多重聚合酶链反应检测法可直接从临床标本中同时检测和区分结核分枝杆菌、鸟分枝杆菌复合体和其他分枝杆菌种。

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens.

机构信息

Division of Clinical Microbiology, Department of Laboratory Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.

出版信息

J Appl Microbiol. 2009 Aug;107(2):425-35. doi: 10.1111/j.1365-2672.2009.04218.x. Epub 2009 Mar 16.

DOI:10.1111/j.1365-2672.2009.04218.x

PMID:19302308

Abstract

AIMS

Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management.

METHODS AND RESULTS

Two hundred and thirty-five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L-J & BACTEC MGIT-960 culture and multiplex PCR tests. The multiplex PCR comprised of genus-specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex-specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex-specific primer pairs targeting 16S-23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3-4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77.24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L-J culture (34.4%) and BACTEC MGIT-960 culture (50.34%) methods. The mean isolation time for M. tuberculosis was 19.03 days in L-J culture and 8.7 days in BACTEC MGIT-960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co-infection in 1.3% HIV-negative and 2.9% HIV-positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38.09% HIV-positive and 15.38% HIV-negative cases.

CONCLUSIONS

The developed in-house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens.

SIGNIFICANCE AND IMPACT OF THE STUDY

The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.

摘要

目的

聚合酶链反应（PCR）是诊断分枝杆菌感染和鉴定病原性分枝杆菌物种的最快速和敏感的方法，以便进行适当的治疗和更好地管理患者。

方法和结果

对来自 145 例临床疑似肺结核的 235 个样本进行了 ZN（齐尔-尼尔森）染色检查、L-J 和 BACTEC MGIT-960 培养和多重 PCR 检测，以检测分枝杆菌感染。多重 PCR 包括针对 hsp65 基因的属特异性引物、针对结核分枝杆菌复合体的 cfp10（Rv3875、esxB）区域的特异性引物和针对 16S-23S 内部转录间隔区序列的鸟分枝杆菌复合体特异性引物对。开发的多重 PCR 具有 10 fg（3-4 个细胞）分枝杆菌 DNA 的分析灵敏度。多重 PCR 检测显示出最高（77.24%）的检测率，而 ZN 染色检查的检测率最低（20%），L-J 培养（34.4%）和 BACTEC MGIT-960 培养（50.34%）的方法有所改善。结核分枝杆菌的平均分离时间在 L-J 培养中为 19.03 天，在 BACTEC MGIT-960 培养中为 8.7 天。使用多重 PCR，我们可以在 1.3%的 HIV 阴性和 2.9%的 HIV 阳性患者中建立结核分枝杆菌+鸟分枝杆菌合并感染。多重 PCR 还可用于诊断 38.09%的 HIV 阳性和 15.38%的 HIV 阴性患者的分枝杆菌血症。