Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids.

The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III (Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/µL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA-Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine.

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

Detecting Melanocortin 1 Receptor Gene's SNPs by CRISPR/enAsCas12a.

Beyond its powerful genome-editing capabilities, the CRISPR/Cas system has opened up a new era of molecular diagnostics due to its highly specific base recognition and trans-cleavage activity. However, most CRISPR/Cas detection systems are mainly used to detect nucleic acids of bacteria or viruses, while the application of single nucleotide polymorphism (SNP) detection is limited. The MC1R SNPs were investigated by CRISPR/enAsCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enAsCas12a has a preference for divalent magnesium ion (Mg2+) and can effectively distinguish the genes with a single base difference in the presence of Mg2+, and the Melanocortin l receptor (MC1R) gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enAsCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enAsCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.

A Simple, Affordable, and Rapid Visual CRISPR-Based Field Test for Sex Determination of Earlier Porcine Embryos and Pork Products.

Sex selection technologies have immensely impacted swine production globally. Conventional earlier embryo sex identification methods require professional technicians and sophisticated laboratory instruments. Rapid on-site gender identification of porcine embryos and pork products remains challenging. In this study, we developed a CRISPR/Cas12a-based fluorescence visualization point-of-care sex determination test that is rapid, accurate and easy to implement on-site. The CRISPR/Cas12a assay coupled with either the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) employs precisely designed primers and single-guide RNAs targeting the sex-determining region Y (SRY) and the zinc finger protein X-linked (ZFX) genes. PCR and LAMP amplicons were cleaved with the subsequent generation of fluorescing products detectable with portable blue and ultraviolet light transilluminators. Approximately two copies per microliter of the ZFX and SRY genes were detected using the RApid VIsual CRISPR (RAVI-CRISPR) assay. This method is a sensitive, inexpensive, versatile, and point-of-care test. The technology has other potential applications like determining the sex of diverse livestock species, detecting livestock disease-causing pathogens and evaluating the quality of meat products.

Construction of Attenuated Strains for Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) via Reverse Genetic System.

The nervous necrosis virus (NNV) mainly attacks the central nervous system of fish to cause viral nervous necrosis, which is an acute and serious prevalent disease in fish. Among different genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) is the most widely reported, with the highest number of susceptible species. To better understand the pathogenicity of RGNNV, we first developed a reverse genetic system for recombinant RGNNV rescue using B7GG and striped snakehead (SSN-1) cells. Furthermore, we constructed attenuated RGNNV strains rRGNNV-B2-M1 and rRGNNV-B2-M2 with the loss of B2 protein expression, which grew slower and induced less Mx1 expression than that of wild-type RGNNV. Moreover, rRGNNV-B2-M1 and rRGNNV-B2-M2 were less virulent than the wild-type RGNNV. Our study provides a potential tool for further research on the viral protein function, virulence pathogenesis, and vaccine development of RGNNV, which is also a template for the rescue of other fish viruses.

A one-tube rapid visual CRISPR assay for the field detection of Japanese encephalitis virus.

Early and rapid detection of Japanese encephalitis virus (JEV) is necessary for timely preventive and control measures. However, JEV RNA detection remains challenging due to the low level of viremia. In this study, a RApid VIsual CRISPR (RAVI-CRISPR) assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a targeting was developed for easy detection of JEV in the field. We showed successful detection of 8.97 or more copies of the C gene sequence of JEV RNA within approximately 60 min. This assay also displayed no cross-reactivity with other porcine pathogens. We applied our one-tube RAVI-CRISPR assay to 18 brain tissue sample for JE diagnosis. The results from both fluorescence intensity measurements and directly naked-eye visualization were consistent with those from real-time PCR analysis. Taken together, our results showed that one-tube RAVI-CRISPR assay is robust, convenient, sensitive, specific, affordable, and potentially adaptable to on-site detection or surveillance of JEV in clinical and vector samples.

Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification.

Lumpy skin disease (LSD) is a severe and highly infectious pox disease of cattle caused by the lumpy skin disease virus (LSDV). To facilitate early control of LSD, this study aimed to develop a new rapid on-site LSDV detection method using an orf068 gene-based recombinase polymerase amplification assay (RPA) coupled with a CRISPR-Cas12a-based fluorescence assay (RPA-Cas12a-fluorescence assay). The results showed that the sensitivity of our RPA-Cas12a-fluorescence assay for detecting LSDV orf068 gene reached 5 copies/µL with plasmid as a template, and 102 TCID50/mL with viral genomic DNA as a template. No cross-reaction with other common bovine viruses was observed. Further, an on-site RPA-Cas12a-fluorescence assay of 40 clinical samples from cattle with or without LSD showed a diagnostic sensitivity of 96.3% (95% CI: 81.0-99.9%) and specificity of 92.31% (95% CI: 62.1-99.6%), which was close to those of the quantitative PCR assay. Therefore, our RPA-Cas12a-fluorescence assay has promising prospects in on-site rapid LSDV detection.

An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products.

The growing demand for and supply of meat and meat products has led to a proportional increase in cases of meat adulteration. Adulterated meat poses serious economic and health consequences globally. Current laboratory methods for meat species identification require specialized equipment with limited field applications. This study developed an inexpensive, point-of-care Loop-Mediated Isothermal Amplification (LAMP)-CRISPR/Cas12a colorimetric assay to detect meat species using a Texas Red-labelled single-strand (ssDNA) reporter. As low as 1.0 pg/µL of the porcine NADH4, the chicken NADH dehydrogenase subunit 2 (ND2) and the duck D-loop genes was detectable under white, blue and ultraviolet light. The test turnaround time from DNA extraction to visualization was approximately 40 min. The assay accurately detected pure and mixed-meat products in the laboratory (n = 15) and during a pilot point-of-care test (n = 8) in a food processing factory. The results are 100% reproducible using lateral flow detection strips and the real-time PCR detection instrument. This technology is fully deployable and usable in any standard room. Thus, our study demonstrates that this method is a straightforward, specific, sensitive, point-of-care test (POCT) adaptable to various outlets such as customs, quarantine units and meat import/export departments.

Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.

Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.

Rapid Visual CRISPR Assay: A Naked-Eye Colorimetric Detection Method for Nucleic Acids Based on CRISPR/Cas12a and a Convolutional Neural Network.

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.

Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of swine that is caused by PRRS virus (PRRSV). In this study, we established a fluorescence assay for highly sensitive detection of PRRSV through integration of the reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled Cas12a system with an optical property of single stranded DNA-fluorescently quenched (ssDNA-FQ) reporter. This technique can achieve isothermal and visual detection of PRRSV in 25 min. In particular, the assay reaction can be completed in a single tube. The limit of sensitivity for PRRSV detection was single copy without cross-reactivity of other porcine viruses. Correlation between 11 PRRSV clinical samples measured by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) and CRISPR/Cas12a assay was determined; the result showed that our results were highly accurate. To sum up, this study developed a visual, sensitive, and specific method of nucleic acid detection based on a CRISPR-Cas12a technique for the on-site detection of PRRSV.

Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/µL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.