荧光PCR高分辨率熔解曲线分析法检测食源性金黄色葡萄球菌

Rapid detection of Staphylococcus aureus by real-time PCR using high resolution melting curve analysis

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。

A rapid real-time PCR method for detecting Staphylococcus aureus （SA） by high resolution melting curve analysis （HRM） was developed. A pair of specific primers was designed targeting the sa442 gene of SA. After optimizing the conditions, the specificity of the method was validated with 8 target SA strains and 8 non-target strains. The solutions with different copies of purified recombinant plasmids PMD18-sa442 were analyzed by the HRM real-time PCR method to evaluate its sensitivity. The reproducibility of the method was also evaluated. The method was also applied to detect artificially- inoculated chicken samples added with 5, 10, 20 cfu/25 g Staphylococcus aureus. The results showed that： （1）The developed HRM real-time PCR method showed a good specificity by detecting only SA with the Tm value of （77.34±0.287） ℃ and was not affected by other normal food-borne pathogenic bacteria such as Salmonela, Listeria monocytogenes, E.coli O157, et al. （2）The sensitivity of this method was 2.00x102 copies/mL. （3）The developed protocol of HRM real-time PCR method showed a high reproducibility with the variations （CVs） of （0.76±0.52）% and （1.34±0.45）% inner and between tests, respectively. （4）The method showed a good performance in the detection of artificially-inoculated chicken samples. The established HRM real-time PCR assay exibited sufficient specificity, high sensitivity and good reproducibility. It is rapid, simple and cheap and would be useful in food microbiology laboratory for detectina SA.