Establishment And Application Of Multiplex-PCR Detection For Staphylococcus Aureus Escherichia Coli And Clostridium Perfringens In Dairy Products

Dairy safety accidents have occurred frequently in recent years ,which are mainly dairy poisoning caused by pathogenic bacteria. Taccidents made dairy safety has become one of the topic problems. The main pathogenic bacteria in the dairy product includes staphylococcus aureus, escherichia coli and compared with perfringens bacteria, so the fast detection technique for them and their toxins has been hotspot of research currently. Thereinto PCR technology has been widely applied for its high specificity, sensitivity and efficiency.Our test isolated staphylococcus aureus, escherichia coli and Clostridium perfringens from clinical dairy and carried out PCR amplification. Meanwhile, the respective standard strains of staphylococcus aureus (ATCC25923), escherichia coli (ATCC25922) and Clostridium perfringens (ATCC13124) were positive control, and salmonella was negative control. With the standard of the respective target strap, the specificities of nuc.16S-23S rRNA and cpa genes were tested in detection of staphylococcus aureus, escherichia coli and Clostridium perfringens. The PCR products were sequenced and compared with the standard sequences of V01281; CU928160.2 and L43546.1 in Genebank, the results showed that three homologies were all above 99.50% that proved the primers'specificities further. By optimizing the factors which effect PCR amplification, such as the concentration of Mg²⁺, primers, dNTP and Taq enzyme, the temperature and time of annealing and extension, the optimum condition was determine: the concentration of Escherichia coli.: 80 nmol/mL, Staphylococcus aureus and Clostridium perfringens were 40 nmol/mL, Mg2+ (25 mmol ) 1.5μL, dNTP (2.5 mmol) 2uL, Taq (2.5 U) 0.5uL, and the cycle parameters were: Pre-degenerated at 94℃for 7 mins, degenerated at 94℃for 30 s, annealing at 55℃for 30 s, extension at 71℃for 90 s and finally extension at 71℃for 5 mins. The whole reaction included 30 cycles. Under the above test condition the sensitivity of bacteria detection was l0cfu/mL. Man-made polluted pasteur milk had been detected the quantity of bacteria by enlarged culturing experiment, and the results displayed that they could be detected in 6 h whenever the original strain concentration was 10²cuf/mL.This test confirmed the specificities of nuc, 16S-23S rRNA and cpa gene in detection of staphylococcus aureus, escherichia coli and Clostridium perfringens in milk respectively. And the sensitivity of PCR detection was ensured by optimizing reaction conditions. We detected some clinical samples with this method and the results proved the feasibility of multiple PCR method using nuc, 16S-23S rRNA and cpa as target genes in practical pathogenic bacteria detection.