建立一个基于双 RPA 与 CRISPR/Cas12a 结合的平台,用于检测肺炎克雷伯氏菌及其 KPC 耐药基因。

IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Frontiers in Bioengineering and Biotechnology Pub Date : 2024-10-02 eCollection Date: 2024-01-01 DOI:10.3389/fbioe.2024.1447963
Meiying Tan, Xueli Yi, Chuan Liao, Zihan Zhou, Baoyan Ren, Lina Liang, Xuebin Li, Guijiang Wei
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引用次数: 0

摘要

耐碳酸培南肺炎克雷伯菌(CRKP)可导致严重的医院和社区获得性感染。CRKP 感染的治疗方法有限,导致住院时间延长和高昂的诊疗费用。KPC 基因型在 CRKP 中的检出率最高,其死亡率也高于 CRKP 的总体死亡率。然而,传统检测方法存在时间长、依赖复杂精密仪器等缺点,不利于快速筛查 CRKP。因此,本研究旨在建立一个早期筛查 CRKP 的检测平台,以便及时进行有效的抗菌治疗,防止 CRKP 的广泛传播。我们将双 RPA 与 CRISPR/Cas12a 相结合,建立了肺炎克菌(Kp)rcsA 特异基因和 KPC 耐药基因的双重检测平台。我们建立了四种结果读取方法,包括荧光检测法(FD)、蓝光照射检测法(BLID)、紫外照射检测法(UID)和侧流检测条(LFTS)。对于 rcsA 基因,荧光检测法的 LOD 为 1 × 10 pg/μL,其他三种方法可检测出 1 × 101 pg/μL 的细菌 DNA。至于 KPC 基因,四种读出方法都能检测出 1 × 102 pg/μL 的细菌 DNA。在检测的 59 株临床菌株中,与培养法相比,RPA-CRISPR/Cas12a 双重检测 rcsA 的灵敏度、特异性和准确性均为 100%。与药敏试验相比,RPA-CRISPR/Cas12a 双重检测 KPC 的灵敏度为 85.71%,特异度为 100%,准确度为 94.92%。总之,事实证明我们的 RPA-CRISPR/Cas12a 双平台快速、精确、方便,可在实验室或医疗点有效检测 Kp 与 KPC。
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Establishment of a platform based on dual RPA combined with CRISPR/Cas12a for the detection of Klebsiella pneumoniae and its KPC resistance gene.

Carbapenem resistant Klebsiella pneumoniae (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 K. pneumoniae (Kp) 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 × 101 pg/μL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 102 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 Kp with KPC in the laboratory or at the point of care.

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来源期刊
Frontiers in Bioengineering and Biotechnology
Frontiers in Bioengineering and Biotechnology Chemical Engineering-Bioengineering
CiteScore
8.30
自引率
5.30%
发文量
2270
审稿时长
12 weeks
期刊介绍: The translation of new discoveries in medicine to clinical routine has never been easy. During the second half of the last century, thanks to the progress in chemistry, biochemistry and pharmacology, we have seen the development and the application of a large number of drugs and devices aimed at the treatment of symptoms, blocking unwanted pathways and, in the case of infectious diseases, fighting the micro-organisms responsible. However, we are facing, today, a dramatic change in the therapeutic approach to pathologies and diseases. Indeed, the challenge of the present and the next decade is to fully restore the physiological status of the diseased organism and to completely regenerate tissue and organs when they are so seriously affected that treatments cannot be limited to the repression of symptoms or to the repair of damage. This is being made possible thanks to the major developments made in basic cell and molecular biology, including stem cell science, growth factor delivery, gene isolation and transfection, the advances in bioengineering and nanotechnology, including development of new biomaterials, biofabrication technologies and use of bioreactors, and the big improvements in diagnostic tools and imaging of cells, tissues and organs. In today`s world, an enhancement of communication between multidisciplinary experts, together with the promotion of joint projects and close collaborations among scientists, engineers, industry people, regulatory agencies and physicians are absolute requirements for the success of any attempt to develop and clinically apply a new biological therapy or an innovative device involving the collective use of biomaterials, cells and/or bioactive molecules. “Frontiers in Bioengineering and Biotechnology” aspires to be a forum for all people involved in the process by bridging the gap too often existing between a discovery in the basic sciences and its clinical application.
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