实时荧光定量PCR检测石蜡标本中麻风菌DNA

Identification of M.leprae specific DNA from paraffin-embedded skin biopsy specimens with real time PCR

目的:评价实时定量荧光PCR(Real-time PCR)法检测石蜡标本中麻风菌DNA的应用价值。方法:根据基因库(GenBank)发表的M.leprae的全基因组序列,以一段重复序列(repetitive element,RLEP)为扩增靶序列,合成引物和探针,构建质粒pGEMT-101作为标准品,用Real-time PCR对石蜡标本进行麻风菌DNA检测,并评价其敏感性。结果:对52例麻风患者的石蜡包埋组织标本麻风菌进行了检测,不同型别麻风(LL:8;BL:10;BT:28;TT:6)的石蜡标本检测阳性率分别为100%(8/8)、80%(8/10)、78.57%(22/28)、50%(3/6)。结论:Real-time PCR方法可在石蜡标本中快速灵敏的检测到麻风菌DNA。

Objective：To assess the application of real time quantitative PCR assay for the detection of M. leprae DNA in paraffin-embedded skin biopsy specimens. Methods： Primers and prob were designed according to the repetitive sequence of M. leprae from gen Bank. The recombinant plasmid pGEMT- 101 was constructed and served as a template to prepare the standard curve for TaqMan real time PCR. The M. Lerae DNA in paraffin-embedded skin biopsy specimens was detected and the sensitivity of the results was assessed. Results： The paraffin-embedded skin biopsy specimens from 52 leprosy patients were tested. The sensitivities of the results varied in different types of the disease： LL 100% （8/8） , BL 80% （8/10）, BT 78.57% （22/28） and TF 50% （3/6）, respectively. Conclusion： Real-time PCR is a rapid, specific and sensitive method for the detection of M. leprae DNA in paraffin-embedded skin bioDsv snecimens.