Tan Meiying, Yi Xueli, Liao Chuan, Zhou Zihan, Ren Baoyan, Liang Lina, Li Xuebin, Wei Guijiang
Center for Medical Laboratory Science, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China.
Baise Key Laboratory for Research and Development on Clinical Molecular Diagnosis for High-Incidence Diseases, Guangxi, China.
Front Bioeng Biotechnol. 2024 Oct 2;12:1447963. doi: 10.3389/fbioe.2024.1447963. eCollection 2024.
Carbapenem resistant (CRKP) can cause serious hospital- and community-acquired infections. Treatment for CRKP infection is limited, resulting in prolonged hospitalization and high consultation costs. The KPC genotype has the highest detection rate of CRKP, and its mortality rate is higher than the overall mortality rate of CRKP. However, traditional testing methods have disadvantages such as long time and reliance on complex and sophisticated instruments, which are not conducive to rapid screening for CRKP. Therefore, this study aimed to establish a detection platform for early screening of CRKP so that effective antimicrobial therapy could be administered promptly to prevent the widespread spread of CRKP. We integrated dual RPA with CRISPR/Cas12a to establish a dual platform for the detection of () rcsA-specific gene and KPC resistance gene. Four result reading methods were established, including fluorescence detection (FD), blue light irradiation detection (BLID), ultraviolet irradiation detection (UID), and lateral flow test strips (LFTS). For the rcsA gene, the LOD of FD was 1 × 10 pg/μL, and the other three methods could detect 1 × 10 pg/μL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 10 pg/μL of bacterial DNA. In 59 clinical strains tested, the dual RPA-CRISPR/Cas12a detection of the rcsA had 100% sensitivity, specificity, and accuracy compared to the culture method. Compared with the drug sensitivity test, the sensitivity of dual RPA-CRISPR/Cas12a detection for the KPC was 85.71%, the specificity was 100%, and the accuracy was 94.92%. In summary, our dual RPA-CRISPR/Cas12a platform proved to be rapid, precise, and convenient for the efficient detection of with KPC in the laboratory or at the point of care.
耐碳青霉烯类肺炎克雷伯菌（CRKP）可引起严重的医院获得性感染和社区获得性感染。CRKP感染的治疗方法有限，导致住院时间延长和会诊费用高昂。KPC基因型在CRKP中的检出率最高，其死亡率高于CRKP的总体死亡率。然而，传统检测方法存在耗时以及依赖复杂精密仪器等缺点，不利于对CRKP进行快速筛查。因此，本研究旨在建立一个用于CRKP早期筛查的检测平台，以便能够及时给予有效的抗菌治疗，防止CRKP广泛传播。我们将双RPA与CRISPR/Cas12a整合，建立了一个用于检测rcsA特异性基因和KPC耐药基因的双平台。建立了四种结果读取方法，包括荧光检测（FD）、蓝光照射检测（BLID）、紫外线照射检测（UID）和侧向流动试纸条（LFTS）。对于rcsA基因，FD的检测限为1×10 pg/μL，其他三种方法可检测到1×10 pg/μL的细菌DNA。至于KPC基因，四种结果读取方法均能够检测到1×10 pg/μL的细菌DNA。在59株临床菌株检测中，与培养法相比，双RPA-CRISPR/Cas12a对rcsA的检测灵敏度、特异性和准确性均为100%。与药敏试验相比，双RPA-CRISPR/Cas12a对KPC的检测灵敏度为85.71%，特异性为100%，准确性为94.92%。总之，我们的双RPA-CRISPR/Cas12a平台在实验室或床边护理中对KPC的高效检测方面被证明是快速、精确且便捷的。
