Establishment of a Novel Detection Platform for Clostridioides difficile Toxin Genes Based on Orthogonal CRISPR

Abstract

C. difficile is currently the primary causative agent of hospital-acquired antibiotic-induced diarrhea, and timely and accurate diagnosis is crucial for hospital-acquired infection control and epidemiological investigation. Here, a new method for the identification of C. difficile was developed based on the recently popular CRISPR technology, and an orthogonal CRISPR dual system was utilized for the simultaneous detection of toxin genes A and B.

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

“…( An et al, 2021 ), Neisseria gonorrhoeae ( Allan-Blitz et al, 2023 ) and Group B Streptococci (GBS; Li Z. et al, 2023 ). Moreover, the CRISPR-Cas13a technology was also utilized to identify multiple genes, such as toxin genes in Clostridioides difficile ( Jiang et al, 2023 ), the mecA and clfA genes in methicillin-resistant S. aureus (MRSA; Liu et al, 2023 ), the blaKPC gene in Enterobacterales ( Liang et al, 2023 ), the lcrV gene in Yersinia pestis ( Schultzhaus et al, 2021 ). Similarly, in this study, we established a one-tube and two-step RPA-Cas13a platform ( Figure 1 ) to detect the mexX gene from P. aeruginosa , with respectable sensitivity and reasonable specificity.…”

Multiplexed Detection Strategies for Biosensors Based on the CRISPR-Cas System

“…110−112 Lateral flow strips with two target lines were designed to accommodate the dual-channel readout CRISPR system. 86,113 Chen et al developed test strip based on CRISPR-Cas system to detect PCA3 and KLK3 simultaneously, to improve the efficiency of detecting prostate cancer. 91 Another strip for the detections of PCA3 and KLK3 was established by combining RT-RAA amplification with the CRISPR-Cas9 system.…”

“…Due to simple requirement of reaction condition control and signal readout, CRISPR-Cas system is suitable to develop point-of-care testing platform. ,− Lateral flow and microfluidic technology are suitable combined with the CRISPR-Cas system to construct POCT with multiplexed detection. Because of the advantages of tractability, portability, affordability and rapid response of paper-based test strips, it had been applied in nucleic acid multiplexed detections. − Lateral flow strips with two target lines were designed to accommodate the dual-channel readout CRISPR system. , Chen et al developed test strip based on CRISPR-Cas system to detect PCA3 and KLK3 simultaneously, to improve the efficiency of detecting prostate cancer . Another strip for the detections of PCA3 and KLK3 was established by combining RT-RAA amplification with the CRISPR-Cas9 system.…”

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS‐CoV‐2 dual gene detection

“…In fact, we recently used Cas12a/Cas13a to simultaneously detect bacterial dual pathogenicity genes with excellent results. 26 In this study, Cas12a/Cas13a double zymography was utilized to…”

“…In a sense, this does not apply to POCT due to the cost of the instruments designed, and it does not apply to a wide range of situations because it is inconvenient and requires consideration of the instrument's condition, power supply, and maintenance. In fact, we recently used Cas12a/Cas13a to simultaneously detect bacterial dual pathogenicity genes with excellent results 26 …”