Real-time PCR检测麻风患者治疗前后及少菌型石蜡标本中麻风菌DNA的价值

The Value of Real-time PCR Amplification of M.leprae Specific DNA from Paraffin-embedded Skin Biopsy Specimens

目的应用已建立的实时定量荧光PCR(real-time PCR)方法检测少菌型麻风(PB)患者的石蜡标本,探讨该方法对BI为0患者的辅助诊断价值。方法从石蜡标本中提取的DNA进行麻风菌重复序列的特异性real-time PCR检测,包括短程联合化疗(MDT)治疗前后标本共92例;2008年和2013年收集的界限类偏瘤型麻风(BL)石蜡标本分别为7例和8例。并检测70例临床诊断PB和未定类麻风,同时将RT-PCR检测结果与病理分析结果进行比较。结果 92例(LL:19例;BL:20例;BB:20例;BT:16例;TT:17例)不同型别麻风患者的石蜡标本检测阳性率治疗前、治疗后分别为100.00%,100.00%,90.00%,87.50%,50.00%与40.00%,40.00%,0,0,22.22%。比较保存5年前后15例BL患者石蜡标本,检测结果差异无统计学意义。70例临床诊断PB和未定类麻风病患者的石蜡标本real-time PCR检测阳性率为71.43%(50/70),病理检测与临床符合率为51.43%(36/70),两者的阳性检测率差异有统计学意义(P<0.05)。结论建立Taq Man(水解探针)技术的real-time PCR方法可以快速、特异、灵敏的检测石蜡标本中的麻风菌DNA,提高抗酸染色阴性标本的检测阳性率,对临床诊断的指导意义重大。

Objective To develop a TaqMan real-time quantitative PCR assay for detection of M. leprae DNA for the di- agnosis of leprosy. Methods Primers and probes were designed according to the repetitive sequence of M. leprae from GeneBank. The recombinant plasmid pGEMT-101 was constructed and served as a template to prepare the standard curve for TaqMan real-time PCR. We detected the paraffin-embedded skin biopsy speci- mens from 90 patients with confirmed diagnosis and 70 patients with clinical diagnosis. Results The real- time PCR positive rate of 92 different types leprosy specimens （ LL： 19 ; BL ：20 ; BB ： 20 ; BT： 16 ;＇IT： 17 ） were 100.00% ,100.00% ,90.00% ,87.50% ,50.00% with before MDT and 40.00% ,40.00% ,0,0,22.22% with after MDT respectively. Before and after 5 years the results with the 15 BL cases were not different. The real-time PCR positive rate of 70 leprosy patients with clinical diagnosis was 71.43 % （50/70）, and the posi- tive results of pathological examination was 51.43 % （36/70）, which have significant difference （P 〈 0.05 ）. Conclusion The results indicated that TaqMan real-time PCR was a rapid, specific and sensitive method for detecting M. leprae DNA.