![]() ![]() ![]() cholerae serogroups O1 and O139 ( Figure 1). RAA is a sensitive, rapid, low-cost isothermal amplification technique that can be completed within 15–30 min at 37 to 42☌ without using large, expensive instruments ( Shen et al., 2019 Wang et al., 2020).īased on the detection principles of CRISPR-Cas12a and RAA, we developed CARID (Cas12a-assisted rapid isothermal detection) for the rapid visual detection of toxigenic V. For double-stranded target DNA activators, the prerequisite for Cas12a to effectively cleave non-target DNA is identification of a short T-rich (5 ‘-TTTN-3’) protospacer-adjacent motif (PAM) in the target strand ( Zetsche et al., 2015 Chen et al., 2018), which provides theoretical guidance for the design of crRNA.īecause the detection ability of CRISPR-Cas systems relies on the template provided, it is usually combined with several amplification methods, such as recombinase polymerase amplification (RPA) ( Chen et al., 2018), PCR ( Li et al., 2018) and recombinase-aided amplification (RAA) ( Xiao et al., 2021). CRISPR-Cas12a systems can cleave single-stranded DNA (ssDNA) indiscriminately when bound to target sequences under the guidance of crRNA in vitro ( Chen et al., 2018) therefore, this principle has been used in the specific detection of pathogens ( Chen et al., 2018 Gootenberg et al., 2018 Li et al., 2018). CRISPR-Cas relies on crRNAs for sequence-specific detection and silencing of foreign nucleic acids, thereby protecting organisms from viruses and phages ( Barrangou et al., 2007 Al-Attar et al., 2011 Bhaya et al., 2011 Wiedenheft et al., 2012). ![]() Therefore, rapid and point-of-care testing methods are essential for timely cholera detection and control.ĬRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems are adaptive immune systems consisting of Cas effector proteins and CRISPR RNAs (crRNAs) that are widely distributed in archaea and bacteria ( Li and Du, 2011 Terns and Terns, 2011). While molecular methods such as polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) have accelerated the diagnosis process, they rely heavily on expensive instruments and require elaborate experimental conditions, which are often difficult to obtain in rural areas. cholerae can take three days or more and require extensive laboratory infrastructure and experienced staff ( Ramamurthy et al., 2020). However, traditional culture methods for isolation and identification of V. cholerae ( Harris et al., 2012 Chowdhury et al., 2017).Įarly detection and confirmation of cholera cases are critical for rapid implementation of interventions. To date, only the toxigenic serogroups O1 and O139 are known to have caused epidemics and pandemics of cholera, even though there are more than 200 serogroups of V. cholerae that carry ctxA and ctxB are referred to as toxigenic strains ( Bharati and Ganguly, 2011). cholerae, is encoded by ctxA and ctxB, and V. Cholera toxin (CT), an important pathogenic factor of V. Vibrio cholerae, the causative agent of cholera, can be transmitted through contaminated water and/or food ( Sack et al., 2004). It has been estimated that there are roughly 1.3 to 4.0 million cases of cholera annually, with 21,000 to 143,000 deaths worldwide ( Ali et al., 2015). cholerae, which makes it suitable for field responses to cholera.Ĭholera remains a threat to public health in many countries with poor sanitation or that are short of safe water ( Deen et al., 2020). In conclusion, CARID is a rapid, sensitive, economically efficient, and portable method for the detection of V. Simulated sample tests showed that CARID is suitable for complex samples. Multiple-CARID was also established for efficiency and economic considerations with an acceptable decrease in sensitivity. cholerae genomic DNA, which is comparable to that of polymerase chain reaction (PCR) and qPCR. The limit of detection of CARID was 20 copies/reaction of V. cholerae strains and 129 strains of other intestinal diarrheagenic bacteria with a 100% coincidence rate. ![]() The results can be determined by fluorescence signal and visualized by lateral flow dipstick. cholerae serogroups O1 and O139 by combining recombinase-aided amplification and CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). In this study, we developed a Cas12a-assisted rapid isothermal detection (CARID) system for the detection of toxigenic V. There is a growing demand for rapid, sensitive, field-deployable nucleic acid tests for cholera, which usually occurs in rural areas. ![]()
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