用巢式聚合酶链式反应从全血扩增麻风菌特异片段提高麻风病确诊率

Study on nested-PCR for amplification of specific of M. leprae from whole blood for diagnosis of leprosy

目的用巢式聚合酶链式反应(Nested-PCR)检测全血中麻风菌特异片段,以提高麻风病诊断水平。方法收集多菌型(multibacillary leprosy,MB)、少菌型(paucibacillary leprosy,PB)麻风病患者、其家庭内接触者以及麻风病流行区、非流行区的正常人全血和血清样本,建立巢式PCR,扩增麻风菌重复序列的特异片段,PCR阳性产物直接测序鉴定扩增片段的特异性。结果在35例MB患者中巢式PCR有34例阳性;26例PB患者中19例阳性;23例家庭内接触者中2例阳性。流行区和非流行区正常人中未见阳性。此结果与血清学结果进行比较,发现血清学检测MB患者阳性率稍高于巢式PCR;值得注意的是,对PB患者中细菌密度指数(BI)为阴性者,巢式PCR阳性检测率高达78.57%。结论巢式PCR对于麻风病MB患者的诊断敏感性不亚于血清学抗体诊断,但可提高麻风病特别是对PB患者诊断的敏感性;对高危人群尤其是家庭内接触者的监测有较大的实用价值。

Objective To detect the specific fragment of M.leprae DNA from whole blood with nested-PCR and improve the ability of leprosy diagnosis.Methods The whole blood and blood serum samples were collected from MB and PB leprosy as well as their household contacts（HHC）and normal persons from endemic and non-endemic villages to amplify repeated specific sequences of M.leprae by PCR and analyze.Results The positive cases for M.leprae DNA in MB,PB and HHC were 34/35,19/26 and 2/19 respectively by nested-PCR.Twenty healthy controls from Kunming and Beijing were all negative（0/20）.The results were compared with serological results.The true positive rate of MB patients with serological tests was slightly higher than that with nested-PCR.It is worthwhile to note that PB patients with negative BI results had the highest positive detection rate（78.57%） by nested-PCR.Conclusion The study shows that the sensitivity of nested-PCR for diagnosing PB patients is not less than serological antibody detection tests,nested-PCR could be used to improve the sensitivity for diagnosing PB patients and applied in surveillance for high risk population especially for household contacts（HHC）.