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.

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.