Ezaki T, Takeuchi N, Liu S L, Kai A, Yamamoto H, Yabuuchi E
Department of Microbiology, Gifu University School of Medicine.
Microbiol Immunol. 1988;32(2):141-50. doi: 10.1111/j.1348-0421.1988.tb01373.x.
A simplified and rapid genetic identification method for Campylobacter species without radioisotope was established. Three different amounts of DNA (200, 50, and 12.5 ng) extracted from each type strain of Campylobacter species with standard Marmur's procedure were spotted on a nitrocellulose filter. DNA obtained from one ml bacterial suspension at a concentration of McFarland standard turbidity No. 1 of Campylobacter fetus, C. jejuni, C. coli, and C. pylori isolates were sufficiently labeled with photo-biotin within 15 min and clearly hybridized with the type strain of the corresponding species within four to six hours. Hybridized spots were visualized with alkaline-phosphatase-conjugated streptavidin color-detection method. The reaction was usually stopped within 30 min. Atypical clinical isolates such as a nitrate-negative C. jejuni, two nalidixic acid-resistant C. jejuni, and two strains of C. fetus able to grow at 42 C, which were tentatively identified as such, were definitely identified by the simplified DNA hybridization method presented here. This method will be applicable routinely for the definite identification of atypical strains of Campylobacter species and other gram-negative bacteria difficult to identify biochemically.
建立了一种无需放射性同位素的用于弯曲杆菌属菌种的简化快速基因鉴定方法。采用标准的Marmur方法从每种弯曲杆菌属标准菌株中提取三种不同量的DNA（200、50和12.5 ng），点样于硝酸纤维素滤膜上。从胎儿弯曲杆菌、空肠弯曲杆菌、结肠弯曲杆菌和幽门螺杆菌分离株的1 ml处于麦氏标准浊度1号的细菌悬液中获得的DNA在15分钟内用光生物素充分标记，并在4至6小时内与相应菌种的标准菌株清晰杂交。用碱性磷酸酶偶联链霉亲和素颜色检测法观察杂交斑点。反应通常在30分钟内终止。通过本文介绍的简化DNA杂交方法明确鉴定了一些非典型临床分离株，如硝酸盐阴性空肠弯曲杆菌、两株耐萘啶酸空肠弯曲杆菌以及两株能在42℃生长的胎儿弯曲杆菌，这些菌株最初是这样初步鉴定的。该方法将常规适用于弯曲杆菌属菌种非典型菌株及其他难以通过生化方法鉴定的革兰氏阴性菌的明确鉴定。
