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志贺氏菌检测和分群实时荧光PCR方法的建立

6940 2020-03-12 14:32:07 王海艳,赵治国,催 强,等 中国动物检疫
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为对志贺氏菌属细菌进行快速、准确检测和分群鉴定,根据志贺氏菌invC、rfc、wbgZ和rfpB基因,分别设计引物和探针,建立了鉴定志贺氏菌属以及福氏志贺氏菌、宋内志贺氏菌和痢疾志贺菌氏菌实时荧光PCR方法,同时将根据ompA基因设计的引物和探针作为内参照,并通过特异性试验和人工接种试验等对该方法进行验证。结果显示,该方法对 17 株志贺氏菌和 19株非志贺氏菌均出现100%的特异性;用增菌肉汤直接进行PCR检测,发现在消毒奶、冰淇淋、酸奶、奶酪、熟肉和香肠等即食食品中的志贺氏菌检出限为1~3 cfu/25 g(mL),在原奶、生肉和奶粉中的检出限分别为≤8 cfu/25 mL、12 cfu/25 g和≤12 cfu/25 g;分离培养后再对可疑菌落进行实时荧光PCR鉴定,发现所有样品的检出限为1~3 cfu/25 g(mL),与传统培养法检出限一致。结果表明,该实时荧光PCR方法是一个快速、敏感、特异的检测方法,适合于志贺氏菌的常规检测分析。

Establishment of a Real-time PCR Assay for Detecting and Classifying Shigella

In order to detect and classify Shigella bacteria rapidly and accurately,the primers and probes were respectively designed according to the genes of invC,rfc,wbgZ and rfpB,and a real-time PCR assay was established to identify Sh. castellani including Sh. flexneri,Sh. sonnei and Sh. dysenteriae,then it was verified by specific test and artificial inoculation test with reference to the results of using the primers and probes designed according to ompA gene. The results showed that the specificity was 100% when all 17 strains of Shigella and 19 non-shigella strains were amplified by the established assay;through PCR assay with enrichment broth,the detection limits of Shigella in pasteurized milk,ice cream,acidophilus milk,cheese,cooked meat,sausage and other instant foods were 1–3 cfu/25 g(mL),and that in raw milk,raw meat and milk powder were≤8 cfu/25 mL,12 cfu/25g and≤12 cfu/25 g,respectively;the suspicious bacterial colonies were isolated and cultivated for real-time PCR assay,it was found that the detection limit of all samples was 1–3 cfu/25 g(mL),which was consistent with the result by traditional cultivation method. In short,the real-time PCR assay was rapid,sensitive and specific,which was appropriate for routine detection and identification of Shigella.

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