嗜肺军团菌聚合酶链反应检测方法及其应用研究

根据嗜肺军团菌基因组ＤＮＡ的种特异性ＤＮＡ片段序列，合成一对引物进行聚合酶链反应（ＰＣＲ）。经琼脂糖凝胶电泳、ＥＢ染色结果表明，一条８７０ｂｐ的核苷酸区带为嗜肺军团菌１～１４血清型菌株所共有。ＰＣＲ检测水中军团菌，其敏感性为３５０ｃｆｕ／ｍｌ，而用同位素标记探针、斑点杂交法检测，其敏感性为４３ｃｆｕ／ｍｌ。ＰＣＲ检测人工感染嗜肺军团菌的豚鼠组织标本，阳性率为８３．３％，而细菌分离培养的阳性率仅为２６．６％。在现场调查中，用ＰＣＲ法初步验证了一起由Ｌｐ１０引起的军团菌病爆发。上述结果表明，ＰＣＲ法能快速、敏感、特异性检测嗜肺军团菌感染。

Studies on the Detection of Legionella pneumophila by the Application of Polymerase Chain Reaction (PCR)

According to the sequence of genomic DNA fragment of Legionella pneumophila, the PCR was performed with a pair of artificial synthesized primer.Results of agarose electrophoresis and EB staining showed that there was a 870 bp band shared by serogroups 1~14 of L.pneumophila. The sensitivity of PCR in detecting Legionella from water was 350cfu/ml, however, the specific DNA probe labeled with 32p was 43cfu/ml by blot hybridization. The positive rate of tissue specimens from infected guinea-pigs with Legionella pneumophila was 83.3% by PCR detection, and only 26.6% by bacteriological culture method. An outbreak caused by Lp10 was verified by PCR. The result showed that the PCR could detect the infection of Legionella rapidly, specifically and sensitively.