Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens
Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens
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用于诊断皮肤活检标本中少杆菌性麻风病的液滴数字 PCR 检测方法的开发和评估
DOI：
10.1371/journal.pntd.0007284
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发表时间：
2019-03-01
影响因子：
3.8
通讯作者：
Zhang, Furen
中科院分区：
文献类型：
--
作者：
Cheng, Xiujun;Sun, Lele;Zhang, Furen
背景麻风杆菌（M. leprae）数量减少。麻风（leprae）在少菌型（PB）患者中的分布情况反映了需要进一步优化麻风诊断方法。越来越多的报道表明，液滴数字聚合酶链反应（ddPCR）是一种很有前途的工具，用于诊断感染性疾病的样本中的低拷贝数。到目前为止，还没有出版物研究ddPCR在检测M.麻风病人本研究的目的是建立和评价一种用于PB型麻风诊断的ddPCR方法。方法采用两种最敏感的DNA靶标检测M.从电子数据库中选择麻风患者，通过定量聚合酶链反应（qPCR）和ddPCR评估敏感性和特异性。患有其他皮肤病的对照患者（n = 59）用于定义双重ddPCR测定的截止值。为了进行比较评价，在44例PB患者和68例多菌（MB）患者中进行了qPCR和ddPCR检测。主要结论M.用麻风特异性重复元件（RLEP）和GroEL（编码65 kDa分子伴侣GroEL）建立ddPCR检测方法，系统分析特异性和敏感性。根据定义的临界值，ddPCR检测试剂盒在检测M时显示出更高的灵敏度。与qPCR相比，PB患者中的麻风病DNA（79.5% vs 36.4%），而两种检测方法在MB患者中的灵敏度均为100%。结论/意义我们开发并评价了一种用于麻风病患者皮肤活检样本中麻风病诊断的双重ddPCR方法。虽然仍然昂贵，但ddPCR可能是检测PB麻风的有前途的诊断工具。
Background The reduced amounts of Mycobacterium leprae (M. leprae) among paucibacillary (PB) patients reflect the need to further optimize methods for leprosy diagnosis. An increasing number of reports have shown that droplet digital polymerase chain reaction (ddPCR) is a promising tool for diagnosis of infectious disease among samples with low copy number. To date, no publications have investigated the utility of ddPCR in the detection of M. leprae. The aim of this study was to develop and evaluate a ddPCR assay for the diagnosis of PB leprosy. Methodology The two most sensitive DNA targets for detection of M. leprae were selected from electronic databases for assessment of sensitivity and specificity by quantitative polymerase chain reaction (qPCR) and ddPCR. Control patients (n = 59) suffering from other dermatological diseases were used to define the cut-off of the duplex ddPCR assay. For comparative evaluation, qPCR and ddPCR assays were performed in 44 PB patients and 68 multibacillary (MB) patients. Principal findings M. leprae-specific repetitive element (RLEP) and groEL (encoding the 65 kDa molecular chaperone GroEL) were used to develop the ddPCR assay by systematically analyzing specificity and sensitivity. Based on the defined cut-off value, the ddPCR assay showed greater sensitivity in detecting M. leprae DNA in PB patients compared with qPCR (79.5% vs 36.4%), while both assays have a 100% sensitivity in MB patients. Conclusions/Significance We developed and evaluated a duplex ddPCR assay for leprosy diagnosis in skin biopsy samples from leprosy patients. While still costly, ddPCR might be a promising diagnostic tool for detection of PB leprosy.