本文關(guān)鍵詞：手足口病原核酸提取不同方法的比較研究　出處：《中國疾病預(yù)防控制中心》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 核酸提取 磁珠法 離心柱吸附法 自動化工作站

【摘要】：背景手足口病是一種由人腸道病毒引起的常見傳染病,其中主要病原體為腸道病毒71(HEV-71)和柯薩奇病毒A16 (CAV-16),世界大部分國家和地區(qū)均有此病流行的報道。手足口病有明顯的季節(jié)性特點,夏秋季多見；患者主要為學(xué)齡前兒童,尤其是5歲以下兒童。由于手足口病的傳染性強、流行強度大且傳播迅速,因此快速檢測腸道病毒對于控制傳染病具有重要意義。而手足口病快速分子生物學(xué)檢測,首先要面臨的問題就是病原體核酸提取。傳統(tǒng)的沉淀法和實驗室常用的離心柱吸附法,雖然可以提取病毒核酸,但是費時費力、有機溶劑可能對實驗人員造成傷害。而自動化的磁珠提取法具有省時省力、防止人員傷害、可實現(xiàn)大批量樣本同時處理的優(yōu)點。目的建立一種人腸道病毒的全自動磁珠提取方法,和離心柱吸附法相比,具有相當(dāng)?shù)撵`敏度和特異性。方法2014年3月-10月共收集河北省手足口輕癥病人的咽拭子或糞便樣品230份,患者均為2月-5歲的嬰幼兒。分別采用兩種磁珠法和離心柱吸附法提取核酸,用同一種逆轉(zhuǎn)錄實時熒光PCR法評價核酸提取效果。以離心柱吸附法的結(jié)果作為參考標(biāo)準(zhǔn),用SPSS 18.0分別計算兩種磁珠法的靈敏度、特異性和一致性。下文用“A”字母代表購自創(chuàng)智力合(北京)科技有限公司的磁珠法病毒DNA/RNA提取試劑盒(Magnetic Viral DNA/RNA Kit);用“B”字母代表購自常州金麥格生物技術(shù)有限公司的病毒DNA/RNA提取試劑盒(磁珠法)。結(jié)果230份樣品用于不同提取方法的比較研究。用離心柱吸附法提取核酸202份樣品檢測為陽性,其中124份為HEV-71陽性,78份為CAV-16。A磁珠法提取核酸后檢測的HEV-71、CAV-16病毒陽性樣品分別為118、74份；B磁珠法提取核酸后檢測的HEV-71、CAV-16陽性樣品分別為121、75份。與離心柱吸附法相比,A磁珠法提取腸道病毒核酸的靈敏度、特異性、符合率分別為95%、86%、94%；提取HEV-71核酸的靈敏度、特異性、符合率分別為95%、87%、94%；提取CAV-16核酸的靈敏度、特異性、符合率分別為96%、80%、95%；提取咽拭子樣品的靈敏度、特異性、符合率分別為95%、89%、95%；提取糞便樣品的靈敏度、特異性、符合率分別為95%、78%、93%。B磁珠法提取腸道病毒核酸的靈敏度、特異性、符合率分別為97%、89%、96%；提取HEV-71核酸的靈敏度、特異性、符合率分別為98%、91%、97%；提取CAV-16核酸的靈敏度、特異性、符合率分別為96%、80%、95%；提取咽拭子樣品的靈敏度、特異性、符合率分別為97%、89%、96%；提取糞便樣品的靈敏度、特異性、符合率分別為97%、89%、96%。A、B磁珠法提取核酸的效果無統(tǒng)計學(xué)差異。結(jié)論A、B兩種全自動磁珠法提取核酸的靈敏度、特異性良好,與離心柱吸附法相比,一致性良好。兩種磁珠法提取CAV-16的特異性略低,可能是因為CAV-16陽性樣品數(shù)偏少和自動化工作站的硬件設(shè)備的調(diào)試造成的,可通過優(yōu)化硬件系統(tǒng)和自動化程序和用更多的樣品驗證來提高特異性。磁珠法全自動提取核酸處理大批量樣本,不僅省時省力、大幅度提高工作效率、減少化學(xué)試劑對工作人員的影響,還可以節(jié)約大量成本,對于大規(guī)模流行病學(xué)調(diào)查和應(yīng)對突發(fā)疫情具有重要意義。
[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.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R512.5

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