目的:引起隐球菌性脑膜炎的机会致病菌是新生隐球菌和加替隐球菌。目前,这种情况的临床检测方法包括培养,墨水染色,和隐球菌抗原检测。此外,酶联免疫吸附测定(ELISA),聚合酶链反应(PCR),实时定量PCR(qPCR)可用于隐球菌的检测。然而,这些方法无法实现即时检测(POCT);因此,迫切需要建立一个快速的,敏感,和有效的检测方法。
方法:重组酶聚合酶扩增(RPA)和成簇规则间隔短回文重复(CRISPR)技术是实现快速POCT的有效工具。在这项研究中,RPA与CRISPR-Cas12a联合建立了一种快速,敏感,以及隐球菌性脑膜炎的特异性检测方法。
结果:本研究包括RPA-Cas12a荧光检测和RPA-Cas12a免疫层析检测,可以在50分钟内进行。此外,检测限低至102拷贝/μL。有趣的是,所开发的方法显示出令人满意的特异性,并且与其他真菌和细菌没有交叉反应性。36个临床样本进行了测试,检测结果与使用常用临床培养方法获得的结果之间的一致性为100%。
结论:在这项研究中,基于CRISPR-Cas12a技术,开发了一种新型隐球菌和加替隐球菌种复合物的快速检测方法,其特点是高灵敏度和特异性,易用性,和成本效益,使其适合现场检测。
OBJECTIVE: The opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method.
METHODS: Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis.
RESULTS: This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 102 copies/μL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %.
CONCLUSIONS: In this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.