An extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of Haemophilus influenzae.

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Yaling Cao, Junwen Wang, Zihao Fan, Ling Xu, Zhenzhen Pan, Yinkang Mo, Qiwen Yang, Jing Huang, Feng Ren
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引用次数: 0

Abstract

Timely and accurate diagnosis is essential for the effective treatment of Haemophilus influenzae (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. The aim of this study was to establish a one-pot detection device for HI DNA detection using CRISPR/Cas13a that would be applicable for POCT. We established a two-step polymerase chain reaction (PCR)-CRISPR assay, a two-step recombinase-aided amplification (RAA)-CRISPR assay, and a one-pot RAA-CRISPR assay. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct an extraction-free one-pot detection device, named EFORCA (extraction-free one-pot RAA-CRISPR/Cas13a assay). Validation was performed on 90 simulated samples and 77 clinical samples. The two-step PCR/RAA-CRISPR assay for HI DNA detection was established and optimized, with a detection limit of 1 copy/μL and 100% specificity. The sensitivity and specificity of the two-step PCR-CRISPR assay for the 90 simulated clinical samples were 96.7% and 95%, respectively, and for the 77 clinical samples, 97.5% and 100%, respectively. The one-pot RAA-CRISPR assay and the EFORCA detected 50 and 25 CFU/mL HI in bacterial suspensions within 30 min, respectively. The valuation results for 77 clinical samples demonstrated that the sensitivity of both methods was 95%, which was greater than the sensitivity of quantitative real-time PCR (qPCR) (92.5%) with the same specificity of 100%. We developed an extraction-free one-pot RAA-CRISPR/Cas13a assay for HI detection, which effectively performs a POCT test and is a valuable tool for the early detection and monitoring of HI infection.IMPORTANCETimely and accurate diagnosis is essential for the effective treatment of Haemophilus influenzae (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. In this study, sensitive two-step polymerase chain reaction (PCR)-CRISPR and recombinase-aided amplification (RAA)-CRISPR assays were developed, followed by the establishment of the one-pot RAA-CRISPR assay through integration of RAA, CRISPR, and rapid lysis for HI detection. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct the extraction-free one-pot RAA-CRISPR/Cas13a assay (EFORCA). With its high sensitivity, specificity, rapid turnaround time, and operational simplicity, this assay shows great potential as a practical diagnostic tool for HI infection in small laboratory settings.

一种用于流感嗜血杆菌即时检测的无萃取物、冻干单罐RAA-CRISPR检测方法。
及时和准确的诊断对于有效治疗流感嗜血杆菌感染至关重要。值得注意的是,诊断HI DNA的快速即时检测(POCT)方法仍然缺乏。本研究的目的是建立一种适用于POCT的CRISPR/Cas13a检测HI DNA的单锅检测装置。我们建立了两步聚合酶链反应(PCR)-CRISPR检测方法、两步重组酶辅助扩增(RAA)-CRISPR检测方法和一锅RAA-CRISPR检测方法。此外,结合试剂冻干、快速裂解技术和一锅检测装置,构建无提取一锅检测装置,命名为EFORCA(无提取一锅RAA-CRISPR/Cas13a assay)。对90个模拟样本和77个临床样本进行了验证。建立并优化了两步PCR/RAA-CRISPR检测HI DNA的方法,检测限为1拷贝/μL,特异性为100%。两步PCR-CRISPR检测对90份模拟临床样本的灵敏度和特异性分别为96.7%和95%,对77份临床样本的灵敏度和特异性分别为97.5%和100%。单锅RAA-CRISPR法和EFORCA法在30 min内分别检测到细菌悬液中50和25 CFU/mL的HI。对77份临床样本的评估结果表明,两种方法的灵敏度均为95%,均高于实时荧光定量PCR (qPCR)的灵敏度(92.5%),其特异性为100%。我们开发了一种无提取的单罐RAA-CRISPR/Cas13a检测HI的方法,该方法有效地执行POCT测试,是早期检测和监测HI感染的有价值的工具。重要意义及时准确的诊断对于流感嗜血杆菌感染的有效治疗至关重要。值得注意的是,诊断HI DNA的快速即时检测(POCT)方法仍然缺乏。本研究首先建立了灵敏的两步聚合酶链反应(PCR)-CRISPR和重组酶辅助扩增(RAA)-CRISPR检测方法,然后通过RAA、CRISPR和快速裂解相结合,建立了一锅RAA-CRISPR检测方法,用于HI检测。此外,结合试剂冻干、快速裂解技术和一罐检测装置构建无提取的一罐RAA-CRISPR/Cas13a检测(EFORCA)。由于其高灵敏度、特异性、快速周转时间和操作简便,该检测方法在小型实验室环境中显示出作为HI感染实用诊断工具的巨大潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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