广东省575株肺炎链球菌的毒力因子psaA、ply基因检测分析  

Detection and analysis of the virulence factors psaA and ply in 575 strains of Streptococcus pneumoniae from Guangdong Province

作　　者：熊平[1] 李柏生[2,3] 江锦志 彭托华 杨彤 刘美真[2,3] 柯碧霞[2,3] 刘闪闪[1] 柯昌文[2,3] 

机构地区：[1]华南农业大学制药工程系,广东广州510640 [2]广东省疾病预防控制中心 [3]广东省应急病原学重点实验室 [4]广东省生物制品和药物研究所

出　　处：《中国病原生物学杂志》2015年第8期693-698,共6页Journal of Pathogen Biology

基　　金：中美新发和再发传染病合作项目;广州市科技计划重点项目(No.11C32100704)

摘　　要：目的采用PCR检测肺炎链球菌(S.pn)毒力基因psaA和ply,研究分析两个毒力基因的特异性,为S.pn临床诊断提供依据。方法对广东省12家医疗机构诊断为S.pn感染患者的654份标本,经过细菌培养和乳胶凝集试验筛选出575株,以psaA、ply基因核心区域序列设计合成扩增引物,以抽提的S.pn分离株DNA为模板,采用常规聚合酶链反应(PCR)扩增目标基因psaA、ply片段,长度分别为838bp和209bp,调查分析不同来源标本的分离株psaA和ply基因检出率;采用乳胶凝集试验与多重PCR分型,比较两种结果中S.pn菌株psaA和ply检出率差异,以及广东S.pn主要血清型与非主要血清型菌株的psaA和ply检出率情况。结果经乳胶凝集试验鉴定为S.pn的560株菌株中,psaA和ply基因检出率分别为78.21%和83.57%,ply基因检出率高于psaA基因,psaA基因阳性的S.pn菌株,其ply基因均阳性;来源于血液和脑脊液标本的S.pn分离株的psaA和ply基因检出率分别为93.18%和95.45%,脓液标本分离的S.pn菌株psaA和ply基因检出率为100%。在荚膜肿胀反应和多重PCR检测为S.pn可分型血清型菌株中,前者psaA和ply基因阳性率分别为93.90%和98.57%,后者为96.34%和98.31%;而在多重PCR检定为不可分型血清型菌株中,psaA和ply基因检出率分别为47.06%和57.84%,显著低于荚膜肿胀试验的68.59%和74.64%,但ply基因检出率也均比psaA高;可分型血清型中主要血清型菌株的psaA和ply基因检出率高于非主要血清型。结论广东地区S.pn临床分离株中ply基因检出率比psaA高,保守性高,但其他链球菌亦能扩增出ply基因从而出现假阳性,因此,对于S.pn菌株的鉴定,采用PCR同时扩增psaA和ply基因,并结合临床常规实验,鉴定更为可靠。此外,血液和脑脊液psaA和ply基因阳性率高可能与psaA和ply是S.pn入侵血和脑的关键因子有关。Invasive infections caused by Streptococcus pneumoniae continue to be a major cause of morbidity and mortality worldwide,especially in children under 5years of age.Pneumococcal surface adhesin A（PsaA）and pneumococcal pneumolysin（Ply）are two important virulence factors of S.pneumoniae. Objective To investigate and analyze the distribution of the virulence factors PsaAand Plyin order to help identify and diagnose S.pneumoniaeinfection. Methods The virulence factors PsaAand Plyof S.pneumoniae were assayed using isolates collected from patients with infections diagnosed as Pneumococcal pneumoniain twelve hospitals of Guangdong.Six hundred and fifty-four specimens of S.pneumoniae obtained between 2005 and 2013were tested.Species identification was confirmed with a latex agglutination assay before PCR analysis.Two pairs of PCR primers were designed and synthesized in accordance with the main regions of the PsaAand Plygene.The DNA of S.pneumonia was extracted from the isolates using QIAamp DNA Mini KitDNA according to the manufacturer＇s instructions.Fragments of the psaAand plytarget genes were respectively amplified with the extracted DNA as a template,and the PCR products were used for further analysis.The serotypes of clinical isolateswere detected with a multiplex PCR assay and Quellung reaction.On the basis of the above findings,the rate at which the psaAand plygenes were detected in isolates from different sources was determined.S.pneumoniaeisolates were classified as typeable or untypeable and more prevalent and less prevalent serotypes were identified. Results Five hundred and seventy-five samples were screened for S.pneumoniae using the usual bacteriological techniques（latex agglutination assay and culture）.The latex agglutination assay identified 560 isolates as strains of S.pneumoniae,including 330 from sputum,17 from cerebrospinal fluid,27 from blood,13 from pus,19 from throat swabs,14 from prostatic fluid,and 127 from other sites.PCR revealed that 78.21% of the isolates had the PsaAgene while

关 键 词：肺炎链球菌 psaA基因 ply基因 PCR 

分 类 号：R378.12[医药卫生—病原生物学]