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[利用聚合酶链反应和非放射性DNA探针检测弯曲杆菌属。III. 用于鉴定拉氏弯曲杆菌的DNA探针]

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. III. DNA probe for identification of C. laridis].

作者信息

Yamashita K, Takarada Y, Otsuka N, Kagawa S, Matsuoka A

机构信息

Clinical Laboratory, Hyogo College of Medicine, Nishinomiya.

出版信息

Rinsho Byori. 1994 Dec;42(12):1294-8.

PMID:7869595
Abstract

The development of a rapid and specific DNA probe assay for identification of Campylobacter species, including C. jejuni, C. coli, C. laridis, C. fetus, and C. hyointestinalis is important in determining the precise diagnosis of Campylobacter infections. Sequence data of our previous studies for a 240-base DNA fragment was used to select primers and probes conjugated to alkaline phosphatase, complementary to a portion of DNA between primers. However, a 21-base probe (CL (1)) tested here for detection of C. laridis was cross-reactive with PCR-amplified fragments of C. jejuni, C. coli and C. hyointestinalis, although it was not reactive with C. fetus and C. fetus subsp. fetus. To solve this problem, further modifications of the probe were therefore made to improve the specificity for those particular species. A second 21-base probe with a single base-substitution (CL (2)) and a third 20-base probe (CL(3)) were ineffective for identification of C. laridis, too. A fourth 20-base probe with a single base substitution (CL(4)) was a significant improvement over the results obtained by other three probes specifically to detect C. laridis, Thus, the alkaline phosphatase-labeled probe method developed so far is an interesting alternative without access to radioisotopes for clinical laboratories for identification of Campylobacter species, including C. jejuni/coli/hyointestinalis, C. laridis, and C. fetus/fetus subsp. fetus.

摘要

开发一种快速、特异的DNA探针检测方法来鉴定弯曲杆菌属菌种,包括空肠弯曲菌、结肠弯曲菌、拉氏弯曲菌、胎儿弯曲菌和猪肠弯曲菌,对于准确诊断弯曲杆菌感染非常重要。我们之前对一个240个碱基的DNA片段的研究中的序列数据被用于选择与碱性磷酸酶偶联的引物和探针,这些引物和探针与引物之间的一部分DNA互补。然而,这里测试的用于检测拉氏弯曲菌的一个21个碱基的探针(CL(1))与空肠弯曲菌、结肠弯曲菌和猪肠弯曲菌的PCR扩增片段有交叉反应,尽管它与胎儿弯曲菌和胎儿弯曲菌胎儿亚种没有反应。为了解决这个问题,因此对探针进行了进一步修饰以提高对那些特定菌种的特异性。一个有单个碱基替换的第二个21个碱基的探针(CL(2))和第三个20个碱基的探针(CL(3))对拉氏弯曲菌的鉴定也无效。一个有单个碱基替换的第四个20个碱基的探针(CL(4))在检测拉氏弯曲菌方面比其他三个探针的结果有显著改进。因此,迄今为止开发的碱性磷酸酶标记探针方法对于临床实验室来说是一种有趣的替代方法,无需使用放射性同位素即可鉴定弯曲杆菌属菌种,包括空肠/结肠/猪肠弯曲菌、拉氏弯曲菌和胎儿/胎儿弯曲菌胎儿亚种。

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