志贺氏菌检测和分群PCR方法的建立

Study on PCR Method for Shigella Detection and Group Differentiation

为弥补目前志贺氏菌分子生物学检测方法的不足,根据志贺氏菌的invC、rfc、wbgZ和rfpB等基因,分别设计引物建立了鉴定志贺氏菌属、福氏志贺氏菌、宋内志贺菌和痢疾志贺氏菌的PCR方法。同时,以根据ompA基因设计的引物作为参照,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:17株志贺氏菌和19株非志贺氏菌均出现100%的特异性反应;对增菌肉汤直接进行PCR检测时,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^(-1)),在原奶、生肉和奶粉中的检出限分别为≤12、27和≤27 cfu/(25 g·mL^(-1));分离培养后再对可疑菌落进行PCR鉴定时,所有样品检出限均为1~3 cfu/(25 g·mL^(-1)),与传统培养法检出限一致。结果表明,该PCR方法快速、敏感、特异,适合用于志贺氏菌的常规检测分析。

In order to improve the current methods for molecular biological detection of Shigella,primers were designed respectively according to the genes of invC,rfc,wbgZ and rfpB of Shigella,and a PCR method was established to identify Shigella,S.fexneri,S. sonnei and S.dysenteriae. At the same time,taking the primers designed from ompA gene as a reference,the method was veri？ed by speci？c and arti？cial inoculation tests. The results showed that speci？c reaction were observed in both 17 Shigella strains and 19 non-Shigella strains. When the enrichment broth was detected directly by PCR,the detection limit of Shigella in instant food was 1~3 cfu/（25 g · mL-1）,including pasteurized milk,ice cream,acidophilus milk,cheese,cooked meat and sau sage,and the detection limits of Shigella in raw milk,raw meat and powdered milk were ≤ 12,27 and ≤ 27 cfu/（25 g · mL-1）,respectively. When PCR was done after isolation and culture of suspected bacteria,the detection limit of all samples was 1-3 cfu/（25 g · mL-1）,which was consistent with the traditional culture method. In conclusion,the PCR method was rapid,sensitive and speci？c,and it was suitable for routine analysis of Shigella.