摘要
目的采用实时荧光定量PCR技术,建立并评价一种快速、准确、特异检测类鼻疽伯克霍尔德菌的定量检测方法。方法以类鼻疽伯克霍尔德菌Ⅲ型分泌系统保守基因序列设计引物和探针,构建重组质粒作为DNA标准品,优化荧光定量PCR体系,同时进行方法学评价,并与以16S rRNA基因为靶序列的荧光定量PCR方法进行对比。结果建立了检测类鼻疽伯克霍尔德菌的荧光定量PCR方法,其线性范围为1×101~1×1010拷贝/μL;其灵敏度为10拷贝/μL;批内变异系数（CV）为1.43%~4.6%;对用24份模拟血液标本和67份稻田土壤标本分别采用本法和对照方法进行检测,本菌配制的模拟标本采用两种方法检测均为阳性,且能准确定量,阳性率为100%,而含有其他常见感染细菌的标本检测结果均为阴性;土壤标本中有12份标本采用本法和与细菌培养法均为阳性,但是对照方法未能检测出其中两份低浓度的土壤标本。结论建立的类鼻疽伯克霍尔德菌定量PCR方法具有快速、准确、灵敏度高等特点,可作为环境中该菌检测和疫区类鼻疽病诊断的有效工具。
Objective To establish and evaluate a real-time PCR for fast,accurate and quantitative detection of Burkholderia pseudomallei（ B. pseudomallei）. Methods Based on B. pseudomallei type Ⅲsecretion system conservative sequence,primers and probe were designed,and then recombinant plasma was constructed as DNA standard. The real-time PCR system was optimized,and compared with other real-time PCR with 16 S rRNA as target gene. Results The inventive real-time PCR showed wide range of linearity（ 1 × 101- 1 × 1010 copies / μL）,high sensitivity（ 10 copies / μL）,and intra-experimental coefficients of variation（ CV） of 1. 43%- 4. 6%. When it was applied directly to identify 24 mocked double-blind blood samples,16 samples were positive to B. pseudomallei,and 8 samples were negative to other pathogens. Then,the inventive real-time PCR was further applied to identify 67 soil samples that underwent parallel bacteriological culture,and 12 soil samples showed B. pseudomallei positive results. There were 2 soil samples with positive results based on direct culture and the inventive real-time PCR,but other real-time PCR detection results were negative. Conclusion The inventive real-time RCR is rapid, accurate and sensitive for quantitative detection of B. pseudomallei,and can be used for diagnosis of melioidosis in epidemic area.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2015年第17期1734-1738,共5页
Journal of Third Military Medical University
基金
国家自然科学基金地区基金项目(81360240)
海南省社会发展科技专项(SF201415)
海南省自然科学基金(813187)~~
