摘要

常规的结核分枝杆菌的全面表征是基于文化的，需要花费数周时间。全基因组测序（WGS）可以产生抗生素敏感性分布图以告知治疗情况，增加全球监测的应变信息;如果在护理点处或附近提供这些数据可能是变革性的。我们展示了直接从患者样本中提取结核分枝杆菌WGS的低成本DNA提取方法。我们最初通过使用Illumina MiSeq测序仪（在常规临床测试后获得的40个涂片阳性呼吸样品和27个匹配的液体培养物）评估了该方法。在成功提取DNA的所有39个样品中鉴定出结核分枝杆菌。从24个样本（62％）获得足够的抗生素敏感性预测数据;所有结果都与参考实验室表型一致。系统发生的位置与直接样本和培养样本一致。使用Illumina MiSeq / MiniSeq，从患者样本到结果的工作流程可以在44/16小时内完成，每个样本的试剂成本为96英镑/ 198英镑。然后，我们在Oxford Nanopore Technologies MinION测序仪上采用非特异性基于PCR的文库制备方法进行测序。我们将其应用于培养的牛分枝杆菌菌株BCG DNA和培养阴性痰DNA和BCG DNA的组合。对于流式细胞R9.4版，从患者到BCG鉴定的预计周转时间，吡嗪酰胺耐药性的检测以及系统发生放置的时间为7.5小时，5小时后具有完全的易感性结果。抗生素敏感性预测是完全一致的。 MinION的一个关键优势是能够继续测序，直到获得足够的覆盖率，为直接样本中可变量结核分枝杆菌DNA的问题提供了一种可能的解决方案。

Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to culturedMycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

http://jcm.asm.org/content/55/5/1285.short