中华皮肤科杂志

• 论著 • 上一篇    下一篇

PCR反向线点杂交鉴定常见念珠菌的研究

向华国1, 熊礼宽2, 周华2, 苏放明3, 涂植光4   

  1. 1. 深圳市宝安区福永人民医院 518103;
    2. 深圳市慢性病防治院性病防治中心;
    3. 深圳市人民医院妇科;
    4. 重庆医科大学医学检验系 400016
  • 收稿日期:2006-09-23 出版日期:2007-08-15 发布日期:2007-08-15
  • 通讯作者: 熊礼宽,email:xionglk@yahoo.com.cn E-mail:xionglk@yahoo.com.cn
  • 基金资助:
    深圳市科研基金(2005116)

PCR-reverse line blot hybridization assay for the detection of common species of Canclida

XI-ANG Hua-guo1, XIONG Li-kuan2, ZHOU Hua2, SU Fang-ming3, TU Zhi-guang4   

  1. Department of Laboratory Medicine, Chongqing University of Medical Sciences, Chongqing 400016, China
  • Received:2006-09-23 Online:2007-08-15 Published:2007-08-15

摘要: 目的 建立PCR反向线点杂交技术(PCR-RLB)快速检测和鉴定常见念珠菌的方法。方法 以念珠菌属间隔序列Ⅱ(ITS2)为靶基因设计通用引物,用生物素标记反义引物,PCR扩增白念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念珠菌DNA,然后与通过氨基标记固定在尼龙膜上的各特异性寡核苷酸探针杂交,并进行临床标本和分离株的检测。结果 念珠菌标准菌株可扩增出302~441bpDNA片段,6种特异性寡核苷酸探针可分别与相应念珠菌PCR产物杂交,其敏感性为10cfu/mL。通过对100例分离株的检测,然后与培养法鉴定(Merieux Vitek)的结果比较,有97株与培养结果一致,另外3株通过DNA测序结果证实与PCR-RLB测定结果一致。通过对200例女性阴道拭子进行检测,PCR-RLB阳性率为49%,而涂片法和培养法阳性率分别为27%和39%,明显高于涂片法和培养法(P<0.05)。结论 该方法可快速、敏感、准确鉴定临床上常见的念珠菌。

关键词: 核酸杂交, 念珠菌属, 寡核苷核探针, 聚合酶链反应

Abstract: Objective To develop and evaluate a PCR-reverse line blot hybridization (RLB) assay for rapid detection and identification of common species of Candida.Methods The common primers,labeled with biotin,were designed targeting the second internal transcribed spacer (ITS2) region between 28S rRNA and 5.8S rRNA of Candida,and used to amplify the DNA of 6 species of Candida (C.alblcans,C. troptcalis,C.krusei,C.glabrata,C.parapsilosis,and C.dubliniensis).The PCR products were identified by hybridization with 6 specific oligonucleotide probes labeled with amidogen and fixed on a nylon membrane. The method was also applied to detect 200 clinical specimens and 100 Candida isolates.Results DNA frag-ments of 302~441 bp were amplified from all the standard strains of 6 species of Candida.The products could specifically hybridize with their corresponding probes.The sensitivity of PCR-RLB was 10 cfu/mL for Candida.The identification results were consistent between this method and culture in 97 isolates.DNA sequencing confirmed the identification results from PCR-RLB of the remaining 3 isolates.Of the 200 clinical specimens,49% were positive for Candida by PCR-RLB,27% by microscopic examination,39% by culture; the positivity rate by PCR-RLB was significantly higher than that by the latter two methods (both P<0.05).Conclusion PCR-RLB is a rapid,specific and sensitive method for the identification of common species of Candida.

Key words: Nucleic acid hybridization, Candida, Oligonucleotide probes, Polymerase chain reaction