AIDS患者合并感染耶氏肺孢子虫的巢式PCR检测及ITS基因的克隆测序  

Nested PCR and clone sequencing of ITS gene of Pneumocystis jirovecii isolated from AIDS patients

作　　者：林睿[1] 黎学铭[1] 张陆娟[1] 秦英梅[2] 张鸿满[1] 欧阳颐[1] 黄绍标[2] 江河[1] 

机构地区：[1]广西壮族自治区疾病预防控制中心,南宁530028 [2]广西南宁市第四人民医院,南宁530023

出　　处：《应用预防医学》2009年第4期198-202,共5页Applied Preventive Medicine

基　　金：广西科学研究与技术开发计划课题(桂科攻0632007-3A);广西留学回国人员科技活动择优资助项目(桂人函[2008]987)

摘　　要：目的探讨ITS巢式PCR检测AIDS患者合并耶氏肺孢子虫感染的应用价值并对ITS1-5.8SrDNA-ITS2基因进行克隆测序。方法收集AIDS患者痰液标本,用二硫苏糖醇(DTT)消化处理后进行六亚甲基四胺银(GMS)染色镜检;提取肺孢子虫DNA后进行巢式PCR扩增,选取GMS染色和PCR同时阳性的112号和仅PCR阳性的185、200号病例的PCR产物进行TA克隆、测序,然后用Blastn程序和DNAMAN软件对所测序列进行同源性比较和序列间比对,并和GenBank登录的耶氏肺孢子虫ITS1-5.8SrDNA-ITS2序列进行比较分析。结果用ITS巢式PCR法检测AIDS患者99例,耶氏肺孢子虫DNA阳性43例,阳性率为43.4%(43/99),用GMS染色法检测,阳性率为4.04%(4/99),两者比较,有非常显著性差异(P<0.01)。TA克隆112、185、200号病例的耶氏肺孢子虫ITS1-5.8SrDNA-ITS2基因序列长度分别为523bp、515bp、511bp,三者之间的基因同源性为95%～97%,与GenBank登录的耶氏肺孢子虫(U07220)、(U07221)、(U07222)和(U07226)的ITS1-5.8SrDNA-ITS2基因的同源性为95%～98%,其变异位点多在ITS1和ITS2基因片段。结论ITS巢式PCR法检测耶氏肺孢子虫敏感性明显高于GMS染色法。ITS巢式PCR法可作为肺孢子虫肺炎早期诊断方法,尤其适用无创性标本的检测,GMS染色法对无创性标本痰液的检测敏感性低,在临床上意义不大;成功获取广西株耶氏肺孢子虫ITS1-5.8SrDNA-ITS2的基因序列,与GenBank登录的外国株耶氏肺孢子虫基因序列高度同源,其中5.8SrDNA高度保守,ITS1和ITS2基因变异较大。Objective To discuss the value of ITS nested PCR to detect Pneumocystis jirovecii （P.jirovecii） and clone the ITS1 - 5.8S rDNA - ITS2 sequence of P.jimvecii from AIDS patients.Me,otis Sputum specimens were collected from AIDS cases and digested with DIT. P. jirovecii was observed by the microscopical examinations of sputum smears stained with Gomori＇s methenamine silver stained （GMS）. The DNAs of P. jirovecii were extracted from sputum specimens of the AIDS cases. ITS 1 - 5.8S rDNA - ITS2 of P. jirovecii were amplified by nested PCR. The PCR products of No. 112, 185 and 200 cases were TA cloned and se quenced. No. 112 case was both positive by PCR and GMS, while No. 185 and 200 cases were only positive by PCR. The sequences were analyzed, aligned and compared with the sequences of P.jirovecii from GenBank （U07220）, （U07221）, （U07222） and （U07226） with Blastn program and DNAMAN software, respectively. Results 43 out of 99 AIDS cases were detected the DNAs of P.jirovecii by Nested PCR with a positive rate of 43.4%, while the positive rate of 4.04% （4/99） was detected by GMS method. Comparing the two methods, the sensi- tivity of nested PCR was far higher than that of GMS（ P 〈 0.01） . The lengths of ITS1 - 5.8S rDNA - ITS2 se- quences of P. jirovecii from No. 112, 185, and 200 cases were 523, 515 and 511bp, respectively. The homologies of rlS1 - 5.8S rDNA - ITS2 gene of P. jirovecii among the three cases were 95% - 97%, those homologies among the cases from Guangxi and GenBank were 95% - 98%, and variation spots mostly existed in ITS genes. Conclusions The sensitivity of ITS nested PCR was far higher than GMS method in detecting P. jirovecii, which indicated ITS nested PCR was good for PCP detection in the early stage, especially suitable for the non - traumatic specimens, GMS method made little sense to detect P. jirovecii through sputum specimens in clinic for low sensitivity. We got the ITS1 - 5.8S rDNA - ITS2 sequence of P. jirovecii from AIDS cases of Guangxi successfully, a

关 键 词：AIDS 耶氏肺孢子虫 内转录间隔区(ITS) 巢式PCR 

分 类 号：R512.91[医药卫生—内科学]