【作者】 刘洪波；

【导师】 沈关心；

【作者基本信息】 华中科技大学 ， 免疫学， 2012， 博士

【摘要】 背景：基于PCR的核酸测定技术已成为流感病毒学监测和诊断的首选方法。然而除了核酸测定方法、测定试剂、使用的引物和探针外,核酸测定分析前的样品储存、转运以及核酸分离纯化方法等变量也对核酸测定结果产生重要影响。此外,核酸测定过程中潜在的生物危害也是核酸测定技术实际应用必须考虑的因素。目的：1)为提高核酸检测样本的制备效率,将基于磁珠技术的自动核酸提取系统Maxwell 16 System应用于流感病毒核酸提取并分析该方法对实时RT-PCR的影响,以评估该自动系统用于流感病毒核酸检测的适宜性。2)了解不同核酸提取试剂的裂解缓冲液稳定流感病毒RNA的能力,为将裂解缓冲液用作流感病毒核酸检测样品的储存和转运介质奠定基础。3)测定常用的病毒核酸提取方法的裂解缓冲液灭活流感病毒的能力,以评估流感病毒核酸提取过程中的生物危害暴露风险。方法：1) Maxwell 16 System提取系列稀释的流感病毒培养液以及咽拭子、支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)、人和禽粪便标本中的流感病毒核酸,以CDC开发的实时RT-PCR (real-time RT-PCR, rRT-PCR)方法检测提取物,所获结果与经典的基于硅胶柱层析的手工核酸提取试剂QIAamp Viral RNA Mini Kit (QIAamp Kit)提取所获得的结果相比较,分析Maxwell 16 System提取流感病毒核酸的敏感性、线性、重复性以及从多种临床和环境样本中提取流感病毒核酸的效率。2)分别以QIAamp Kit中的Buffer AVL和Maxwell 16 System中的Lysis Solution两种裂解缓冲液处理流感病毒溶液,处理后的裂解缓冲液/病毒混合物分别保存于室温、4℃和-20℃或者进行多次冻融,以rRT-PCR分析病毒RNA量的变化,以扩增流感病毒基因组RNA第7节段全长的传统RT-PCR (conventional RT-PCR, cRT-PCR)评估病毒RNA的完整性。3)以Maxwell 16 System的Lysis Solution QIAamp Kit的Buffer AVL和改良的异硫氰酸胍法试剂的RNA提取试剂A液(RNA Extraction Reagent A, ERA)等三种常用的病毒RNA提取方法的裂解缓冲液处理高滴度的流感病毒,系列对数稀释病毒处理液后接种于MDCK细胞进行分离培养,以倒置显微镜观察细胞生长状态,血凝实验确定流感病毒存在与否。结果：1)与QIAamp Kit比较,Maxwell 16 System的标准程序(Maxwell 16-S)有良好的线性、重复性以及更高的分析敏感性；Maxwell 16-S程序从咽拭子和BALF标本中可分离到更多的病毒RNA和/或更少的PCR抑制物,然而在粪便标本中的效率刚好相反,不能获得满意的效果；通过改良Maxwell 16程序,Maxwell 16 System清除粪便基质中PCR抑制物的能力得到改善,提取禽粪便标本病毒核酸的效率与QIAamp方法可比,而提取人粪便标本中病毒核酸的效率仍不及QIAamp方法。2) Lysis Solution和Buffer AVL两种裂解缓冲液处理的病毒样品,-20℃放置160天和冻融5次均仍能收获量没有明显减少的完整的病毒基因组RNA；室温条件下,病毒RNA降解明显,两缓冲液处理后放置3天均不能确定收获到完整的流感病毒RNA分子；4℃条件下保存一定时间能收获到全长的流感病毒RNA分子；而在4℃和室温条件下,Buffer AVL处理较Lysis Solution处理后样品中的病毒RNA降解速度更快。3) Lysis Solution、Buffer AVL和ERA三种裂解缓冲液处理病毒所获得的裂解溶液/病毒混合物,通过对数稀释结果发现,在低稀释度时和试剂对照一样,接种24h内引起MDCK细胞的完全死亡；在高稀释度时,三种裂解溶液/病毒混合物没有引起MDCK细胞病变,且培养物血凝实验阴性,而同时设置的病毒对照出现了明显的细胞病变,且培养物血凝实验阳性。结论：1) Maxwell 16 System结合rRT-PCR可以用于流感病毒的定量和定性测定；其清除不同样本基质中的PCR抑制物的能力不同,从而对rRT-PCR检测不同样本的敏感性产生影响,使其适用于咽拭子、BALF和禽粪标本中流感病毒RNA的提取,而不适于人粪便标本病毒核酸的提取；对于一些复杂的样本基质,适当地减少样本输入量可以提高检出。2)不同的核酸提取试剂的裂解缓冲液用作流感病毒样本的保存剂都有助于病毒RNA的稳定性,可抵抗多次冻融对病毒RNA的损伤,冷藏或冷冻的低温条件下(特别是-20℃或更低温度)较长时间仍可收获到完整的流感病毒基因组RNA；但不同方法学的裂解缓冲液稳定病毒RNA的能力不尽相同,实际应用之前需进行验证。3) Lysis Solution、Buffer AVL和ERA三种裂解溶液均可完全灭活流感病毒,经裂解缓冲液初步裂解后的核酸提取操作的生物危害风险大大降低。这对于将流感病毒核酸的自动提取仪器移出生物安全柜,或者在低防护水平区域或实验室进行核酸提取的初始裂解后操作,以及对于将裂解缓冲液用作流感病毒样品的稳定剂都有指导意义。更多还原

【Abstract】 Background PCR-based nucleic acid (NA) testing is increasingly the first-choice method for virological surveillence and diagnosis of influenza. However, not only used NA assay protocols, reagents, primers and probes, but also pre-analytical variables, including the storage and transport of samples and NA purification methods, have major effects on the performance of NA assay. And, potential biohazard exposure to personnel during NA assay must be taken into consideration and would affect practical use of NA assay.Objective1) To apply Maxwell 16 System, an automated NA extraction platform based-on magnetic bead technology, to automated extraction of influenza virus (flu-v) RNA for diagnosis of flu using PCR-based testing and evaluated its performance and suitability for detection of flu-v from various samples.2) To assess the effects of lysis buffers of different NA extraction methods on the stability of flu-v RNA in order to lay a foundation for the utilization of lysis buffer as preservative of flu-v samples.3) To evaluate the biohazard risk during extraction of flu-v RNA by examining the ability of lysis buffers of frequently used NA extraction methods to inactivate flu-v.Methods1) Following extraction by Maxwell 16 System and QIAamp Viral RNA Mini Kit (QIAamp Kit) from flu-v stock dilution series, throat swabs, bronchoalveolar lavage fluid (BALF) and fecal samples of human and poultry origin, the extracted RNA was assayed by the CDC-developed real-time RT-PCR (rRT-PCR) protocols. The performance of Maxwell 16 System, including sensitivity, linearity, precision and extraction efficiency of flu-v RNA from the various clinical and field samples, was evaluated by comparison with QIAamp Kit based on silica gel column chromatography, a common used method for extraction of flu-v RNA.2) Influenza virus stocks were treated respectively with two lysis buffers, Lysis Solution (Lysis Buffer+Proteinase K) from Maxwell 16 System and Buffer AVL with carrier RNA from QIAamp Kit, and the lysis buffer-virus mixtures were kept at 22℃,4℃,—20℃or underwent cyclic freeze-thaw. The relative quantity and quality of viral RNA after storage or freeze-thaw were evaluated with an rRT-PCR assay and a conventional RT-PCR assay targeting full-length matrix gene of flu-v.3) Three lysis buffers, Lysis Solution, Buffer AVL and RNA Extraction Reagent A (ERA) from a modified guanidinium isothiocyanate kit, which based on three different methods commonly used in viral RNA extraction, were selected to process high-titer flu-v stocks (105.7TCID50/ml) according to manufacturer’s instructions and preliminary research of ours. The lysis buffer-virus combinations were then serially tenfold diluted using virus growth medium and the dilutions were inoculated onto MDCK cells for virus isolation. Cell growth was observed daily under an inverted microscope. Cultures were harvested and hemagglutination tests were done with the supernatant when cytopathic effect (CPE) of cultured cells developed more than 75%.Results1) Extraction with Maxwell 16 System standard procedure (Maxwell 16-S) resulted in good linearity across a wide range, low inter-and intra-run variation and higher analysis sensitivity compared to those with the QIAamp extraction. Maxwell 16-S extraction yielded more RNA and/or fewer PCR inhibitors from throat swabs and BALF samples than QIAamp extraction but they had opposite performance for extraction from fecal samples. When extraction with a modified procedure (with reduced input and increased output) of Maxwell 16 System (Maxwell 16-M), the improved capacity to remove PCR inhibitors contributed to an enhanced sensitivity for detection of flu-v in poultry fecal samples although still a relative low sensitivity in human fecal samples compared to those with QIAamp Kit.2) Entire matrix gene was amplified from samples treated with either of the lysis buffers after storage of 160 days at -20℃or five times of freeze-thaw, and the relative intensity of amplification products did not show to decline. Viral RNA in Buffer AVL degraded more rapidly than in Lysis Solution when kept at 22℃and 4℃, while the integrity of viral RNA could be maintain for certain time intervals at 4℃in both of the lysis buffers.3) Massive cell death occurred within 24 hours after inoculation of lysis buffer-virus mixtures at low dilutions as well as the corresponding reagent controls at low dilutions. Cytopathic effect did not develop even after three blind passages of all the lysis buffer-virus mixtures at high dilutions and no viable virus in the cultures was detected by the hemagglutination tests, while infectious virus was isolated in the virus controls diluted at the same concentrations.Conclusion1) Qualitative and quantitative detection of flu-v is feasible using Maxwell 16 System in combination with rRT-PCR. But it differs in capacities of removing PCR inhibitors from different sample matrices, thereby having impacts on the sensitivity of rRT-PCR assay. A properly reduction of input volume of sample extracted with Maxwell 16 System for challenging matrices may improve detection.2) The Maxwell 16-S procedure is suitable for extraction of flu-v RNA from throat swab and BALF samples, and the Maxwell 16-M procedure is suitable for extraction from poultry fecal samples, while a more optimized Maxwell 16 procedure is needed to develop for extraction from human fecal matrix.3) Intact and non-decreased influenza virus RNA could obtained from samples preserved in lysis buffer underwent cyclic freeze-thaw or a long period of storage at refrigeration and frozen temperatures. However, lysis buffers different in methodology have not always the same ability to stabilize viral RNA.4) The lysis buffers of the three nucleic acid extraction methods could render high-titer influenza virus non-infectious according to the procedures recommended by the manufacturers. Thus the risk of biohazard exposure during NA extraction was significantly reduced after initial lysis of flu-v samples, which has implications for moving processing of flu-v samples within an automated extractor outside of a biosafety cabinet or under low containment laboratories, and for application of lysis buffer as stabilizing agent of flu-v samples during storage and transport.更多还原