双重实时荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌  

Identification of Candida krusei and Candida glabrata by duplex quantitative real-time PCR

作　　者：杨淳[1] 黄红川[1] 李冰[2] 刘启才[2] 彭燕[2] 袁锦屏[1] 杨灵[1] 苏丹红[1] 邱贵霞[1] 杨其霖[1] 黎毅敏[1] 

机构地区：[1]广州医学院第一附属医院广州呼吸疾病研究所,510120 [2]广州医学院实验医学研究中心

出　　处：《中华生物医学工程杂志》2007年第6期349-352,共4页Chinese Journal of Biomedical Engineering

基　　金：广州市科技局科研基金（2006J1-C0031）

摘　　要：目的建立一种快速、灵敏、特异的鉴定克柔念珠菌和光滑念珠菌的双重实时荧光定量PCR方法。方法以核糖体基因内转录间隔区Ⅱ（ITSⅡ）为靶目标，设计并合成分别针对克柔念珠菌、光滑念珠菌的种特异引物和探针。建立双重实时荧光定量PCR反应体系，并用该体系对呼吸道相关致病菌进行检测。鉴定结果与临床常规鉴定方法对照，评价其敏感度、特异度及重复性。结果通过对100例样品的检测，结果显示该双重实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照，特异度为100％，敏感度为100％；最小能检测到10个拷贝数的重组质粒；批内重复实验和批间重复实验结果均与常规鉴定方法结果相符。结论双重荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌，特异度和敏感度高，重复性好，且快速、简便，该方法将有助于念珠菌病的早期诊断和针对性治疗。Objective To set up a duplex quantitative real-time PCR （QPCR） assay with high sensitivity, specificity and rapidity to detect Candida krusei and Candida glabrata. Methods A pair of species-specific primers, combined with two probes which were selected from the internally transcribed spacers Ⅱ （ ITS Ⅱ）, were used to amplify the common pathogen associated with the respiratory system in order to evaluate its sensitivity, specificity and repeatability, Results The results obtained from the duplex QPCR assay and the general culture were the same by detecting the samples of 100 eases . Its specificity was 100% ,and its sensitivity was 100%. The lower limit of detection of this duplex QPCR assay was 10 eopys of recombinant plasmid DNA. The result within group and between groups were the same as the general culture. Conclusion The duplex quantitative real-time PCR assay can detect Candida krusei and Candida glabrata sensitively, specifically, rapidly, simply and stably, and is useful to early diagnosis and target treatment of the diseases caused by the Candida.

关 键 词：克柔念珠菌 念珠菌 光滑 聚含酶链反应 

分 类 号：R4[医药卫生—临床医学]