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以sa442为靶基因,结合特异性引物,建立了一种快速检测鉴定食品中常见的金黄色葡萄球菌的HRM(高分辨率熔解曲线)real-time PCR法,对该法进行特异性验证,敏感性分析及重复性评价,并实现了其在人工染菌鸡肉样本检测的初步应用。结果表明,该方法具有较强的特异性,对8株金黄色葡萄球菌目标菌株均产生特异性溶解曲线,Tm值为(77.34±0.287)℃,而沙门氏菌、单增李斯特菌、大肠杆菌O157等8种肉产品中常见的食源性非目标病原菌均不产生扩增曲线;灵敏度高,该法对阳性重组质粒PMD18-sa442的检测限为2.00×102 copies/mL;重复性强,同一样品于试验内及试验间的变异系数分别为(0.76±0.52)%和(1.34±0.45)%;且该法可初步应用于人工染菌鸡肉样(染菌量5、10、20 cfu/25 g样品匀浆)的检测。所建立的金黄色葡萄球菌HRM real-time PCR法具有特异性好、灵敏度高、重复性强的特点,能应用于食品样本的检测,为金黄色葡萄球菌的快速检测提供了新的方法。
Using sa442 as target gene and combining with specific primers, HRM (high resolution melting curve) real-time PCR method for rapid detection and identification of common Staphylococcus aureus in food was established. The method was specifically verified and sensitive Sex analysis and repeatability evaluation, and to achieve its preliminary application in the detection of artificial bacteria-contaminated chicken samples. The results showed that the method was highly specific, and all of the eight Staphylococcus aureus strains had a specific melting curve with a Tm value of (77.34 ± 0.287) ℃. Salmonella, Listeria monocytogenes, Escherichia coli O157 And other 8 kinds of meat products common non-target pathogenic bacteria food did not produce amplification curve; high sensitivity, the method of positive recombinant plasmid PMD18-sa442 detection limit of 2.00 × 102 copies / mL; repeatability, the same sample (0.76 ± 0.52)% and (1.34 ± 0.45)%, respectively. The method could be applied to the artificial staining of chicken meat (5, 10, 20 cfu / 25 g Sample homogenate) test. The established Staphylococcus aureus HRM real-time PCR method has the characteristics of good specificity, high sensitivity and repeatability, and can be applied to the detection of food samples, thereby providing a new method for the rapid detection of Staphylococcus aureus.