Optimizing DNA Extraction from Pediatric Stool for Diagnosis of Tuberculosis and Use in Next-Generation Sequencing Applications.
Optimizing DNA Extraction from Pediatric Stool for Diagnosis of Tuberculosis and Use in Next-Generation Sequencing Applications.
复制标题
DOI：
10.1128/spectrum.02269-22
复制
发表时间：
2023-02-14
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
世卫组织已批准使用粪便样本诊断儿童结核病（TB），粪便的靶向下一代测序（tNGS）已被证明可支持诊断并提供有关药物敏感性（DS）的信息。优化从粪便中提取DNA进行测序对于确保高诊断灵敏度和准确的DS信息至关重要。用不同浓度的牛分枝杆菌卡介苗（BCG）加标人粪便样本，并使用四种不同的DNA提取试剂盒从样本中提取DNA。对每个样品进行定量PCR以鉴定结核分枝杆菌复合菌，并进行进一步分析以评估总DNA产率、片段长度和纯度。对10名诊断为肺结核的儿科参与者进行了相同的过程，并对样本进行了tNGS。与评估的其他提取方法相比，用于土壤的FastDNA旋转试剂盒在加标已知量BCG的模型样品上显示出最佳结果。对于临床样本，FastDNA和PowerFecal Pro DNA（PowerFecal）试剂盒均显示总体DNA量增加，M.结核病特异性DNA的数量，并成功的靶向测序时，对粪便样本进行测试，相比其他两个试剂盒。通过PowerFecal提取的三个样本和通过FastDNA提取的三个样本（来自不同患者）提供了成功的测序数据，FastDNA的rpoB区域的平均覆盖深度为298（范围，107至550），PowerFecal为310（范围，182至474），结果彼此相当（P = 0.946）。PowerFecal Pro和FastDNA旋转试剂盒在从儿科粪便样本中提取DNA用于tNGS方面具有上级优势。重要性这是第一项比较从儿科粪便样本中提取结核分枝杆菌DNA技术与测序技术的研究。它为其他研究人员提供了一个重要的起点，以分离出高质量的DNA，从而进一步推动该领域的发展，并继续优化方案和方法。
The WHO has endorsed the use of stool samples for diagnosis of tuberculosis (TB) in children, and targeted next-generation sequencing (tNGS) of stool has been shown to support diagnosis and provide information about drug susceptibility (DS). Optimizing extraction of DNA from stool for sequencing is critical to ensure high diagnostic sensitivity and accurate DS information. Human stool samples were spiked with various concentrations of Mycobacterium bovis bacillus Calmette-Guérin (BCG), and DNA was extracted from the samples using four different DNA extraction kits. Each sample was subjected to quantitative PCR for identifying Mycobacterium tuberculosis complex bacteria and underwent further analysis to assess the overall DNA yield, fragment length, and purity. This same process was performed with 10 pediatric participants diagnosed with pulmonary TB, and the samples underwent tNGS. The FastDNA spin kit for soil showed the best results on model samples spiked with known quantities of BCG, compared to the other extraction methods evaluated. For clinical samples, the FastDNA and PowerFecal Pro DNA (PowerFecal) kits both showed an increase in the overall DNA quantity, M. tuberculosis-specific DNA quantity, and successful targeted sequencing when testing was performed on stool samples, compared to the two other kits. Three samples extracted via PowerFecal and three samples extracted via FastDNA (from different patients) provided successful sequencing data, with an average depth of coverage of the rpoB region for FastDNA of 298 (range, 107 to 550) and for PowerFecal of 310 (range, 182 to 474), results that were comparable to one another (P = 0.946). The PowerFecal Pro and FastDNA spin kits were superior for extracting DNA from pediatric stool samples for tNGS. IMPORTANCE This is the first study to compare Mycobacterium tuberculosis DNA extraction techniques from pediatric stool samples for use with sequencing technologies. It provides an important starting point for other researchers to isolate quality DNA for this purpose to further the field and to continue to optimize protocols and approaches.
登录
查看更多内容
影响因子：
9.4
作者：
Cabibbe AM;Spitaleri A;Battaglia S;Colman RE;Suresh A;Uplekar S;Rodwell TC;Cirillo DM
通讯作者：
Cirillo DM
影响因子：
15.5
作者：
Wesolowska-Andersen A;Bahl MI;Carvalho V;Kristiansen K;Sicheritz-Pontén T;Gupta R;Licht TR
通讯作者：
Licht TR
影响因子：
3.8
作者：
Ayana, Mio;Cools, Piet;Levecke, Bruno
通讯作者：
Levecke, Bruno
影响因子：
4.6
作者：
Videnska, Petra;Smerkova, Kristyna;Budinska, Eva
通讯作者：
Budinska, Eva
影响因子：
46.9
作者：
Costea, Paul I.;Zeller, Georg;Bork, Peer
通讯作者：
Bork, Peer