爲對(duì)志賀氏菌屬細菌進(jìn)行快速、愛生準确檢測和分群鑒定，根據志賀氏菌invC、rfc、wbgZ和rfpB基因，樂是分别設計引物和探針，建立了鑒定志賀氏菌屬以及福氏志賀氏菌、爸子宋内志賀氏菌和痢疾志賀菌氏菌實時(shí)熒光PCR方法，同時(shí)將(工票jiāng)根據ompA基因設計的引物和探針作爲内參照，并離們通過(guò)特異性試驗和人工接種(zhǒng)試驗等對(duì)該方法進(j離業ìn)行驗證。結果顯示，該方法對(山亮duì) 17 株志賀氏菌和 19株非志賀氏菌均出現100又站%的特異性；用增菌肉湯直接進(jìn)行PCR檢測，發(fā)現在消毒奶得暗、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爲1~3 cfu/關議25 g（mL），在原奶、生肉和奶粉中的檢出限分别爲≤8 生區cfu/25 mL、12 cfu/25 g和≤1的煙2 cfu/25 g；分離培養後(hòu)再對(duì)可疑菌落進東動(jìn)行實時(shí)熒光跳年PCR鑒定，發(fā)現所有樣(yàng)品的檢出限爲1~3 cf高有u/25 g（mL），與傳統培養法檢出限一緻。結舞雪果表明，該實時(shí)熒光PCR方法是一個快速、敏感、特異的視那檢測方法，适合于志賀氏菌的常規務老檢測分析。
Establishment of a Real-time PCR As到體say for Detecti黃事ng and Classifying Shigella
In order to detect a到視nd classify Shigella bact子現eria rapidly and accurately，the 市現primers and probes were respecti跳站vely designed acc雨多ording to the genes of筆年 invC，rfc，wbgZ and rfpB，and a r兒司eal-time PCR assay was es舊路tablished to ident多了ify Sh. castella為女ni including Sh. flexneri，Sh. sonne要姐i and Sh. dysenteriae，then it w舞懂as verified by specific te火師st and artifici你體al inoculation tes公黑t with reference to男機 the results of using the primers and p樂睡robes designed according to o著妹mpA gene. The results 分文showed that the specificity was 100通些% when all 17 strains of Shige開又lla and 19 non-shigella strains w到業ere amplified by the esta頻東blished assay；through PCR assay with en東土richment broth，th開市e detection lim通店its of Shigella in pasteurized milk筆書，ice cream，acidophilus milk，cheese，co雨見oked meat，sausage 文雨and other instant 事器foods were 1–3 cfu/25 g（mL），a近熱nd that in raw milk山讀，raw meat and milk 什地powder were≤8 cfu/25 mL，12 cf好知u/25g and≤12 cfu/25 g，respectively；做黑the suspicious b能請acterial colonies w跳妹ere isolated and cultiva吧算ted for real-time PCR assay，it was f黑妹ound that the detection l開舊imit of all samp跳月les was 1–3 cfu/25 g（mL），什線which was consistent with the result村物 by traditional cultivation房但 method. In short，the real-time PCR ass習下ay was rapid，sensitive長雨 and specific，which was appropriate fo月個r routine detection and identif草照ication of Shigella.
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