用于坎帕拉城郊结核分枝杆菌复合群基因分型的单核苷酸多态性实时聚合酶链反应检测法

A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala.

作者信息

Wampande Eddie M, Hatzios Stavroula K, Achan Beatrice, Mupere Ezekiel, Nsereko Mary, Mayanja Harriet K, Eisenach Kathleen, Boom W Henry, Gagneux Sebastien, Joloba Moses L

机构信息

Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda.

Department of Bio-molecular Resources and Biolab Sciences, College of Veterinary Medicine, Animal Resources and Bio Security, Makerere University, Kampala, Uganda.

出版信息

BMC Infect Dis. 2015 Sep 30;15:396. doi: 10.1186/s12879-015-1121-7.

DOI:10.1186/s12879-015-1121-7

PMID:26423522

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4590274/

Abstract

BACKGROUND

Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients.

METHOD

Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing.

RESULTS

The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage.

CONCLUSION

The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.

摘要

背景

结核分枝杆菌复合群（MTBC）的准确且高通量基因分型对于理解结核病（TB）的流行病学和发病机制可能至关重要。在本研究中，我们报告了一种LightCycler®实时荧光定量PCR单核苷酸多态性（LRPS）检测方法的开发，用于快速确定结核病患者经最少处理的痰液样本中的MTBC谱系/亚谱系。

方法

使用长序列多态性PCR（LSP-PCR）技术和LRPS检测方法对70株MTBC菌株进行平行基因分型分析。为进行靶向测序，分析了9株MTBC分离株（每个MTBC谱系3株）在以下三个基因中的谱系特异性单核苷酸多态性（SNP）以验证LRPS结果：MTB乌干达家族的Rv004c、MTB谱系4的Rv2962和MTB谱系3的Rv0129c。然后通过经过验证的LRPS方法在不进行预先培养的情况下确定300份涂片阳性痰液样本中存在的MTBC谱系。

结果

LSP-PCR和LRPS检测方法对所有70株MTBC菌株产生了一致的基因分型数据；然而，LSP-PCR检测方法的灵敏度比LRPS方法低10倍，并且需要更高的DNA浓度才能成功鉴定某些样本的MTBC谱系。对含有谱系特异性SNP的基因进行靶向测序与基因分型结果100%一致，并进一步验证了LRPS检测方法。在分析的300份痰液样本中，58%含有来自MTBC-乌干达家族的MTBC，27%来自MTBC谱系4（不包括MTBC乌干达家族），13%来自MTBC谱系3，其余2%的谱系不确定。