Rapid Detection Of Pathogenic Bacteria In Infant Milk Powder By Triplex PCR

Food-borne diseases constantly threatening people’s health and lives is a major public health problem in food security. Especially, bacterial food poisoning is the main factor which causes the food poisoning. At present, the contaminated infant milk powder scandal owing to Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus happens occasionally. So the detection of food-borne pathogenic bacteria is a great of part in food and health security.At present, the detection of food-borne pathogenic bacteria mainly uses the traditional isolation and biochemical identification method, the immunological methods and the methods in molecular biology. It is complicated operation, time consuming and low-sensitive for the traditional isolation and biochemical identification method. The specificity and sensibility of immunological methods are unsatisfactory. The methods in molecular biology are higher-specific, shorter-time and higher-sensitive.Triplex PCR is an amplification method that can amplify three target sequences through adding to three pairs of specific primers. In this study, three pairs of specific primers were designed according to Enterobacter sakazakii ompA gene, Staphylococcus aureus nuc gene and Bacillus cereus hblA gene and triplex PCR was established to simultaneous diagnose three kinds of food-borne pathogens. The detecting results of 15 strains of bacteria are that 4 strains of Enterobacter sakazakii, 2 strains of Staphylococcus aureus and 1 strain of Bacillus cereus had amplified specific target channels, and another 8 strains of bacteria could not have amplified channels. The sequencing results of Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus were compared with the target sequences at GenBank and homologies of three pathogenic bacteria were all above 99%. The reaction system and reaction procedure of triplex PCR were determined through optimizing annealing temperature, three pairs of primers, Mg²⁺, dNTPs and EasyTaq DNA Polymerase. The reaction system was 25μL: 10×Easy Taq Buffer(-Mg²⁺) 2.5μL, 2.5 mM dNTPs 2.5μL, 50 mM MgSO4 1.5μL, each 10μM ompA primer 0.6μL, each 10μM nuc primer 1.0μL, each 10μM hblA primer 1.0μL, each template DNA 2μL, 5 U/μL EasyTaq DNA Polymerase 0.3μL, ddH20 7μL. The reaction procedure was pre-degenerated at 95℃for 5 min, degenerated at 94℃for 45 s, annealing at 61℃for 45 s, extention at 72℃for 45 s, run 30 cycles, final extention at 72℃for 10 min.Specific target channels could be amplified with random combinations of Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus. Under the above optimum experiment condition, the sensitivities of the triplex PCR for Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus were 103 CFU/mL. Extracting genomic DNA with absolute ethyl alcohol, ammonia water, petroleum ether and kit extraction, the detection limits of the triplex PCR for Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus in infant milk powder were 104 CFU/g. Compared with the national standard method, the sensitivities of triplex PCR to detect the actual samples for the three pathogenic bacteria were all 100%; the specificities of triplex PCR to detect the actual samples for the three pathogenic bacteria were 96.4%, 96.6%, 100%, respectively; the coincidence rate of triplex PCR to detect the actual samples for the three pathogenic bacteria were 96.7%, 96.7%, 100%, respectively.In the study, triplex PCR assay had been established according to the food-borne pathogenic bacteria appeared in infant milk powder and it would provide a new method to simultaneously detect Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus.