手足口病原核酸提取不同方法的比较研究

发布时间：2018-01-16 05:10

本文关键词：手足口病原核酸提取不同方法的比较研究　出处：《中国疾病预防控制中心》2015年硕士论文　论文类型：学位论文

【摘要】：背景手足口病是一种由人肠道病毒引起的常见传染病,其中主要病原体为肠道病毒71(HEV-71)和柯萨奇病毒A16 (CAV-16),世界大部分国家和地区均有此病流行的报道。手足口病有明显的季节性特点,夏秋季多见；患者主要为学龄前儿童,尤其是5岁以下儿童。由于手足口病的传染性强、流行强度大且传播迅速,因此快速检测肠道病毒对于控制传染病具有重要意义。而手足口病快速分子生物学检测,首先要面临的问题就是病原体核酸提取。传统的沉淀法和实验室常用的离心柱吸附法,虽然可以提取病毒核酸,但是费时费力、有机溶剂可能对实验人员造成伤害。而自动化的磁珠提取法具有省时省力、防止人员伤害、可实现大批量样本同时处理的优点。目的建立一种人肠道病毒的全自动磁珠提取方法,和离心柱吸附法相比,具有相当的灵敏度和特异性。方法2014年3月-10月共收集河北省手足口轻症病人的咽拭子或粪便样品230份,患者均为2月-5岁的婴幼儿。分别采用两种磁珠法和离心柱吸附法提取核酸,用同一种逆转录实时荧光PCR法评价核酸提取效果。以离心柱吸附法的结果作为参考标准,用SPSS 18.0分别计算两种磁珠法的灵敏度、特异性和一致性。下文用“A”字母代表购自创智力合(北京)科技有限公司的磁珠法病毒DNA/RNA提取试剂盒(Magnetic Viral DNA/RNA Kit);用“B”字母代表购自常州金麦格生物技术有限公司的病毒DNA/RNA提取试剂盒(磁珠法)。结果230份样品用于不同提取方法的比较研究。用离心柱吸附法提取核酸202份样品检测为阳性,其中124份为HEV-71阳性,78份为CAV-16。A磁珠法提取核酸后检测的HEV-71、CAV-16病毒阳性样品分别为118、74份；B磁珠法提取核酸后检测的HEV-71、CAV-16阳性样品分别为121、75份。与离心柱吸附法相比,A磁珠法提取肠道病毒核酸的灵敏度、特异性、符合率分别为95%、86%、94%；提取HEV-71核酸的灵敏度、特异性、符合率分别为95%、87%、94%；提取CAV-16核酸的灵敏度、特异性、符合率分别为96%、80%、95%；提取咽拭子样品的灵敏度、特异性、符合率分别为95%、89%、95%；提取粪便样品的灵敏度、特异性、符合率分别为95%、78%、93%。B磁珠法提取肠道病毒核酸的灵敏度、特异性、符合率分别为97%、89%、96%；提取HEV-71核酸的灵敏度、特异性、符合率分别为98%、91%、97%；提取CAV-16核酸的灵敏度、特异性、符合率分别为96%、80%、95%；提取咽拭子样品的灵敏度、特异性、符合率分别为97%、89%、96%；提取粪便样品的灵敏度、特异性、符合率分别为97%、89%、96%。A、B磁珠法提取核酸的效果无统计学差异。结论A、B两种全自动磁珠法提取核酸的灵敏度、特异性良好,与离心柱吸附法相比,一致性良好。两种磁珠法提取CAV-16的特异性略低,可能是因为CAV-16阳性样品数偏少和自动化工作站的硬件设备的调试造成的,可通过优化硬件系统和自动化程序和用更多的样品验证来提高特异性。磁珠法全自动提取核酸处理大批量样本,不仅省时省力、大幅度提高工作效率、减少化学试剂对工作人员的影响,还可以节约大量成本,对于大规模流行病学调查和应对突发疫情具有重要意义。
[Abstract]:The background of HFMD is a common infectious disease caused by enterovirus, which were the major pathogens of enterovirus 71 (HEV-71) and Coxsackie virus A16 (CAV-16), most of the countries and regions in the world have reported the disease epidemic. HFMD has obvious seasonal characteristics, summer and autumn to see more patients; mainly for Preschool children, especially children under 5 years old. Because of HFMD contagious, epidemic and spread rapidly, so the rapid detection of enteroviruses is important for the control of infectious diseases. The foot and mouth disease rapid molecular biological detection, extraction of the first problem is the traditional precipitation pathogen nucleic acid. The commonly used laboratory adsorption method and centrifugal column method, although can extract the viral nucleic acid, but time-consuming, organic solvents may cause harm to the staff of the experiment. And automatic bead extraction method has the advantages of saving time Force, prevent injuries, the advantages can be processed at the same time achieve the bulk sample. Automatic bead extraction method to establish a human intestinal virus, compared with adsorption column centrifugation method, with a sensitivity and specificity comparable. Methods -10 months of March 2014 were collected in Hebei province and young patients swabs or stool samples in 230, the patients were aged -5 years in February infants. By using two kinds of magnetic beads and the adsorption column centrifugation method for nucleic acid extraction, evaluation with the same kind of reverse transcriptase real-time fluorescence PCR method of nucleic acid extraction effect. The adsorption column centrifugation results as the reference standard, two kinds of magnetic sensitivity calculation method respectively using SPSS 18, specific and consistent with the letter "A" on behalf of the following purchase of own intelligence (Beijing) and magnetic beads method virus DNA/RNA science and technology limited company Kit (Magnetic Viral DNA/RNA Kit); with the letter "B" on behalf of often purchased from State gold MEG Biotechnology Co. Ltd. virus DNA/RNA Extraction Kit (MACS). Results 230 samples for comparison of different extraction methods. The adsorption by centrifugal column method for extracting nucleic acid of 202 samples tested positive, 124 of them were positive for HEV-71, 78 for CAV-16.A magnetic beads method for extracting nucleic acid detection HEV-71 CAV-16, the virus positive samples were 118,74; nucleic acid extraction B magnetic beads method after the detection of HEV-71, CAV-16 positive samples were 121,75. Compared with the adsorption column centrifugation method, extraction sensitivity, enterovirus A magnetic beads method specificity, coincidence rate was 95%, 86%, 94%; the sensitivity of HEV-71 nucleic acid extraction. The specificity and accuracy were 95%, 87%, 94%; extraction of CAV-16 nucleic acid sensitivity, specificity and accuracy were 96%, 80%, 95%; extraction sensitivity, swab sample specificity and coincidence rate was 95%, 89%, 95%;. The sensitivity, specificity and fecal samples, the coincidence rate were 95%, 78%, extraction sensitivity, enterovirus 93%.B magnetic beads method specificity, coincidence rate was 97%, 89%, 96%; extraction of HEV-71 nucleic acid sensitivity, specificity and accuracy were 98%, 91%, 97%; extraction of CAV-16 nucleic acid sensitivity. The specificity and accuracy were 96%, 80%, 95%; extraction sensitivity, swab sample specificity and coincidence rate was 97%, 89%, 96%; the extraction of fecal samples sensitivity, specificity, accuracy were 97%, 89%, 96%.A, nucleic acid extraction effect of B magnetic beads method showed no significant difference conclusion. A, nucleic acid extraction sensitivity of B two automatic magnetic beads method, good specificity, compared with adsorption centrifugal column method, good consistency. Two kinds of beads extraction of specific CAV-16 slightly lower, probably because of fewer CAV-16 positive samples and automation workstation hardware Debugging cause, through the optimization of hardware system and automation program and sample validation more to improve specificity. Automatic extraction of nucleic acid processing of large quantities of samples by magnetic bead, not only save time, improve work efficiency, reduce the chemical reagents on the staff's influence, but also save a lot of cost, is of great significance for the large-scale epidemiological investigation and emergency response to the epidemic.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R512.5

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