大鼠卡氏肺孢子虫ITS1-5.8S rDNA-ITS2的巢式PCR检测及基因序列的测定

Nested PCR and sequencing of ITS1-5.8S rDNA-ITS2 gene of Pneumocystis carinii isolated from SD rats

目的探讨ITS1-5.8Sr DNA-ITS2巢式PCR检测卡氏肺孢子虫的应用价值并测定其基因序列。方法采用地塞米松免疫抑制法诱导SD大鼠感染肺孢子虫;制作肺印片、肺组织匀浆液及支气管肺泡灌洗液(BAL)涂片进行六亚甲基四胺银(GMS)染色镜检;提取肺孢子虫DNA进行巢式PCR扩增,测序后与GenBank的不同宿主肺孢子虫相关序列进行比较分析。结果用ITS1-5.8Sr DNA-ITS2巢式PCR法均能检测出实验感染大鼠BAL和肺组织卡氏肺孢子虫DNA,阳性率为100%(10/10),比较BAL标本、肺印片和肺组织匀浆液GMS染色法的检出效率,BAL最低,肺组织匀浆液最高。其中20%(2/10)大鼠的BAL标本用GMS染色法未能检测出,但用巢式PCR方法均能成功扩增;所测SD大鼠卡氏肺孢子虫ITS1-5.8S rDNA-ITS2基因序列长度为516bp,ITS1、5.8S rDNA、ITS2基因片段分别为198、158、160bp,与GenBank的大鼠(L27658)、小鼠(AY532651)和长爪沙鼠(AY875972)的ITS1-5.8S rDNA-ITS2基因的同源性分别为96%(452/467)、88%(275/310)、95%(152/159),其变异位点多在ITS1和ITS2基因片段。结论ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫敏感性高、特异性强,可作为早期诊断肺孢子虫肺炎方法,特别适用无创性标本的检测;成功获取SD大鼠卡氏肺孢子虫ITS1-5.8S rDNA-ITS2的基因序列,与不同宿主比较其5.8S rDNA较保守,ITS1和ITS2基因变异较大,适合基因分型和系统进化研究。

Objective To discuss the value of ITS1 - 5.8S rDNA - ITS2 nested PCR to detect Pneumocystis carinii（P.carinii） and get the ITS1 - 5.8S rDNA - ITS2 sequence of P.carinii from SD rots.Methods Rats were rendered immunodepressant by subcutaneous injection of dexamethasone, Pneumocystis carinii were observed by the microscopical examinations of bronchoalveolar lavage（BAL）, lung impression smears and lung tissue smears stained with Gomori＇ s methenamine silver stained （GMS）, respectively. The DNAs of P. carinii were extracted from the BAL and lung tissues of the infected rats. ITS1 - 5.8S rDNA - ITS2 of P. carinii were amplified by nested PCR. The PCR products were purified and sequenced. The sequences were analyzed, aligned and compared with the sequences of Pneumocystis wakefieldiae （L27658）, Pneumocystis murina （AY532651） and Pneumocystis gerbil（AY875972） in GenBank respectively. Results The DNAs of P.carinii both from BAL and lung tissue in SD rats were detected by nested PCR with a positive rate of 100% . Comparing the detection efficiency of P. carinii among BAL, lung impression smears and lung tissue smears by GMS method, we found that the detection efficiency of BAL was the lowest, lung tissue smears was the highest. 20% of specimens of BAL couldn＇t be detected by GMS method, while these specimens could be detected by nested PCR . The lengths of ITS1, 5.8S rDNA and ITS2 sequences of P. carinii from SD rats were 198, 158 and 160 bp, with total 516 bp. The homologies of ITS 1 - 5.8S rDNA - ITS2 gene of P. carinii among SD rats, Wakefieldiae, Murina and Gerbil were 96% （452/467）, 88% （275/310）and 95% （152/ 159） respectively, those variation spots mostly existed in ITS genes. Conclusions ITS1 - 5.8S rDNA - ITS2 nested PCR is a high sensitive and specific method, which can detect PCP in the early stage, especially suitable for the non - traumatic specimens. We have got the ITS1 - 5.8S rDNA - ITS2 sequence of P. carinii from SD rats successfully, and found that the 5.8S rDNA gene is conservative, both ITS 1 and ITS2 gene are variant, indicating that ITS gene is suitable for the gene variation and phylogenetic evolution study.