用PCR SSCP方法检测念珠菌和隐球菌的实验研究

<Abstract>ng of Cryptococcus and Candida with PCRSSCP

目的 根据医学真菌大亚单位核糖体（ｌｓｕ ｒ Ｒ Ｎ Ａ） 基因的高度保守区设计广谱扩增引物，建立应用 Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ 技术检测医学重要真菌的方法，并应用于临床。方法 首先用 Ｐ Ｃ Ｒ 技术扩增白色念珠菌、热带念珠菌、假热带念珠菌、近平滑念珠菌、光滑念珠菌、解脂念珠菌、新型隐球菌的ｌｓｕｒ Ｒ Ｎ Ａ 基因，然后根据７ 种标准菌株 Ｄ Ｎ Ａ 的单链构象多态性特征将上述医学真菌鉴定到菌种；观察引物对常见细菌和哺乳动物细胞 Ｄ Ｎ Ａ 的扩增结果； 于生理盐水和血液中加入念珠菌，观察方法的灵敏度；对扩增产物进行序列分析；方法建立后用于临床标本的检测。结果 不同真菌菌种的 Ｄ Ｎ Ａ 具有可以区分的单链构象多态性，扩增片段长度为２１９ ～２８５ｂｐ ，可将白色念珠菌、热带念珠菌和新型隐球菌等医学重要真菌鉴定到菌种；此引物对大肠埃希氏菌、铜绿假单胞菌和金黄色葡萄球菌等１２ 种常见细菌和病毒 Ｄ Ｎ Ａ 扩增均为阴性结果， 人血液白细胞、 Ｈｅ Ｌａ 细胞、 Ｋ Ｂ 细胞、 Ｓ１８０ 细胞和３ Ｔ３ 细胞亦无阳性 Ｄ Ｎ Ａ 带出现。于生理盐水和血液中加入白色念珠菌，检测灵敏度分别为５ 个和１０ 个真菌细胞。 Ｐ Ｃ Ｒ 产物的序列分析结果表明。

Objective According to the conservative region of r R N Agene of fungi with medicalimportance,a pair of universal primers were designed to amplify the 7 species of the medical fungi. Amethod of differentiating the fungi was developed using P C R S S C Ptechnique. Methods The D N Aof 7species of the fungi( Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parap silosis , Candida glabrata , Candidalipolytica , Cryptococcus neoformans) were amplified with the univer sal primers mentioned above . The P C Rproducts were denatured and electrophoresised . Compared with thepatterns of standard strains, the electrophoresis patterns of specimens were classified . The D N A of 12species of microorganisms and 5 celllines were tested to study the specificity ofthetechnique. The sensitivi ty ofthis method was tested by adding Candida albicansinto normal saline and human blood . The P C Rproducts were sequenced to verify the reliability ofthe technique. The methods was used to differentiate theclinicalfungus specimens. Results The fungi had distinguishable single strand conformational polymor phism which could be used to differentiate the 25 S r R N Agene offungi. Thusthe fungicould be identifiedat thespecieslevel.positiveresults were obtained using the method was 5 fungus cellsin the N Sand was 10cellsin the blood . The results ofsequence analysis of P C Rproducts were coincident with the sequences pro vided in Gen Bank . The study of clinical application suggested that the sensitivity of the P C R S S C Ptech niques was higher than that of routine culture method . Conclusions The P C R S S C P method is a rapid ,accurate,reliable and a simple method ,and has a good prospect ofclinical application . Subject words Candida ; Cryptococcus r R N A Polymerade chain reactionTesting of Cryptococcus and Candida with P C R S S C P C H E N Jiankui, M U Zhaoqin , Y I N Xiuyun , etal. Research Clinic , Academy of Military Medical Sciences, Beijing 100039 Abstract Objective According to the conservative region of r R N Agene of fungi with medicalimportance,a pair of universal primers were designed to amplify the 7 species of the medical fungi. Amethod of differentiating the fungi was developed using P C R S S C Ptechnique. Methods The D N Aof 7species of the fungi( Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parap silosis , Candida glabrata , Candidalipolytica , Cryptococcus neoformans) were amplified with the univer sal primers mentioned above . The P C Rproducts were denatured and electrophoresised . Compared with thepatterns of standard strains, the electrophoresis patterns of specimens were classified . The D N A of 12species of microorganisms and 5 celllines were tested to study the specificity ofthetechnique. The sensitivi ty ofthis method was tested by adding Candida albicansinto normal saline and human blood . The P C Rproducts were sequenced to verify the reliability ofthe technique. The methods was used to differentiate theclinicalfungus specimens. Results The fungi had distinguishable single strand conformational polymor phism which could be used to differentiate the 25 S r R N Agene offungi. Thusthe fungicould be identifiedat thespecieslevel.positiveresults were obtained using the method was 5 fungus cellsin the N Sand was 10cellsin the blood . The results ofsequence analysis of P C Rproducts were coincident with the sequences pro vided in Gen Bank . The study of clinical application suggested that the sensitivity of the P C R S S C Ptech niques was higher than that of routine culture method . Conclusions The P C R S S C P method is a rapid ,accurate,reliable and a simple method ,and has a good prospect ofclinical application .