等温扩增技术在儿童肺炎中四种常见呼吸道病毒检测中的临床应用与评价
发布时间：2018-11-21 11:24
【摘要】：背景:呼吸道病毒感染是在下呼吸道感染中引起严重发病率和死亡率的重要原因之一。等温扩增检测技术与传统的分子诊断方法相比具有快速、简单和成本低等优势,该技术越来越多地应用在病毒检测领域。然而,这些方法在临床应用中的系统评价很少有报道。方法:本研究通过检索在MEDLINE(Pubmed)摘要中涉及到等温扩增检测技术和呼吸道病毒等主题词和关键词找到相关文献。选择目前已建立环介导等温扩增技术(LAMP)检测呼吸道合胞病毒A型(RSVA)和B型(RSVB),人偏肺病毒(HMPV),腺病毒(Adv)的方法和逆转录基因组指数扩增反应(RT-GEAR)检测人鼻病毒(HRV)的方法,与基于Qiaxcel系统的双管多重逆转录PCR测定法同时检测进行比较。本研究通过对来自中国不同地区的634份肺炎儿童呼吸道临床鼻咽吸出物样本的检测,对所选等温扩增方法在省份、性别和年龄组之间的灵敏度差异进行系统性评价。等温扩增方法和两管测定法之间有差异的结果通过一代测序进行判断分析。结果:本研究对来自河北省儿童医院和湖南省疾病预防控制中心共634份呼吸道标本进行检测。通过逆转录环介导等温扩增方法(RT-LAMP)/LAMP/RT-GEAR检测病毒的总检出率(阳性样品的数量/总样品)为35.9%,而通过双管多重逆转录PCR测定的检测率为46.2%。等温扩增检测出RSV,HMPV,ADV和HRV的灵敏度分别为88.4%,74.3%,100%和73.6%。在等温扩增检测结果中没有发现假阳性。测定结果中所有有差异的阴性结果通过sanger 一代测序确认为假阴性。LAMP法检测ADV的实验结果显示与双管多重PCR测定法完全一致。湖南省临床样本中RSV检测灵敏度高于河北省(P=0.01)。在年龄组中,1岁以上儿童的RSV检测灵敏度也高于1岁以下(P=0.01)。结论:检测每种不同病毒的等温扩增方法的临床表现不同。系统性评价等温扩增方法检测,尤其是对RNA病毒的检测,是评价其在临床中是否能够广泛使用的关键。本研究中用于检测ADV的LAMP法测定结果显示是可靠的,在临床中具有巨大的潜在用途,而RT-LAMP/RT-GEAR测定用于检测RSV,HMPV和HRV的敏感性则需要进一步改善。
[Abstract]:Background: respiratory tract virus infection is an important cause of severe morbidity and mortality in lower respiratory tract infections. Compared with traditional molecular diagnostic methods, isothermal amplification technique has the advantages of rapidity, simplicity and low cost. It is more and more widely used in the field of virus detection. However, the systematic evaluation of these methods in clinical application is rarely reported. Methods: in this study, the relevant literature was found by retrieving the subject words and keywords of isothermal amplification technique and respiratory tract virus in MEDLINE (Pubmed) abstract. Detection of respiratory syncytial virus type A (RSVA) and B type (RSVB), human metapulmonary virus (HMPV),) by ring mediated isothermal amplification (LAMP) has been established. The methods of adenovirus (Adv) and reverse transcriptase genomic index amplification (RT-GEAR) for detection of human rhinovirus (HRV) were compared with double-tube multiplex PCR assay based on Qiaxcel system. In this study, 634 samples of clinical nasopharyngeal aspirates from children with pneumonia from different regions of China were detected to evaluate the sensitivity differences among provinces, sex and age groups by the method of isothermal amplification. The results of isothermal amplification and two tube determination were analyzed by first generation sequencing. Results: 634 respiratory tract samples from Hebei Children's Hospital and Hunan Center for Disease Control and Prevention were detected. The total positive rate (the number of positive samples / total sample) was 35.9by reverse transcription-mediated isothermal amplification (RT-LAMP) / LAMP/RT-GEAR, while the detection rate by double-tube multiplex reverse transcription PCR was 46.2%. The sensitivity of RSV,HMPV,ADV and HRV by isothermal amplification was 88.4% and 73.6%, respectively. No false positive results were found in isothermal amplification. All the different negative results were confirmed as false negative by sanger generation sequencing. The results of ADV detection by LAMP method were consistent with that of double tube multiplex PCR assay. The sensitivity of RSV detection in clinical samples of Hunan Province was higher than that in Hebei Province (P0. 01). In the age group, the sensitivity of RSV in children over 1 year old was also higher than that in children under 1 year old (P0. 01). Conclusion: the clinical manifestations of isothermal amplification methods for detecting different viruses are different. Systematic evaluation of isothermal amplification, especially the detection of RNA virus, is the key to evaluate whether it can be widely used in clinical practice. The results of LAMP method for detecting ADV in this study show that it is reliable and has great potential use in clinic, but the sensitivity of RT-LAMP/RT-GEAR for detecting RSV,HMPV and HRV needs to be further improved.
【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】：2017
【分类号】：R725.6
本文编号：2346821
[Abstract]:Background: respiratory tract virus infection is an important cause of severe morbidity and mortality in lower respiratory tract infections. Compared with traditional molecular diagnostic methods, isothermal amplification technique has the advantages of rapidity, simplicity and low cost. It is more and more widely used in the field of virus detection. However, the systematic evaluation of these methods in clinical application is rarely reported. Methods: in this study, the relevant literature was found by retrieving the subject words and keywords of isothermal amplification technique and respiratory tract virus in MEDLINE (Pubmed) abstract. Detection of respiratory syncytial virus type A (RSVA) and B type (RSVB), human metapulmonary virus (HMPV),) by ring mediated isothermal amplification (LAMP) has been established. The methods of adenovirus (Adv) and reverse transcriptase genomic index amplification (RT-GEAR) for detection of human rhinovirus (HRV) were compared with double-tube multiplex PCR assay based on Qiaxcel system. In this study, 634 samples of clinical nasopharyngeal aspirates from children with pneumonia from different regions of China were detected to evaluate the sensitivity differences among provinces, sex and age groups by the method of isothermal amplification. The results of isothermal amplification and two tube determination were analyzed by first generation sequencing. Results: 634 respiratory tract samples from Hebei Children's Hospital and Hunan Center for Disease Control and Prevention were detected. The total positive rate (the number of positive samples / total sample) was 35.9by reverse transcription-mediated isothermal amplification (RT-LAMP) / LAMP/RT-GEAR, while the detection rate by double-tube multiplex reverse transcription PCR was 46.2%. The sensitivity of RSV,HMPV,ADV and HRV by isothermal amplification was 88.4% and 73.6%, respectively. No false positive results were found in isothermal amplification. All the different negative results were confirmed as false negative by sanger generation sequencing. The results of ADV detection by LAMP method were consistent with that of double tube multiplex PCR assay. The sensitivity of RSV detection in clinical samples of Hunan Province was higher than that in Hebei Province (P0. 01). In the age group, the sensitivity of RSV in children over 1 year old was also higher than that in children under 1 year old (P0. 01). Conclusion: the clinical manifestations of isothermal amplification methods for detecting different viruses are different. Systematic evaluation of isothermal amplification, especially the detection of RNA virus, is the key to evaluate whether it can be widely used in clinical practice. The results of LAMP method for detecting ADV in this study show that it is reliable and has great potential use in clinic, but the sensitivity of RT-LAMP/RT-GEAR for detecting RSV,HMPV and HRV needs to be further improved.
【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】：2017
【分类号】：R725.6
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