Shangkuan Y H, Show Y S, Wang T M
Division of Epidemiology, National Defense Medical Center, Sanhsia, Taipei, Taiwan, ROC.
J Appl Bacteriol. 1995 Sep;79(3):264-73. doi: 10.1111/j.1365-2672.1995.tb03136.x.
A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hylA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.
开发了一种多重聚合酶链反应（PCR），用于鉴定产霍乱毒素的霍乱弧菌并对霍乱弧菌O1进行生物分型。使用一对引物扩增ctxA2-B基因片段来鉴定产肠毒素的霍乱弧菌菌株。在同一反应中，使用针对hylA基因的三种引物将霍乱弧菌O1菌株同时区分为不同的生物型。多重PCR中的hlyA扩增子在检测产毒素菌株时用作内部对照，因为所有测试的霍乱弧菌菌株中都存在hlyA基因序列。在用ctxA2-B基因内部引物进行巢式PCR检测之前，将牡蛎上存在的霍乱弧菌在碱性蛋白胨水中富集6小时，检测限低于每克食物3个菌落形成单位（cfu）。
