流感嗜血杆菌耐药菌株基因分型的初步研究

Use of a randomly amplified polymorphic DNA technique to genotype resistant strains of Haemophilus influenza

目的建立随机扩增DNA多态性(RAPD)反应体系,对海口市流感嗜血杆菌(Hi)耐药菌株进行基因分型。方法对临床分离的62株Hi进行药物敏感试验。同时采用RAPD技术对耐药菌株进行基因分型,并对分型结果进行聚类分析。结果 62株Hi中有23株耐药,其中7株(第10～16菌株)对氨苄西林、氯霉素、四环素、复方新诺明耐药,2株(第17～18菌株)对四环素、复方新诺明耐药,1株(第19菌株)对复方新诺明、阿奇霉素耐药,9株(第1～9菌株)对氨苄西林耐药,4株(第20～23株)只对阿奇霉素耐药。采用引物(5′-ACGTATCTGC-3′)构建的RAPD-PCR反应体系检测Hi基因,扩增出15条带,大小在300～2 000bp之间。所构建的亲缘关系聚类图中,第1～9菌株聚成一簇,第10～16菌株聚成一簇,第17～18菌株聚成一簇,第19菌株为一簇,第20～23菌株聚成一簇。结论采用RAPD-PCR反应体系可对Hi耐药菌株进行基因分型。

Objective To analyze the genotype of resistant strains of Haemophilus influenza in Haikou using randomly amplified polymorphic DNA（RAPD）.Methods Drug sensitivity tests were conducted for 62 H.influenza strains isolated from clinical samples.RAPD was used to genotype 62 resistant strains,and cluster analysis was performed on RAPD results.Results Among the 62 H.influenza strains,23 strains were drug-resistant.Seven strains（No.10-16） were resistant to ampicillin,chloromycetin,tetracycline,and a sulfamethoxazole compound,2 strains（No.17-18） were resistant to tetracycline and a sulfamethoxazole compound,1 strain（No.19） was resistant to a sulfamethoxazole compound and azithromycin,9 strains（No.1-9） were resistant to ampicillin,and 4 strains（No.20-23） were resistant to azithromycin.Quality maps were obtained with RAPD-PCR using a random primer（5′-ACGTATCTGC-3′）.The relationship dendrogram mapped with the random primer showed that the 23 drug-resistant strains were divided into 4 clusters,including a cluster with strain Nos.1-9,a cluster with Nos.10-18,a cluster with No.19,and a cluster with Nos.20-23.Conclusion RAPD was used to genotype H.influenza strains to determine their genetic homology.