聚合酶链反应检测致病性钩端螺旋体微量DNA的研究

作者通过对黄疸出血群钩端螺旋体DNA的分子克隆和筛选,获得了两个重组DNA片段,其中一个具有广泛针对各群致病性构体的特性,另一个仅对黄疸出血群钩体起杂交反应。经DNA序列分析和限制性谱测定,分别合成两对聚合酶链反应寡核苷酸引物——引物B1、2和引物B3、4。以该引物对各群、型钩体微量DNA(0.1 ng)行聚合酶链扩增反应,引物B1、2能引导扩增全部致病性钩体DNA,引物B3、4则特异性地扩增黄疸出血群钩体DNA,非致病性钩体和其他微生物DNA均无扩增反应。结果表明,聚合酶链反应是一种灵敏度极高、针对性很强的DNA扩增技术,可用于钩体病早期诊断和流行病学分类鉴定。

Detection of the Microquantity DNA of Leptospira Interrogans by Polymerase Chain Reaction

Leptospirosis is a severe zoonosis inthe world. The methods for detectingleptospira are not sensitive and specificso far. The problems in early diagnosisand epidemiological identification of Lep-tospirosis remain unsolved. Two recom-binant DNA fragments of serogroup Icter-ohaemorrhagiae, Leptospira were selectedby repeated molecular cloning and screen-ing in this study firstly. One of themcan hybridize with the DNA of variousserogroups of Leptospira interrogans; theother can only hybridize with the DNA ofserogroup Icterohaemorragiae. After thenu cleotides sequence analysis, from these2 recombinant DNA fragments, 2 pairs ofpolymerase chain reaction(PCR) oligonu-cleotide primers were synthesized,namedprimer B 1, 2 and primer B 3, 4 PCRswere carried out with these 2 primers fordetecting the microquantity (0.1 ng) ofvarious serovars, serogroup of leptospires.All the DNA of Leptospira interroganscan bo amplified by primer B 1, 2, andonly the DNA of serogroup icterohaemor-rhagiae, Leptospira reacted specially withprimer B 3, 4. The DNA of non--patho-genetic Leptospira and some other micro-bes, however. had no amplification at all.This study is first reported at home andabroad. The results demonstrate that PCRis a very sensitive and specific techniqueof DNA amplification, which can be usedas a powerful tool in the early diagnosisand epidemiological idenification ofleptospirosis.