為對志賀氏菌屬細(xì)菌進行快速、準(zhǔn)確檢測和分群鑒定，根據(jù)志賀氏菌invC、rfc、wbgZ和rfpB基因，分別設(shè)計引物和探針，建立了鑒定志賀氏菌屬以及福氏志賀氏菌、宋內(nèi)志賀氏菌和痢疾志賀菌氏菌實時熒光PCR方法，同時將根據(jù)ompA基因設(shè)計的引物和探針作為內(nèi)參照，并通過特異性試驗和人工接種試驗等對該方法進行驗證。結(jié)果顯示，該方法對 17 株志賀氏菌和 19株非志賀氏菌均出現(xiàn)100%的特異性；用增菌肉湯直接進行PCR檢測，發(fā)現(xiàn)在消毒奶、冰淇淋、酸奶、奶酪、熟肉和香腸等即食食品中的志賀氏菌檢出限為1~3 cfu/25 g（mL），在原奶、生肉和奶粉中的檢出限分別為≤8 cfu/25 mL、12 cfu/25 g和≤12 cfu/25 g；分離培養(yǎng)后再對可疑菌落進行實時熒光PCR鑒定，發(fā)現(xiàn)所有樣品的檢出限為1~3 cfu/25 g（mL），與傳統(tǒng)培養(yǎng)法檢出限一致。結(jié)果表明，該實時熒光PCR方法是一個快速、敏感、特異的檢測方法，適合于志賀氏菌的常規(guī)檢測分析。
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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