二聚体蝎型探针荧光定量PCR快速检测临床标本结核分枝杆菌DNA  

Rapid detection of Mycobacterium tuberculosis DNA by fluorescent quantitative PCR assay using duplex scorpion primers

作　　者：王虹[1] 钟敏[2] 舒朝忠[1] 陈淑惠[1] 尹一兵[1] 

机构地区：[1]重庆医科大学医学检验系重庆医科大学临床检验诊断学省部共建教育部重点实验室重庆市重点实验室,重庆400016 [2]重庆市肺科医院重庆市结核菌基因诊断中心实验室,400020

出　　处：《中国人兽共患病学报》2007年第7期661-663,666,共4页Chinese Journal of Zoonoses

摘　　要：目的比较二聚体蝎型探针荧光定量PCR与抗酸染色、L-J培养法、结核分枝杆菌直接试验(MTD)的检出率、检测时间等,探讨二聚体蝎型探针荧光定量PCR检测临床标本中结核分枝杆菌DNA(TB-DNA)的实际应用价值。方法以建立的结核二聚体蝎型探针荧光定量PCR方法为基础,对43例结核确诊患者的标本及11例非肺结核患者的痰标本进行检测,并与抗酸染色、L-J培养法、结核分枝杆菌直接试验(MTD)进行比较。结果二聚体蝎型探针荧光定量PCR能快速准确的检测临床标本中的TB-DNA。其标准品浓度在102～106copies/μl时,方法能够保持良好的线性关系(相关系数为0.9994),阳性检出率(100%)显著高于抗酸染色法(16.28%)和培养法(37.21%)的检出率,与MTD试验的检出率(100%)一致。MTD试验检测时间约4～5h,而二聚体蝎型探针FQ-PCR检测约需80min即可得到检测结果,检测费用仅为MTD的1/4。结论二聚体蝎型探针荧光定量PCR用于临床标本结核分枝杆菌DNA的检测具有快速价廉、敏感特异的特点,该方法作为定量检测结核分枝杆菌的分子诊断新途径值得临床推广应用。The practical value of the fluorescent quantitative PCR assay （FQ-PCR） using duplex scorpion primers to detect the Mycobacterium tuberculosis DNA in clinical specimens was evaluated in the present study, in which this assay was compared with direct acid last staining , cultivation on Loewenstain Jensen （L-J） medium and the Mycobacterium tuberculosis direct test （MTD） to evaluate their detection rate and detection time in the clinical specimens of 43 patients with tuberculosis and 11 non-tuberculosis patients. The FQ-PCR assay was performed on the quantitative PCR amplifier ABI-7000 using primers targeting an 55 bp fragment of the intergenic region TB senX3-regX3. It was found that the M. tuberculosis DNA could be rapidly detected by PCR assay with a excellent linear relationship （correlation coefficient =0. 9994） under the concentration of the standard samples at 10^2- 10^6 copies/μl. The positive detection rates by using direct acid last staining, cultivation on L-J medium, MTD and FQ-PCR assay were 16.28%, 37.21%, 100% and 100% respectively. Although the detection rates of both MTD and FQ-PCR assay were attained up to 100 %, but the detection time was quite different, for FQ-PCR assay it only required approximately 80 rain, in contrast to 4-5 hrs for MTD,. In addition, the cost of testing for FQ-PCR assay was just only 1/4 for MTD. From the above observations,it is apparent that the fluorescent quantitative PCR assay appears to be a sensitive, specific and time-saving method to detect the presence of M. tuberculosis in clinical samples.

关 键 词：结核分枝杆菌 荧光定量PCR 二聚体蝎型探针 

分 类 号：R520.4[医药卫生—内科学]