核酸序列依赖扩增技术检测隐球菌的方法建立和评价

Development and evaluation of nucleic acid sequence-based amplification for detection of Cryptococcus neoformans

目的:建立核酸序列依赖扩增技术(NASBA)检测隐球菌RNA的方法,提高隐球菌感染诊断的准确性。方法:设计一对具有隐球菌属特异性的引物,构建NASBA扩增隐球菌RNA的方法并评价该方法的灵敏度和特异性。按欧洲癌症研究和治疗组织/侵袭性真菌感染协作组和美国变态反应和感染病研究院真菌病研究组(EORTC/MSG)2008年修订的侵袭性真菌病定义标准收集隐球菌感染的阳性病例和排除隐球菌感染的阴性病例,分别应用隐球菌荚膜多糖抗原检测(胶体金法)、PCR及本研究建立的NASBA方法进行检测。应用配对诊断试验设计、McNemar检验分别对3种方法进行比较以评价其诊断效能。结果:NASBA产物电泳结果显示仅新型隐球菌RNA扩增产物出现了特异性条带,而其他对照菌(烟曲霉、串珠镰刀菌、金黄色葡萄球、铜绿假单胞菌、大肠埃希菌、白色念珠菌、近平滑念珠菌)均没有出现电泳条带;NASBA结合电泳检测结果表明该方法灵敏度可达10 cfu/mL。NASBA、荚膜多糖抗原胶体金法及PCR灵敏度分别是87.5%(95%CI=66.53%~96.71%)、100%(95%CI=82.83%~100%)、66.67%(95%CI=44.69%~83.57%),特异性分别是94.59%(95%CI=80.47%~99.06%)、81.08%(95%CI=64.29%~91.44%)、78.37%(95%CI=61.34%~81.58%)。结论:应用NASBA检测隐球菌RNA具有快速、准确高、操作简便的优点。并且整个实验过程无须特殊仪器,适合在普通的临床实验室开展,有望成为新的隐球菌感染的常规诊断方法。

Objective:To develop a nucleic acid sequence-based amplification(NASBA)protocol for detecting Cryptococcus neoformans RNA and improving the accuracy of cryptococcal infection diagnosis.Methods:A pair of primers for Cryptococcus neoformans were designed to develop the method for amplifying Cryptococcus neoformans RNA by NASBA,and the sensitivity and specificity of this method were evaluated.By using the 2008 revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group(EORTC/MSG),the positive cases of cryptococcal infection were enrolled and the negative cases were excluded.Cryptococcal capsular polysaccharide antigen test(colloidal gold method),common polymerase chain reaction(PCR),and NASBA method developed in this study were applied for detection.Three methods were compared by paired design for diagnostic test and McNemar’s test to evaluate the diagnostic efficacy.Results:The electrophoresis results of NASBA products showed that only specific bands of Cryptococcus neoformans RNA amplification products appeared,while no electrophoretic bands of other control bacteria(Aspergillus fumigatus,Fusarium moniliforme,Staphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli,Candida albicans,and Candida parapsilosis)appeared.The sensitivity test of NASBA combined with electrophoresis showed that the detection limit of the method was as low as 10 cfu/mL.The sensitivities of NASBA,capsular polysaccharide antigen colloidal gold method,and PCR were 87.5%(95%CI=66.53%-96.71%),100%(95%CI=82.83%-100%),and 66.67%(95%CI=44.69%-83.57%),respectively;the specificities were 94.59%(95%CI=80.47%-99.06%),81.08%(95%CI=64.29%-91.44%),and 78.37%(95%CI=61.34%-81.58%),respectively.Conclusion:The application of NASBA to detect cryptococcal RNA is fast,accurate,and easy to use.The whole process does not require special instruments,which makes it suitable for application in general clinical laboratories and likely become a new routine diagnostic method for cryptococcal infection.