嗜肺军团菌毒力岛基因聚合酶链反应分型鉴定

Identification of pathogenicity island genotyping for Legionella pneumophila by polymerase chain reaction

目的建立嗜肺军团菌毒力岛基因分型鉴定方法,探讨其致病特性和分布状况。方法根据GenBank公布的嗜肺军团菌核苷酸序列设计和合成嗜肺军团菌种和毒力岛基因鉴定引物,采用PCR法对25株军团菌属标准参考株,3株国内和20株广东分离嗜肺军团菌,53份临床标本和57份水样、进行嗜肺军团菌种和毒力岛基因分型鉴定。结果5株嗜肺军团菌标准株只有嗜肺军团菌1型(Philadelphia-1 ATCC 33152)12个毒力岛基因全阳性,其他4株只检出11个毒力岛基因。20株军团菌属中其他菌株,分别检出2-11个毒力岛基因;3株国内嗜肺军团菌检出11个毒力岛基因;广东地区分离的20株嗜肺军团菌,7株检出12个毒力岛基因,12株检出11个毒力岛基因,1株未检到毒力岛基因。53例病源不明性肺炎患者支气管洗液和痰液,PCR检测嗜肺军团菌DNA阳性5例(9．4％)。结论广东地区嗜肺军团菌广泛存在水源环境中,而且是医院感染性肺炎的重要病源之一。不同血清型嗜肺军团菌所含毒力岛基因不同,毒力岛基因与致病性相关。

Objective To establish a genotype method for newly discovered L. pneumophila pathogenicity island（PAI） and to study the characteristics of its pathogenicity and distribution. Methods Primer design for PCR amplification was based on the oligonucleotide sequence published by C, eneBank Specieses identification and genotyping for pathogenicicty island were conducted by PCR for twenty five reference stains , three strains isolated from China, 20 strains of L. pneumophila isolated from Guangdong region, and 53 clinical specimens, and 37 samples of cooling tower water. Results Twelve putative pathogenicity island genes （raA, iraB, mimp, irvA, ivhB, ivhD, cpxR, cpxA, dotA, rpoB, icmC, icmD） were detected from only Philadelphia-1 strains of serogroup 1 L. pneumophila （ ATCC-33152 ） out of 5 reference strains , 11 PAI genes were detected from 4 other reference strains of L. pneumophila Knozvi （lle- 1, Togus-1, Knox, LDB7 ） . Among 20 strains of reference legionella spp. 2 tol 1 PAI genes could be detected. 11 PAI genes were detected from three strains of L. pneumophila isolated from China. 12, 11 and 0 PAI genes were detected from 8, 11 and 1 strains of L. pneumophila respectively from 20 Guangdong region isolates Five （9. 4% ） of 53 clinical specimens of nontyping pneumoph ila patients were LP-DNA positive. Twenty five （67.6%） samples of 37 cooling tower water were LP-DNA positive, while 7 samples （18. 9% ） were culture positive. Sensitivity of the PCR assay is higher than that of the culture , DNA contained in 0. 6- 6 organisms may be detected by PCR. Conclusions L. pneumophila exists broadly in water environment in Guangdong, It＇s an important pathogen causing nosocomially acquired pneumonia. The PAI genes of L. pneumophila were different in various serogroups , and the PAI genes were related to pathogenicity.