新生隐球菌(C.新生动物)/C.gattii可以很容易地侵入人体中枢神经系统并引起隐球菌性脑膜炎(CM)。这些真菌的临床死亡率极高,每年在全球范围内造成180,000多人死亡。目前,常见的临床鉴定方法有传统的培养方法和印度墨水染色法。此外,酶联免疫吸附测定(ELISA),聚合酶链反应(PCR),实时定量PCR检测系统(qPCR),质谱,和宏基因组下一代测序(mNGS)也已用于检测这些真菌。由于新型梭菌/C引起的脑膜炎的快速进展。gattii感染,迫切需要快速,敏感,和现场检测方法,以满足临床诊断。重组酶聚合酶扩增(RPA)是一种有前途的等温扩增技术,可以弥补上述技术的缺点,反应时间短,高特异性,灵敏度高,从而满足了对新型C.C.的现场检测的需求。Gattii.在我们的研究中,RPA-侧向流条(LFS)用于扩增胶囊相关基因,CAP64,C.neoformans/C.加蒂,并通过引入碱基错配对引物-探针设计进行优化,以获得用于临床测试的特异性和灵敏的引物-探针组合,并确定了26种临床常见病原体的检测系统的特异性。开发该系统以在37°C的等温温度下在20分钟内获得结果,检测下限低至10CFU/μL或1fg/μL。对从多中心多路复用器收集的487个临床样本进行了测试,以评估RPA-LFS系统的检测性能,这表明该系统可以特异性检测C.neoformans/C.加蒂,满足快速的需求,具体,和灵敏的检测。
Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans/C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans/C. gattii. In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64, of C. neoformans/C. gattii, and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/μL or 1 fg/μL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans/C. gattii, meeting the need for rapid, specific, and sensitive detection.