阳阳科普馆 (nat样本液)

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与传统的病毒分离培养相比,核酸分子检测技术灵敏度和特异性高,可识别病毒的不同亚型,可实现快速准确的检测,且因为检测对象为核酸而不必保持病毒颗粒完整性和感染活性[1,2],因此大多数诊断实验室已从传统方法向核酸检测过渡,此外,传统热灭活或乙醇灭活的方式容易造成核酸破坏,出现检测结果假阴性[3,4],在这样的背景下,灭活型样本保存液应运而生。

灭活型样本保存液多是基于核酸提取试剂中的裂解液优化开发而来,裂解液主要成分为高浓度异硫氰酸胍[5]。异硫氰酸胍为一种强烈的蛋白质变性剂,已有研究表明高浓度胍盐或核酸提取试剂裂解液可有效灭活病毒[6]。高浓度胍盐结合其他化学试剂可抑制样本中DNase和RNase活性,可稳定保存样本核酸[7]。

灭活型样本保存液除了能灭活病毒保护核酸外,相较于非灭活型样本保存液,由于采集样本后可常温运输,因此更加便于标本从各种偏远、交通不便的采集点运送至检测实验室。DAUM等[8]研究了一种混合优化的分子转运培养基MTM,结果表明该培养基能有效地杀死病原体,包括高致病性H5流感病毒,并在环境温度下保存释放的RNA长达30天。

一方面,随着疾病筛查、流行病学等研究的普遍开展,核酸检测标本量日益增加;另一方面,全球对实验室生物安全的管理日益严格,未达相关安全等级的实验室无法开展活病毒的培养等相关检测[9]。综合两方面因素,临床对灭活型样本保存液的需求日益增加。

但关于灭活型样本保存液的相关研究表明[10],常用的病毒裂解剂AVL和RLT缓冲液处理后的病毒仍然存活,这提示我们对灭活型样本保存液针对特定病毒的灭活能力应当进行相应的临床评估。还有研究表明[11],一旦加入乙醇,AVL就可以完全灭活EBOV病毒,但这与下游自动核酸提取系统不兼容。这提示我们改进灭活型样本保存液的同时还应验证对下游核酸提取及检测的影响。

参考文献

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[3] Pan Y, Long L, Zhang D, et al. Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads[J]. Clinical Chemistry, 2020.

[4] Yueying W A, Wei S A, Zuguo Z B C, et al. The impacts of viral inactivating methods on quantitative RT-PCR for COVID-19[J]. Virus Research, 2020,285.

[5] Hill V R, Narayanan J, Gallen R R, et al. Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water[J]. Pathogens (Basel, Switzerland), 2015,4(2):335-354.

[6] Blow J A, Dohm D J, Negley D L, et al. Virus inactivation by nucleic acid extraction reagents[J]. Journal of Virological Methods, 2004,119(2):195-198.

[7] Blow J A, Mores C N, Dyer J, et al. Viral nucleic acid stabilization by RNA extraction reagent[J]. Journal of Virological Methods, 2008,150(1-2):41-44.

[8] Daum L T, Worthy S A, Yim K C, et al. A clinical specimen collection and transport medium for molecular diagnostic and genomic applications[J]. Epidemiology & Infection, 2011,139(11):1764-1773.

[9] WHO W H O. Laboratory biosafety guidance related to coronavirus disease (COVID-19)[EB/OL]. https://www.who.int/publications/i/item/laboratory-biosafety-guidance-related-to-coronavirus-disease-(covid-19).

[10] Kirsten, St., George, et al. Unreliable Inactivation of Viruses by Commonly Used Lysis Buffers[J]. Applied Biosafety Journal of the American Biological Safety Association, 2017.

[11] Burton J E, Easterbrook L, Pitman J, et al. The effect of a non-denaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples[J]. Journal of Virological Methods, 2017,250:34 -40.