本文關(guān)鍵詞： 肺炎鏈球菌 psaA基因 ply基因 PCR　出處：《中國病原生物學(xué)雜志》2015年08期 　論文類型：期刊論文

【摘要】：目的采用PCR檢測肺炎鏈球菌(S.pn)毒力基因psaA和ply,研究分析兩個毒力基因的特異性,為S.pn臨床診斷提供依據(jù)。方法對廣東省12家醫(yī)療機構(gòu)診斷為S.pn感染患者的654份標(biāo)本,經(jīng)過細(xì)菌培養(yǎng)和乳膠凝集試驗篩選出575株,以psaA、ply基因核心區(qū)域序列設(shè)計合成擴增引物,以抽提的S.pn分離株DNA為模板,采用常規(guī)聚合酶鏈反應(yīng)(PCR)擴增目標(biāo)基因psaA、ply片段,長度分別為838bp和209bp,調(diào)查分析不同來源標(biāo)本的分離株psaA和ply基因檢出率;采用乳膠凝集試驗與多重PCR分型,比較兩種結(jié)果中S.pn菌株psaA和ply檢出率差異,以及廣東S.pn主要血清型與非主要血清型菌株的psaA和ply檢出率情況。結(jié)果經(jīng)乳膠凝集試驗鑒定為S.pn的560株菌株中,psaA和ply基因檢出率分別為78.21%和83.57%,ply基因檢出率高于psaA基因,psaA基因陽性的S.pn菌株,其ply基因均陽性;來源于血液和腦脊液標(biāo)本的S.pn分離株的psaA和ply基因檢出率分別為93.18%和95.45%,膿液標(biāo)本分離的S.pn菌株psaA和ply基因檢出率為100%。在莢膜腫脹反應(yīng)和多重PCR檢測為S.pn可分型血清型菌株中,前者psaA和ply基因陽性率分別為93.90%和98.57%,后者為96.34%和98.31%;而在多重PCR檢定為不可分型血清型菌株中,psaA和ply基因檢出率分別為47.06%和57.84%,顯著低于莢膜腫脹試驗的68.59%和74.64%,但ply基因檢出率也均比psaA高;可分型血清型中主要血清型菌株的psaA和ply基因檢出率高于非主要血清型。結(jié)論廣東地區(qū)S.pn臨床分離株中ply基因檢出率比psaA高,保守性高,但其他鏈球菌亦能擴增出ply基因從而出現(xiàn)假陽性,因此,對于S.pn菌株的鑒定,采用PCR同時擴增psaA和ply基因,并結(jié)合臨床常規(guī)實驗,鑒定更為可靠。此外,血液和腦脊液psaA和ply基因陽性率高可能與psaA和ply是S.pn入侵血和腦的關(guān)鍵因子有關(guān)。
[Abstract]:Objective to detect the virulence genes psaA and plyl of S. pneumoniae by PCR, and to analyze the specificity of the two virulence genes in order to provide the basis for the clinical diagnosis of S.pn. Methods 654 samples of patients with S.pn infection were collected from 12 medical institutions in Guangdong Province. 575 strains were screened by bacterial culture and latex agglutination test. Primers were designed and synthesized from the core region of psaAply gene, and the target gene psaAply fragment was amplified by routine polymerase chain reaction (PCR) with the extracted S.pn isolate DNA as template. The length was 838bp and 209bp, respectively. The detection rate of psaA and ply genes in different samples were investigated and analyzed, and the detection rates of S.pn strain psaA and ply were compared by latex agglutination test and multiple PCR typing. The detection rate of psaA and ply in the main serotype and non-serotype strains of S.pn in Guangdong Province were 78.21% and 83.57ply, respectively, which were higher than that of psaA in the 560strains identified as S.pn by latex agglutination test. S.pn, which is positive for psaA gene, All of them were positive for ply gene. The detection rates of psaA and ply genes of S.pn isolates from blood and cerebrospinal fluid samples were 93.18% and 95.45, respectively. The detection rates of psaA and ply genes of S.pn strains isolated from pus samples were 100. The positive rates of psaA and ply genes were 93.90% and 98.57 in the former, 96.34% and 98.31 in the latter, and 47.06% and 57.84, respectively, in the multiplex PCR untyped serotype strains, which were significantly lower than 68.59% and 74.64 in the capsule swelling test, but ply. The detection rate of gene was higher than that of psaA. The detection rate of psaA and ply genes in the main serotype strains was higher than that in non-major serotypes. Conclusion the detection rate of ply gene in clinical isolates of S.pn in Guangdong is higher than that in psaA, and the detection rate of ply gene is higher than that of psaA. However, other streptococcus could amplify the ply gene, so it is more reliable to use PCR to amplify both psaA and ply genes of S.pn strain, and to combine with clinical routine experiments to identify the S.pn strain. In addition, the identification of S.pn is more reliable. The high positive rate of psaA and ply gene in blood and cerebrospinal fluid may be related to psaA and ply which are the key factors of S.pn invading blood and brain.
【作者單位】： 華南農(nóng)業(yè)大學(xué)制藥工程系;廣東省疾病預(yù)防控制中心;廣東省應(yīng)急病原學(xué)重點實驗室;廣東省生物制品和藥物研究所;
【基金】：中美新發(fā)和再發(fā)傳染病合作項目 廣州市科技計劃重點項目(No.11C32100704)
【分類號】：R378.12
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本文編號：1508867