四种常见呼吸道病毒靶序列富集多重PCR-液相芯片检测方法的建立及初步应用

【作者】 王洁；

【导师】 李晓军； 王卫萍；

【作者基本信息】 南京师范大学 ， 微生物学， 2014， 硕士

【摘要】 呼吸道病毒是一组能够通过呼吸道传播引起呼吸系统及其他系统疾病的病原微生物。呼吸道病毒具有发病急、传染性强等特点,可造成局部暴发和世界大范围流行,严重影响人类健康。由于呼吸道致病微生物感染后的临床症状大多相似,缺乏特异的影像学表现,因此病原学证据在临床诊断和流行病监控中十分重要。但目前的检测方法,如病毒分离、核酸检测、病毒抗原检测和血清学检测等方法,尚不能满足高效、快速和高通量的要求。本研究选择甲型流感病毒、乙型流感病毒、呼吸道合胞病毒A和B是传播迅速和致病性的4种常见呼吸道病毒,亟需高效、快速、特异的诊断方法。目的：本研究将靶序列富集多重-聚合酶链反应(target enrich multiple polymerase chain reaction, Tem-PCR)和Luminex液相芯片(multiple analytes profiling, xMAP)技术相结合,建立一种可在单个反应体系中同时检测4种常见呼吸道病毒,包括甲型流感病毒(IFNA)、乙型流感病毒(IFNB)、呼吸道合胞病毒A (RSVA)和呼吸道合胞病毒B (RSVB)基因的快速分子检测方法。方法：1.重组质粒DNA的制备:IFNA、IFNB、RSVA和RSVB的基因组均为RNA,选择可代表上述4种呼吸道病原体的特征保守基因序列作为靶标病毒序列,根据RNA模板的碱基序列首先合成DNA片段,将合成的DNA片段克隆到pCRRⅡ载体,鉴定重组质粒中靶标序列正确性。2.建立并优化Tem-PCR技术,对IFNA、IFNB、RSVA和RSVB4种病毒基因进行同时扩增。3.建立可同时检测4种常见呼吸道病毒的液相芯片杂交反应体系,并评价其特异性、灵敏性及重复性。4.收集2013年3月至2014年3月在南京军区南京总医院儿科就诊的以急性上呼吸道感染为主要表现的门诊和住院患者咽拭子标本。提取病毒RNA备用。5.应用Tem-PCR联合液相芯片技术对上述咽拭子标本进行IFNA、IFNB、 RSVA和RSVB4种病毒基因检测,分别与单病毒基因荧光定量PCR检测方法及xTAG液相芯片法进行比对检测,比较两种方法的检测效能。结果：1.使用pCRR Ⅱ载体,构建包含IFNA、IFNB、RSVA和RSVB4种靶标病毒序列的重组质粒,经过测定和稀释,制备了100～105拷贝/μL浓度梯度的重组质粒DNA。2.建立了可特异性区分IFNA、IFNB、RSVA和RSVB4种呼吸道病毒的Tem-PCR联合液相芯片方法,检测数据表明该方法具备了较好的特异性和灵敏度(可达10拷贝/μL)。3.单病毒基因荧光定量PCR检测方法与上述建立的快速分子检测方法对临床样本进行检测,经配对计数的χ2检验两种方法的检测效能无显著性差异,同时进行一致性检验提示两种检测方法一致性较强且有统计学意义；同时选取部分标本采用xTAG液相芯片法检测,经统计分析显示两种检测方法一致性较好且有统计学意义。结论：本课题构建的基于Tem-PCR和液相芯片系统的分子检测方法可用于呼吸道感染患者四种常见呼吸道病毒(IFNA、IFNB、RSVA和RSVB)的快速甄别。更多还原

【Abstract】 Respiratory viruses are a series of pathogens,which can evoke respiratory infection diseases,and have been increasingly shown to play a role in the morbidity and mortality of elder or immunosuppressed patients. Besides, they are highly contagious in the populations and can easily cause world-wild pandemic. Because it’s very difficult to identify the specific kind of respiratory virus by clinical symptom or radiological indication, the test of pathogenic microorganisms is extremely important in disease diagnoses and supervise. However, traditional diagnostic methods have limitation in test sensitivity and feasibility in many contexts, and focus only on partial viral pathogens. Influenza A virus (INFA), influenza B virus (INFB), respiratory syncytial viruses types A and B are four kinds of important common respiratory RNA viruses, all of which are hard to detect. In this study, we tried to develop a novel molecular method to identify these viruses rapidly and accurately.Objective:The aim of the study was to establish a rapid molecular assay to detect four kinds of common respiratory viruses simultaneously by combining advanced Tem-PCR technique with xMAP method, and to evaluate the reliability and application of the novel assay in clinical diagnosis.Methods:1. Preparation of recombinant plasmid DNA:The genomes of Influenza A and B viruses, respiratory syncytial viruses types A and B were RNA. The conserved gene sequences represented the4respiratory pathogens were selected as the target sequences. DNA were firstly synthesized according to the nucleotide sequences of RNA templates, then the synthesized DNA fragments were cloned into PCRR Ⅱ vector and the accuracy of target sequences in plasmid were identified.2. Tem-PCR technique was built and optimized to amplify the sequences of the above viruses simultaneously.3. We establish and optimize the Luminex x-MAP system, which can simultaneously detect four common respiratory viruses. And we evaluate the specificity, sensitivity and repeatability.4. A total of150throat swabs from respiratory tract infection patients were collected from Nanjing General Hospital of Nanjing Military Command from March2013to March2014. The viral RNA were extracted for further experiment.5. Tem-PCR technique combined with Luminex x-TAG method were adopted to detect the viral sequences of IFNA, IFNB, RSVA and RSVB from the samples of above throat swabs. The results were compared with the single viral gene quantitative PCR or Luminex x-MAP method.Results:1. The use of pCRR Ⅱ vector, recombinant plasmid containing target viral sequence IFNA, IFNB,RSVA and RSVB four kinds of target viral sequence, through determination and dilution,100~105concentration gradient of DNA prepared.2. There was no cross-reactivity among the four respiratory viral target DNAs. The all of detection limits for the four viral DNAs was10copies.The method has good specificity and sensitivity.3. The clinical samples were detected by the single viral gene quantitative PCR assay and the novel built rapid test. No significant difference was observed by Chi-square test, whereas the consistency test result indicated that the two methods were consistent and with significant difference. Part samples were selected by xTAG assay, statistical analysis revealed that the two tests showed good consistency and significant difference.Conclusion:We establish a sensitive and specific detection system suitable for multiple respiratory viruses for clinical specimens.更多还原