A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)
A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)
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DOI：
10.3791/60460
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发表时间：
2020-04-01
影响因子：
1.2
通讯作者：
Gillespie, Stephen H.
中科院分区：
文献类型：
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作者：
Sabiiti, Wilber;Mtafya, Bariki;Gillespie, Stephen H.
结核病是由结核分枝杆菌（Mt B）引起的，这是一种被联合国（UN）列为危险B类生物物质的病原体。为了工作人员的安全起见，所有假定携带结核分枝杆菌的样本的处理必须在3级隔离实验室进行。TB分子细菌负荷试验（TB-MBLA）是一种逆转录酶定量聚合酶链反应（RT-qPCR）试验，使用引物和16 S rRNA双标记探针定量Mtb杆菌负荷。我们描述了使用热灭活使TB样品无感染性，同时保留TB-MBLA的RNA。将1 mL等份痰液样本置于密闭的15 mL离心管中，在80 ℃、85 ℃或95 ℃下煮沸20 min，以除去Mtb杆菌。热灭活和对照（活）样品培养42天证实TB死亡。然后用100 μ L提取对照品加标灭活样品，并按照标准RNA分离程序提取RNA。在热处理样品的培养物中未观察到生长。对分离的RNA进行实时RT-qPCR，扩增Mtb 16 S rRNA基因中的特定靶标，产生定量循环（Cq）形式的结果。使用标准曲线将Cq转化为细菌负荷，或估计的菌落形成单位/mL（eCFU/mL）。Cq与样品的细菌载量之间存在反比关系。局限性在于热失活会溶解一些细胞，使RNA暴露于RNA酶，从而导致RNA的丢失。
Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers' safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 degrees C, 85 degrees C, or 95 degrees C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 mu L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of