Methodological Evaluation of Carbapenemase Detection by Different Methods.

The global proliferation of carbapenemase-producing bacteria (CPB) has garnered significant attention worldwide. Early diagnosis of CPB and accurate identification of carbapenemases are crucial for preventing the spread of CPB and ensuring targeted antibiotic therapy. Therefore, efficient and accurate identification of carbapenemases is paramount in clinically treating diseases associated with CPB. In this study, 58 CPB strains were collected and detected using the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, a rapid detection platform based on CRISPR-Cas12a gene editing and isothermal amplification. Additionally, four conventional methods (the APB/EDTA method, PCR, NG-test Carba 5, and GeneXpert Carba-R) were employed and compared against whole genome sequencing (WGS) results, considered the gold standard, to evaluate their efficacy in detecting carbapenemases. Detection by the APB/EDTA method revealed that 29 strains were positive for Class A serine endopeptidases, while 29 strains were positive for Class B metalloenzymes. The classification of these zymotypes was consistent with the sequencing result. All target carbapenemases for KPC were identified with 100% sensitivity using NG-test Carba 5, PCR, DETECTR, and GeneXpert Carba-R. In the case of NDM, both Xpert Carba-R and DETECTR showed a sensitivity of 100%. In contrast, NG-test Carba 5 and PCR had a slightly lower sensitivity of 96.7%, each missing one target carbapenemase. n this study, the APB/EDTA method is capable of identifying the zymotype classification but not the specific resistant genes, while Xpert Carba-R and DETECTR are able to detect all target carbapenemases.

A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for Vibrio vulnificus Detection.

Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40-60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.

Convergence of Surface-Enhanced Raman Scattering with Molecular Diagnostics: A Perspective on Future Directions.

Molecular diagnostics (MD) is widely employed in multiple scientific disciplines, such as oncology, pathogen detection, forensic investigations, and the pharmaceutical industry. Techniques such as polymerase chain reaction (PCR) revolutionized the rapid and accurate identification of nucleic acids (DNA, RNA). More recently, CRISPR and its CRISPR-associated protein (Cas) have been a ground-breaking discovery that is the latest revolution in molecular biology, including MD. Surface-enhanced Raman scattering (SERS) is a very attractive alternative to fluorescence as the currently most widely used optical readout in MD. In this Perspective, milestones in the development of MD, SERS-PCR, and next-generation approaches to MD, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), are briefly summarized. Our perspective on the future convergence of SERS with MD is focused on SERS-based CRISPR/Cas (SERS-CRISPR) since we anticipate many promising applications in this rapidly emerging field. We predict that major future developments will exploit the advantages of real-time monitoring with the superior brightness, photostability, and spectral multiplexing potential of SERS nanotags in an automated workflow for rapid assays under isothermal, amplification-free conditions.

ISOTHERMAL RECOMBINANT POLYMERASE AMPLIFICATION AND CRIPSR(CAS12A) ASSAY DETECTION OF RENIBACTERIUM SALMONINARUM AS AN EXAMPLE FOR WILDLIFE PATHOGEN DETECTION IN ENVIRONMENTAL DNA SAMPLES.

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon (Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum, is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20-40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection.

In the realm of pathogen detection, isothermal amplification technology has emerged as a swift, precise, and sensitive alternative to conventional PCR. This paper explores the fundamental principles of recombinase polymerase amplification (RPA) and recombinase-aid amplification (RAA) and reviews the current status of integrating the CRISPR-Cas system with RPA/RAA techniques. Furthermore, this paper explores the confluence of isothermal amplification and CRISPR-Cas technology, providing a comprehensive review and enhancements of existing combined methodologies such as SHERLOCK and DETECTR. We investigate the practical applications of RPA/RAA in conjunction with CRISPR-Cas for pathogen detection, highlighting how this integrated approach significantly advances both research and clinical implementation in the field. This paper aims to provide readers with a concise understanding of the fusion of RPA/RAA and CRISPR-Cas technology, offering insights into their clinical utility, ongoing enhancements, and the promising prospects of this integrated approach in pathogen detection.

A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V.

The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 102 copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR.

CRISPR-Cas Systems: Programmable Nuclease Revolutionizing the Molecular Diagnosis.

CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.

Point-of-Care Testing for Infectious Diseases Based on Class 2 CRISPR/Cas Technology.

The early detection of infectious diseases and microorganisms is critical for effective disease treatment, control, and prevention. Currently, nucleic acid testing and antigen-antibody serum reaction are the two methods most commonly used for the detection of infectious diseases. The former is highly accurate, specific, and sensitive, but it is time-consuming, expensive, and has special technician and instrument requirements. The latter is rapid and economical, but it may not be accurate and sensitive enough. Therefore, it is necessary to develop a quick and on-site diagnostic test for point-of-care testing (POCT) to enable the clinical detection of infectious diseases that is accurate, sensitive, convenient, cheap, and portable. Here, CRISPR/Cas-based detection methods are detailed and discussed in depth. The powerful capacity of these methods will facilitate the development of diagnostic tools for POCT, though they still have some limitations. This review explores and highlights POCT based on the class 2 CRISPR/Cas assay, such as Cas12 and Cas13 proteins, for the detection of infectious diseases. We also provide an outlook on perspectives, multi-application scenarios, clinical applications, and limitations for POCT based on class 2 CRISPR/Cas technology.

Variety Differentiation: Development of a CRISPR DETECTR Method for the Detection of Single Nucleotide Polymorphisms (SNPs) in Cacao (Theobroma cacao) and Almonds (Prunus dulcis).

To prevent food fraud, products can be monitored by various chemical-analytical techniques. In this study, we present a CRISPR-Cpf1 DETECTR-based assay for the differentiation of plant ingredients in sweet confectionary like fine and bulk-cocoa, or bitter and sweet almonds. To enable rapid in-field analysis, the trans-cleavage activity of the Cpf1 enzyme was used to develop a DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay for simple, highly specific fluorometric detection of single nucleotide polymorphisms (SNPs). The endonuclease Cpf1 requires the protospacer adjacent motif (PAM) 5'-TTTV-3' for activation, but the recognition sequence is freely programmable. The SNPs were selected to alter the Cpf1 specific PAM sequence. As a result, sequences that do not carry the canonical PAM sequence are not detected and thus not cut. The optimized system was used for both raw material and processed products such as cocoa masses or marzipan with a limit of detection of 3 ng template DNA. In addition, we were able to implement the system in the context of an LFA (lateral flow assay) to serve as a basis for the development of rapid test systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s12161-023-02500-w.

A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18.

CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.

Development and application of DETECTR-based rapid detection for pathogenic Bacillusanthracis.

Bacillus anthracis (B. anthracis) is a gram-positive bacterium responsible for the acute disease anthrax. Rapid and reliable identification of pathogenic B. anthracis is important in the detection of natural infectious disease cases or bio-threats. Herein, a DNA endonuclease targeted CRISPR trans reporter (DETECTR) detection platform based on recombinase polymerase amplification (RPA) was studied. The DETECTR system targeted three sequences from B. anthracis (the BA_5345 chromosomal specific marker, the protective antigen gene pag A from pXO1 plasmid and the capsule-biosynthesis-related gene cap A from pXO2 plasmid). We developed a rapid (<40 min), easy-to-implement and accurate identification method for of B. anthracis nucleic acid with near two-copies sensitivity. The combination of tripartite primer sets is effective for the reliable identification of B. anthracis but also for fast screening of pathogenic strains. More importantly, DETECTR correctly detected simulated clinical blood samples and firstly detected positive samples collected from the location of world War-II site, preserved at north-east China (45°36'55.940″ N, 126°38'33.738″ E) with high sensitivity and specificity. Our study provides insight into the DETECTR-based detection of B. anthracis. We present a novel screening and diagnostic option for pathogenic B. anthracis that can be performed on a user-friendly portable device. Based on its proven reliability, sensitivity, specificity and simplicity, our proposed method can be readily adapted to detect pathogenic B. anthracis, anthrax and biothreats.

A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA). The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9, Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins and their applications, which have the potential to be used in next-generation precise genome engineering.

CRISPR/Cas technology: Opportunities for phytopathogenic viruses detection.

Detection and monitoring of viruses are essential for healthy plants and prosperity. Recent development in CRISPR/Cas system in diagnosis has open an avenue well suited for pathogen detection. Variety of CRISPR associated proteins are being discovered, suggesting array of application and detection strategies in diagnosis. Phytopathogenic viruses are diverse with respect to their nucleic acid compositions, which presents a challenge in developing a single device applicable for almost all viruses. The review describes about the efficient use of CRISPR/Cas Technology in diagnosis, such as SHERLOCK, DETECTR and SATORI. These methods are different in their characteristic to identify specific nucleic acids and processing the detectable signals. These technologies are in their infancy and lot of scope is there to develop commercial kits. Plant tissue culture-based industries, climate control green houses, indoor cultivation facilities etc. has been considered as few examples. This review will be beneficial for researchers seeking to develop detection mechanism based on CRISPR/Cas technology. The outcome in the form of cost-effective detection of viruses will be boon for agro-based industries, which are facing challenges through virus contamination.

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a.

Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification. Using the CRISPR-ENHANCE platform and following the methodology detailed in this paper, nucleic acid detection for low copy numbers can be achieved in less than an hour through either a fluorescence-based detection or a lateral flow assay. The step-by-step instructions provided, in addition to describing how to perform both assays, incorporate details on a LAMP/RT-LAMP-based target amplification step to enable detection of RNA, ssDNA and dsDNA. Furthermore, a protocol for in-house expression and purification of LbCas12a using CL7/lm7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided.

CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout.

The emergence of SARS-CoV-2 has brought the world to a standstill, and till date, effective treatments and diagnostics against this idiosyncratic pathogen are lacking. As compared to the standard WHO/CDC qPCR detection method, which consumes several hours for detection, CRISPR-based SHERLOCK, DETECTR, and FELUDA have emerged as rapid diagnostic tools for the detection of the RNA genome of SARS-CoV-2 within an hour with 100% accuracy, specificity, and sensitivity. These attributes of CRISPR-based detection technologies have taken themselves one step ahead of available detection systems and are emerging as an inevitable tool for quick detection of the virus. Further, the discovery of Cas13s nucleases and their orthologs has opened a new corridor for exploitation of Cas13s as an antiviral therapy against SARS-CoV-2 and other viral diseases. One such approach is Prophylactic Antiviral CRISPR in huMAN cells (PACMAN), which needs a long haul to bring into therapy. The approval of SHERLOCK as the first CRISPR-based SARS-CoV-2 test kit by the FDA, for emergency diagnosis of COVID-19 patients, has given positive hope to scientists that sooner human trials of CRISPR-based therapy will be ratified. In this review, we have extensively reviewed the present CRISPR-based approaches, challenges, and future prospects in the light of diagnostics and therapeutics against SARS-CoV-2. KEY POINTS: • The discovery of Cas12 and Cas13 siblings allowed scientists to detect the viral genes. • Cas13d's identification aided scientists in precisely cleaving the SARS-CoV-2 ssRNA. • CRISPR-Cas system acts as "molecular detector and antiviral proctor."

Potential of CRISPR/Cas system in the diagnosis of COVID-19 infection.

INTRODUCTION: Emerging novel infectious diseases and persistent pandemics with potential to destabilize normal life remain a public health concern for the whole world. The recent outbreak of pneumonia caused by Coronavirus infectious disease-2019 (COVID-19) resulted in high mortality due to a lack of effective drugs or vaccines. With a constantly increasing number of infections with mutated strains and deaths across the globe, rapid, affordable and specific detections with more accurate diagnosis and improved health treatments are needed to combat the spread of this novel pathogen COVID-19. AREAS COVERED: Researchers have started to utilize the recently invented clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas)-based tools for the rapid detection of novel COVID-19. In this review, we summarize the potential of CRISPR/Cas system for the diagnosis and enablement of efficient control of COVID-19. EXPERT OPINION: Multiple groups have demonstrated the potential of utilizing CRISPR-based diagnosis tools for the detection of SARS-CoV-2. In coming months, we expect more novel and rapid CRISPR-based kits for mass detection of COVID-19-infected persons within a fraction of a second. Therefore, we believe science will conquer COVID-19 in the near future.

Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay.

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Rapid, Sensitive, and Specific Severe Acute Respiratory Syndrome Coronavirus 2 Detection: A Multicenter Comparison Between Standard Quantitative Reverse-Transcriptase Polymerase Chain Reaction and CRISPR-Based DETECTR.

BACKGROUND: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. METHODS: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. RESULTS: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. CONCLUSIONS: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.