核酸序列依赖扩增技术检测侵袭性曲霉菌感染的实验研究

发布时间：2018-07-28 20:27

【摘要】：目的：侵袭性曲霉菌感染（invasive aspergillosis，IA）是一种严重威胁免疫力低下病人生命的感染，早期准确的诊断和治疗是良好预后的关键。本研究首先建立核酸序列依赖扩增（nucleic acid sequence-basedamplification，，NASBA）检测曲霉菌的方法，再结合荧光定量PCR（qPCR）及GM试验应用于IA的临床诊断，确立IA的最佳实验室诊断策略。方法：首先在实验室内利用烟曲霉菌标准株（CMCC A1a）建立NASBA结合电泳检测曲霉菌的方法，并对其灵敏度、特异性进行了评估。然后对NASBA结合分子信标（MB）用于定量检测曲霉菌的可行性进行了探索。最后分别利用NASBA、荧光定量PCR及GM试验三种方法对80例IA高危病患的血液标本进行检测，计算三种诊断方法的各项性能指标，并对各种诊断方案进行比较，最终确定IA实验室诊断的最佳方案。结果：在实验室内成功建立NASBA检测曲霉菌的方法，其特异性良好，灵敏度达到了1Cfu；曲霉菌孢子量的对数与MB荧光信号达到设定阈值时的时间存在线性相关关系（y=-10.7x+81.6，r=0.9889）。 NASBA、qPCR及GM试验三种方法的灵敏度分别为76.47%、67.65%、52.94%，特异度分别为80.43%、89.13%、80.43%。联合诊断结果显示，NASBA与qPCR串联方案有最好的特异度（100%）及阳性预测值（100%）；NASBA与qPCR并联方案则最为灵敏（94.12%）。结论：NASBA结合MB可用于定量检测曲霉菌。NASBA用于诊断IA最为敏感，而qPCR则最为特异，GM试验的表现差于前两者。联合应用的诊断策略能够提高诊断效能，提高临床早期诊断IA的准确性。
[Abstract]:Objective: invasive aspergillus infection (invasive aspergillosis IA) is a serious threat to the life of patients with low immunity infection, early accurate diagnosis and treatment is the key to a good prognosis. In this study, a method for detecting Aspergillus by sequence-dependent amplification (nucleic acid sequence-amplified amplification (NASBA) was established, and then the best laboratory diagnostic strategy was established by using fluorescence quantitative PCR (qPCR) and GM test in the clinical diagnosis of IA. Methods: first of all, the standard strain of Aspergillus fumigatus (CMCC A1a) was used to establish a method for the detection of Aspergillus fumigatus by NASBA combined with electrophoretic electrophoresis, and its sensitivity and specificity were evaluated. Then the feasibility of quantitative detection of Aspergillus by NASBA combined with molecular beacon (MB) was explored. Finally, three methods, NASBA, fluorescent quantitative PCR and GM test, were used to detect the blood samples of 80 patients with high risk of IA, to calculate the performance indexes of the three diagnostic methods, and to compare the various diagnostic schemes. Finally, the best scheme for IA laboratory diagnosis was determined. Results: the method of detecting Aspergillus by NASBA was successfully established in laboratory. The specificity of the method was good and the sensitivity reached 1Cfu. There was a linear correlation between the logarithm of aspergillus spores and the time when the MB fluorescence signal reached the threshold (Y-10.7x 81.6rr = 0.9889). The sensitivity of NASBA qPCR and GM test was 76.477.65 and 52.94, respectively, and the specificity was 80.4389.133.The specificity of the three methods was 80.43 89. 13 and 80.43, respectively. The results of combined diagnosis showed that the best specificity (100%) and positive predictive value (100%) of NASBA and qPCR tandem protocol were the most sensitive (94.12%). Conclusion the ratio NASBA combined with MB can be used to detect aspergillus. NASBA is the most sensitive in the diagnosis of IA, while qPCR is the most specific for GM test than the former two. The combined diagnostic strategy can improve the diagnostic efficacy and improve the accuracy of early diagnosis of IA.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R519;R446

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