of Aquatic Product Security

As one of the biggest countries of aquatic foods producing, consuming and exporting in the world, the security of the aquatic products is very important, it concerns the health and life safety of people, the development of economy and stabilization of society, and the reputation of government and nation. At the same time, with the the laggard model of the traditional development, the aquaculture is development in low productive forces, the high-density culture method has caused many bacteria to breed rapidly. In order to prevent and cure these diseases, some farmers abuse antibiotics, added to pollute water increasingly, poisons are accumulated in aquatic's body through food chain, which has already been a big threat to the benefit of the people. So, during a long time in the future, detecting security and controlling quality in aquatic products will be important to us.The present study is mainly divided into two parts. The first part of the study is mainly aimed at the mixture of the pollution in aquatic products nowadays, set up a method to detecte the food-borne pathogens simultaneously. Salmonella, Listeria monocytogenes, Staphylococcus aureus and Vibrio parahaemolyticus are four types of main food-borne pathogens. The traditional microbiology identification need many steps, such as isolating culture,biochemical reaction and serological identification. However, these methods are all complex, time-consuming and less sensitive in a certain extent, furthermore, they only detecte one pathogen at a time. In this study, four pairs of specific primer were designed according to the invasion protein A gene (invA), invasion associated protein gene (iap),thermostable nuclease gene (nuc) and thermostable direct hemolysin gene (tdh) from Salmonella, L. monocytogenes, S. aureus and V. parahaemolyticus, respectively. A fragment of conserved sequence of 16SrRNA gene was used as an internal control of amplifiable bacterial DNA. An optimal multiplex PCR method was established by adjusting and screening reaction parameters such as annealing temperature and primer concentration et al. The optimized reaction mixture for PCR was as follows: 10×PCR buffer (including Mg²⁺) 2.0μL, dNTP 200μmol/L, DNA template 1μL, Taq DNA polymerase 2.5U, and the concentrations of primers were 200nmol/L for 16SrRNA, 250nmol/L for tdh, 300nmol/L for nuc, 350nmol/L for iap and 250nmol/L for invA, the final volume was adjusted to 20μL with sterilized water. Every cycle consisted of denaturation at 94℃for 30s, annealing at 57℃for 40s, and extension at 72℃for 1min. This cycle was repeated 30 times with a final extension step at 72℃for 10min. In order to test feasibility of the multiplex PCR method established, Penaeus vannamei were inoculated with the four types of pathogens, and suffered from multiplex PCR detection. The results showed that the various target DNA fragments can be amplified by multiplex PCR in the cases of both unenrichment and pre-enrichment for Penaeus vannamei artificially inoculated with pathogens. However, the detecting sensitivity of multiplex PCR can be significantly increased by incubating contaminated sample at 37℃. Thus, the multiplex PCR method established in this study offers an efficient and rapid way for high sensitive detection of pathogens in foods including aquatic products.The second part of the study is mainly aimed at the phenomenon of abusing antibiotics. We prepared antimicrobial peptide by recombinant DNA technology. Antimicrobial peptides are used for new feed additives which could instead of traditional antibiotics for animal feeds without drug-resistant and residue problems, generally produced by the cells of living organisms with some small molecule peptides, and play an important role in the host's immune response against microbial invasion. In this study, cloning mature peptide gene from liver-expressed antimicrobial peptide of Ictalurus punctatus, then sequenced and constructing a recombinant expression plasmid, laid a lot of important research foundation for preparing antimicrobial peptide. At first, with the full-length LEAP-2 gene obtained from antimicrobial peptide fragment, mature peptide with 140bp was amplified by PCR using a pair of primer carrying EcoRⅠand HindⅢrestriction enzyme sites, named mleap-2. The product of PCR was cloned into the plasmid pSURE-T, and then sequenced, found that there was no point mutation. We selected pET-30a as expression plasmid, pET-30a and mleap-2 were all dealt with EcoRⅠand HindⅢrestriction enzyme, the linear plasmid and the target designed fragment (mleap-2) were ligated with T4 DNA ligase. It was demonstrated by insert check and double restriction enzyme that the recombinant expression vector pET30a-mleap-2 was constructed.The recombinant plasmid was transformed into the E.coli BL21 (DE3) and sequenced, no point mutation was found in the mleap-2 gene. This study laid an important research foundation for recombinant expression of mature peptide and preparation of antibody.