【摘要】：背景:呼吸道病毒感染是在下呼吸道感染中引起嚴重發(fā)病率和死亡率的重要原因之一。等溫擴增檢測技術(shù)與傳統(tǒng)的分子診斷方法相比具有快速、簡單和成本低等優(yōu)勢,該技術(shù)越來越多地應用在病毒檢測領域。然而,這些方法在臨床應用中的系統(tǒng)評價很少有報道。方法:本研究通過檢索在MEDLINE(Pubmed)摘要中涉及到等溫擴增檢測技術(shù)和呼吸道病毒等主題詞和關鍵詞找到相關文獻。選擇目前已建立環(huán)介導等溫擴增技術(shù)(LAMP)檢測呼吸道合胞病毒A型(RSVA)和B型(RSVB),人偏肺病毒(HMPV),腺病毒(Adv)的方法和逆轉(zhuǎn)錄基因組指數(shù)擴增反應(RT-GEAR)檢測人鼻病毒(HRV)的方法,與基于Qiaxcel系統(tǒng)的雙管多重逆轉(zhuǎn)錄PCR測定法同時檢測進行比較。本研究通過對來自中國不同地區(qū)的634份肺炎兒童呼吸道臨床鼻咽吸出物樣本的檢測,對所選等溫擴增方法在省份、性別和年齡組之間的靈敏度差異進行系統(tǒng)性評價。等溫擴增方法和兩管測定法之間有差異的結(jié)果通過一代測序進行判斷分析。結(jié)果:本研究對來自河北省兒童醫(yī)院和湖南省疾病預防控制中心共634份呼吸道標本進行檢測。通過逆轉(zhuǎn)錄環(huán)介導等溫擴增方法(RT-LAMP)/LAMP/RT-GEAR檢測病毒的總檢出率(陽性樣品的數(shù)量/總樣品)為35.9%,而通過雙管多重逆轉(zhuǎn)錄PCR測定的檢測率為46.2%。等溫擴增檢測出RSV,HMPV,ADV和HRV的靈敏度分別為88.4%,74.3%,100%和73.6%。在等溫擴增檢測結(jié)果中沒有發(fā)現(xiàn)假陽性。測定結(jié)果中所有有差異的陰性結(jié)果通過sanger 一代測序確認為假陰性。LAMP法檢測ADV的實驗結(jié)果顯示與雙管多重PCR測定法完全一致。湖南省臨床樣本中RSV檢測靈敏度高于河北省(P=0.01)。在年齡組中,1歲以上兒童的RSV檢測靈敏度也高于1歲以下(P=0.01)。結(jié)論:檢測每種不同病毒的等溫擴增方法的臨床表現(xiàn)不同。系統(tǒng)性評價等溫擴增方法檢測,尤其是對RNA病毒的檢測,是評價其在臨床中是否能夠廣泛使用的關鍵。本研究中用于檢測ADV的LAMP法測定結(jié)果顯示是可靠的,在臨床中具有巨大的潛在用途,而RT-LAMP/RT-GEAR測定用于檢測RSV,HMPV和HRV的敏感性則需要進一步改善。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R725.6

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