Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a

Abstract

PurposeClostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes.MethodsRecently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a.ResultsThe platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/μL and 1 copy/μL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection.ConclusionIn conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.

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

“…This study is an upgrade of the previously established C. difficile CRISPR diagnostic platform ( 24 ) based on the orthogonal Cas12a and Cas13a dual systems combined with multiplex isothermal amplification technology. It is expected to be an ideal C. difficile diagnostic platform with the following unique advantages.…”

“…Later, we transferred the amplicons to the Cas12a system for individual cleavage. This platform successfully detected C. difficile toxin genes using CRISPR technology ( 24 ). However, although the development of multiplex RPA can amplify double genes concurrently, the nonspecific cleavage activity of the CRISPR system makes it impossible to perform multiplex detection in one tube.…”

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a

“…Upon the activation of Cas12a by a specific target sequence, nonspecific shear is available for arbitrary single-stranded DNA. Current isothermal amplification approaches such as RPA and LAMP can be applied in conjunction with CRISPR-Cas12a for diverse in vitro diagnostics and bioassays such as COVID-19 ( Broughton et al 2020 ), Clostridioides difficile toxins ( Jiang et al 2023 ), carbap-enemase genes ( Xu et al 2022 ). This study combined the two techniques to establish a GII Norovirus detection method on the strength of RPA-Cas12a-lateral flow immunochromatographic strips.…”

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

“…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 …”