Experimental Research Center, Capital Institute of Pediatrics, Beijing, China.
Department of Critical Medicine, Children's Hospital Affiliated Capital Institute of Pediatrics, Beijing, China.
Front Cell Infect Microbiol. 2023 Jul 27;13:1147142. doi: 10.3389/fcimb.2023.1147142. eCollection 2023.
(MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.
（MP）是所有年龄段人群发病和死亡的重要原因，尤其是在极端年龄的患者中。非常需要改进和易于获得的测试方法，以实现对 MP 感染的准确、敏感和快速诊断。在这里，我们在重组酶聚合酶扩增（RPA）的基础上开发了一种基于 CRISPR-Cas12b 的检测平台，用于快速、简单和准确地诊断 MP 感染，命名为 MP-RPA-CRISPR。该 RPA 用于在最佳反应温度 37°C 下扩增社区获得性呼吸窘迫综合征（CARDS）毒素基因的 MP 株。通过基于 CRISPR-Cas12b 的检测平台对产生的扩增子进行解码，该检测平台通过实时 PCR 系统和在蓝光下肉眼进行解释。MP-RPA-CRISPR 可以检测到低至 5 fg 的 MP 株基因组 DNA 模板，并具有高度的准确性，可区分 MP 株和非 MP 株，没有任何交叉反应。总共使用 96 份来自疑似呼吸道感染患者的支气管肺泡灌洗液（BALF）样本评估了 MP-RPA-CRISPR 检测方法的临床性能。结果，我们的检测方法准确地诊断了 45 个 MP 感染样本和 51 个非 MP 感染样本，并且 MP-RPA-CRISPR 的结果与微流控芯片技术一致。总之，我们的 MP-RPA-CRISPR 检测方法是一种简单、快速、便携和高度敏感的方法，可用于诊断 MP 感染，可作为临床、现场和资源有限地区的有前途的工具。
