Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.
Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.
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DOI：
10.1038/srep19541
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发表时间：
2016-01-20
影响因子：
4.6
通讯作者：
Niemz A
中科院分区：
文献类型：
--
作者：
Ferguson TM;Weigel KM;Lakey Becker A;Ontengco D;Narita M;Tolstorukov I;Doebler R;Cangelosi GA;Niemz A
核酸扩增检测（NAAT）能够快速、灵敏地诊断结核病，从而促进治疗并减少传播。从痰液中提取核酸构成了结核病NAAT在近患者环境中的最大技术挑战。本报告提供了半自动化样本处理方法的初步数据，其中对痰液进行消毒和液化，然后在小型化电池驱动的珠混合器中进行PureLyse®机械裂解和固相核酸提取。痰液液化和消毒使得掺入人痰液中的培养的结核分枝杆菌（M.tb）的活菌负荷减少>104倍，这减轻了生物危害问题。通过PureLyse®方法和经临床验证的手动方法制备样本，可对加标104和105菌落形成单位（cfu）/mL M. tb的痰液进行基于PCR的阳性检测。在103 cfu/mL痰液浓度下，分别使用对照方法和PureLyse®方法扩增了6份样本中的4份和2份。对于来自TB病例和对照的临床标本，对于1 mL输入体积的样本，两种方法提供了100%一致的结果（N = 41）。因此，半自动化PureLyse®方法与经验证的手动比较方法相似，但更快，仪器最少，并且可以集成到为接近患者的低资源环境设计的TB分子诊断平台中。
Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse® mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >104 fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse® method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 104 and 105 colony forming units (cfu)/mL M.tb. At 103 cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse® method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with 1 mL input volume (N = 41). The semi-automated PureLyse® method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented, and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.