CRISPR/Cas12a Technology Combined with RT-ERA for Rapid and Portable SARS-CoV-2 Detection.
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ABSTRACT:
SUBMITTER: Liu S
PROVIDER: S-EPMC8169787 | biostudies-literature |
REPOSITORIES: biostudies-literature
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| S-EPMC10269670 | biostudies-literature
Project description:The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.
| S-EPMC7737895 | biostudies-literature
Project description:African swine fever virus (ASFV), the aetiological agent of African swine fever (ASF), causes lethal haemorrhagic fever in domestic pigs with high mortality and morbidity and has devastating consequences on the global swine industry. On-site rapid and sensitive detection of ASFV is key to the timely implementation of control. In this study, we developed a rapid, sensitive and instrument-free ASFV detection method based on CRISPR/Cas12a technology and lateral flow detection (named CRISPR/Cas12a-LFD). The limit of detection of CRISPR/Cas12a-LFD is 20 copies of ASFV genomic DNA per reaction, and the detection process can be completed in an hour. The assay showed no cross-reactivity with other swine DNA viruses, and has 100% agreement with real-time PCR detection of ASFV in 149 clinical samples. Overall, the CRISPR/Cas12a-LFD method provides a novel alternative for the portable, simple, sensitive, and specific detection of ASFV and may contribute to the prevention and control of ASF outbreaks.
| S-EPMC7012833 | biostudies-literature
Project description:The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.
| S-EPMC9769947 | biostudies-literature
Project description:BackgroundCOVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.MethodsWe tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.ResultsThe OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.ConclusionsThe OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.
| S-EPMC7884969 | biostudies-literature
Project description:Extended community testing constitutes one of the main strategic pillars in controlling the COVID-19 pandemic. Reverse transcription PCR (RT-PCR) targeting the SARS-CoV-2 genome on nasopharyngeal swab samples is currently the reference test. While displaying excellent analytical sensitivity and specificity, this test is costly, often requires a substantial turnaround time, and, more importantly, is subject to reagent and other material shortages. To complement this technology, rapid antigen tests have been developed and made available worldwide, allowing cheap, quick, and decentralized SARS-CoV-2 testing. The main drawback of these tests is the reduced sensitivity when RT-PCR is the gold standard. In this study, we evaluate Visby an innovative, portable, easy-to-use RT-PCR point-of-care (POC) diagnostic device. Our retrospective analysis shows that overall, compared to the Cobas 6800 RT-qPCR assay (Roche), this RT-PCR POC technology detects SARS-CoV-2 RNA with 95% sensitivity (95%CI = 86.3-99%) and 100% specificity (95% CI = 80.5-100%). For samples with cycle-threshold values below 31, we observed 100% sensitivity (95% CI = 66.4-100%). While showing an analytical sensitivity slightly below that of a standard RT-qPCR system, the evaluated Visby RT-PCR POC device may prove to be an interesting diagnostic alternative in the COVID-19 pandemic, potentially combining the practical advantages of rapid antigen tests and the robust analytical performances of nucleic acid detection systems.
| S-EPMC8146445 | biostudies-literature
Project description:Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which is time-consuming and requires expensive instrumentation, and the confirmation of variants relies on further sequencing techniques. Herein, we first proposed a robust technique-methodology of electrochemical CRISPR sensing with the advantages of rapid, highly sensitivity and specificity for the detection of SARS-CoV-2 variant. To enhance the sensing capability, gold electrodes are uniformly decorated with electro-deposited gold nanoparticles. Using DNA template identical to SARS-CoV-2 Delta spike gene sequence as model, our biosensor exhibits excellent analytical detection limit (50 fM) and high linearity (R2 = 0.987) over six orders of magnitude dynamic range from 100 fM to 10 nM without any nucleic-acid-amplification assays. The detection can be completed within 1 h with high stability and specificity which benefits from the CRISPR-Cas system. Furthermore, based on the wireless micro-electrochemical platform, the proposed biosensor reveals promising application ability in point-of-care testing.
| S-EPMC9352833 | biostudies-literature
Project description:BackgroundThe newly emerged SARS-CoV-2 variant of concern (VOC) Omicron is spreading quickly worldwide, which manifests an urgent need of simple and rapid assay to detect and diagnose Omicron infection and track its spread.MethodsTo design allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of Omicron variant, and to develop a CRISPR-Cas12a-based assay to specifically detect Omicron variant.ResultsOur system showed a low limit of detection of 2 copies per reaction for the plasmid DNA of Omicron variant, and could readily detect Omicron variant in 5 laboratory-confirmed clinical samples and distinguish them from 57 SARS-CoV-2 positive clinical samples (4 virus isolates and 53 oropharyngeal swab specimens) infected with wild-type (N = 8) and the variants of Alpha (N = 17), Beta (N = 17) and Delta (N = 15). The testing results could be measured by fluorescent detector or judged by naked eyes. In addition, no cross-reaction was observed when detecting 16 clinical samples infected with 9 common respiratory pathogens.ConclusionsThe rapid assay could be easily set up in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests and implemented routinely in resource-limited settings to monitor and track the spread of Omicron variant.
| S-EPMC8849905 | biostudies-literature
Project description:BackgroundThe emergence of novel variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands fast and reliable detection of such variants in local populations.MethodsHere we present a cost-efficient and fast workflow combining a prescreening of SARS-CoV-2-positive samples using reverse transcription polymerase chain reaction melting curve analysis with multiplexed IP-RP-HPLC-based single nucleotide primer extensions.ResultsThe entire workflow from positive SARS-CoV-2 testing to base-specific identification of variants requires about 24 hours.ConclusionsWe applied the sensitive method to monitor local variant of concern outbreaks in SARS-CoV-2-positive samples collected in a confined region of Germany.
| S-EPMC8364759 | biostudies-literature
Project description:Rapid point-of-care tests for infectious diseases are essential, especially in pandemic conditions. We have developed a point-of-care electromechanical device to detect SARS-CoV-2 viral RNA using the reverse-transcription loop-mediated isothermal amplification (RT-LAMP) principle. The developed device can detect SARS-CoV-2 viral RNA down to 103 copies/mL and from a low amount of sample volumes (2 μL) in less than an hour of standalone operation without the need for professional labor and equipment. Integrated Peltier elements in the device keep the sample at a constant temperature, and an integrated camera allows automated monitoring of LAMP reaction in a stirring sample by using colorimetric analysis of unfocused sample images in the hue/saturation/value color space. This palm-fitting, portable and low-cost device does not require a fully focused sample image for analysis, and the operation could be stopped automatically through image analysis when the positive test results are obtained. Hence, viral infections can be detected with the portable device produced without the need for long, expensive, and labor-intensive tests and equipment, which can make the viral tests disseminated at the point-of-care.
| S-EPMC9733968 | biostudies-literature