Institute of Maternity and Child Medical Research, Shenzhen Maternity and Child Healthcare Hospital, Southern Medical University, No.2004 Hongli Road, Shenzhen, 518028, Guangdong, China.
Clinical Laboratory, Shenzhen Third People's Hospital, No.29 Bulan Road, Shenzhen, 518112, Guangdong, China.
BMC Infect Dis. 2019 Feb 4;19(1):108. doi: 10.1186/s12879-019-3744-6.
For definitive diagnosis of cryptococcal meningitis, Cryptococcus neoformans and/or C. gattii must be identified within cerebral spinal fluid from the patients. The traditional methods for detecting Cryptococcus spp. such as India ink staining and culture are not ideal. Although sensitive and specific enough, detection of cryptococcal antigen polysaccharide has a high dose hook effect. Therefore, the aim of this study was to introduce a new rapid and simple detection method of Cryptococcus neoformans and C. gattii in cerebral spinal fluid.
The lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assay was constructed to detect the specific DNA sequences of C. neoformans and C. gattii. The detection limit was evaluated using serial dilutions of C. neoformans and C. gattii genomic DNA. The specificity was assessed by excessive amount of other pathogens genomic DNA. The optimal detection time and amplification temperature were also analyzed. The diagnostic parameters were first calculated using 114 clinical specimens and then compared with that of other diagnostic method. A brief analysis and comparison of different DNA extraction methods was discussed, too.
The LF-RPA assay could detect 0.64 pg of genomic DNA of C. neoformans per reaction within 10 min and was highly specific for Cryptococcus spp.. The system could work well at a wide range of temperature from 25 to 45 °C. The overall sensitivity and specificity were 95.2 and 95.8% respectively. As amplification template for LF-RPA assay, both cell lysates and genomic DNA produce similar experimental results.
The LF-RPA system described here is shown to be a sensitive and specific method for the visible, rapid, and accurate detection of Cryptococcus spp. in cerebral spinal fluid and might be useful for clinical preliminary screening of cryptococcal meningitis.
为了明确诊断隐球菌性脑膜炎，必须从患者的脑脊液中鉴定出新型隐球菌和/或格特隐球菌。传统的检测隐球菌属的方法，如印度墨水染色和培养，并不理想。虽然检测隐球菌抗原多糖的方法足够敏感和特异，但存在高剂量钩状效应。因此，本研究旨在介绍一种新的快速、简便的检测脑脊液中新形隐球菌和格特隐球菌的方法。
构建了侧向流动条结合重组酶聚合酶扩增（LF-RPA）检测新型隐球菌和格特隐球菌的特异 DNA 序列。通过新型隐球菌和格特隐球菌基因组 DNA 的系列稀释来评估检测限。通过过量的其他病原体基因组 DNA 评估特异性。还分析了最佳检测时间和扩增温度。首先使用 114 份临床标本计算诊断参数，然后与其他诊断方法进行比较。还对不同 DNA 提取方法进行了简要分析和比较。
LF-RPA 检测反应中 0.64pg 的新型隐球菌基因组 DNA 仅需 10 分钟，对隐球菌属具有高度特异性。该系统在 25-45°C 的宽温度范围内工作良好。总的敏感性和特异性分别为 95.2%和 95.8%。作为 LF-RPA 检测的扩增模板，细胞裂解物和基因组 DNA 均可产生相似的实验结果。
这里描述的 LF-RPA 系统被证明是一种快速、准确、灵敏的检测脑脊液中隐球菌属的方法，可能有助于临床初步筛查隐球菌性脑膜炎。
