用Nested聚合酶链反应检测嗜肺军团菌的实验研究

THE EXPERIMENTAL STUDY IN DETECTION OF LEGIONELLA PNEUMOPHILA USING NESTED POLYMERASE CHAIN REACTION

用Nested聚合酶链反应(Nested PCR)法检测嗜肺军团菌种。Nested PCR法所用的两对寡核苷酸引物按编码24 kDa嗜肺军团菌表面抗原的基团片段序列设计的。用此两对引物对实验的军团菌种和非军团菌种细菌DNA进行扩增,只有嗜肺军团菌种DNA被扩增,而其他军团菌种和非军团菌属细菌没有被扩增。第1步和第2步PCR分别检测到最小量为10pg和10 fg的靶DNA。对不同量的嗜肺军团菌配制成的痰标本进行检测,能检测到0.1cfu/ml的嗜肺军团菌,只需12h。这些结果显示Nested PCR法检测嗜肺军团菌特异性强,敏感性高。此方法为从临床标本和环境材料中早期、快速检测到嗜肺军团菌提供了有效的工具。

The nested polymerase chain reaction (Nested PCR) was used to detect legionella pneumophila. The oligonucleotide primers derived from the sequence of a gene that codes for the 24-kilodalton surface antigen of legionella] pneumophila amplified DNA from all legionella species and non-legionella species tested. Only the DNA from legionella pneumophila was. amplified. The first and second step PCR achived the sensitivity as small as 10 Pg and 10 fg of the target DNA, respectively. In the detection from legionella pneumophila seeded sputa, 0.1 cfu/ml of the bacteria could be detected and it took about 12 hours. The results showed Nested PCR was sensitive and specific in detecting legionella pneumophila. The method may become a useful tool in the early and rapid detection of legionella pneumophila in clinical specimens and enviromental materials.