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Background and aim: Transmissible gastroenteritis virus (TGEV) is a highly contagious gastrointestinal virus that causes diarrhea, vomiting, anorexia, dehydration, and weight loss in piglets. In clinical practice, it often occurs in mixed infections with other pathogens, and is therefore difficult to diagnose and prevent. It mainly harms piglets of about 2 weeks old, causing huge losses on farms. The clinical confirmation of TGEV usually requires a laboratory diagnosis, but traditional PCR and immunofluorescence assays have some limitations. Moreover, most farms in China are ill-equipped to accurately diagnose the disease. Therefore, a new detection method with high sensitivity and specificity and less dependence on instrumentation is required. Methods: We used recombinase polymerase amplification (RPA), combined with the nuclease characteristics of the activated Cas13a protein to establish a visual CRISPR-Cas13a-assisted detection method for TGEV by adding a reporter RNA with fluorescent and quenching moieties to the system. Result: We selected the optimal RPA primer and best CRISPR RNA (crRNA). The reaction system was optimized and its repeatability, specificity, and sensitivity verified. The TGEV detection system did not cross-react with other common diarrhea viruses, and its detection limit was 101 copies, which is similar with the sensitivity of qPCR. We successfully established an RPA-CRISPR-Cas13a-assisted detection method, and used this detection system to analyze 123 pig blood samples. qPCR was used as the gold standard method. The sensitivity, specificity, positive coincidence rate, and negative coincidence rate of the new method were 100, 98.93, 96.66, and 100%, respectively. [ABSTRACT FROM AUTHOR]

Red-spotted grouper nervous necrosis virus (RGNNV) is a serious pathogen that causes high mortality in many species of fish in marine aquaculture. Larvae and juveniles are more easily infected by RGNNV, and the cumulative mortality can reach 100% post-infection. To date, a simple, convenient, and low complexity method to detect this virus does not exist. Therefore, we developed a rapid detection method for RGNNV based on the CRISPR-Cas13a system. We optimized each of the four parts of the detection system: selection of efficient CRISPR-derived RNA(crRNA), reaction temperature, control of reporter probe content, and reaction concentration ratio of Cas13a to crRNA. Our detection system was sensitive enough to detect single-stranded RGNNV at a minimum of 102 fM. We evaluated the effectiveness of the detection system by testing it on different NNVs and RGNNV-infected RGNNV fish from different regions of China and found that the system had high specificity. Overall, our results showed that the CRISPR-Cas13a detection system was a robust, specific, and validated method for RGNNV detection. Thus, it provided a theoretical basis for the field detection of RGNNV. • Engineered Crispr/Cas13a system successfully dedect RGNNV. • 2.Designed and selected specific crRNA that accurately target RGNNV. • 3.Our detection system was sensitive enough to detect RGNNV at a minimum of 102 fM [ABSTRACT FROM AUTHOR]

Salmonella, being one of the most widespread foodborne pathogens, is a compulsory test item required by national food safety standard of China and many other countries. More sensitive and specific Salmonella detection method is still needed since traditional methods are time consuming and highly dependent on enormous manpower and material resources. In this research, a bacteria detection method based on CRISPR-Cas13a system (where CRISPR is Clustered Regularly Interspaced Short Palindromic Repeats) was proposed. The target DNA was amplified by PCR and transcribed into RNA by T7 transcriptase, which can activate the RNase activity of the Cas13a protein. The self-folding quenched fluorescent probe can be cleaved by the activated Cas13a protein to generate fluorescent signal. We named this method as PCF detection (PCR-CRISPR-Fluorescence based nucleic acid detection). In this study, PCF detection showed excellent sensitivity, which can detect Salmonella genomic DNA with a minimum of 10 1 aM or 10° CFU/ml Salmonella bacteria in 2 hr. It also showed good specificity with no cross-reaction with other common foodborne bacteria. PRACTICAL APPLICATION: The PCF detection method proposed in this article can detect Salmonella sensitively and specifically, providing a novel strategy for the detection of foodborne pathogens in food and has great application potential in other microbial detection fields., (© 2021 Institute of Food Technologists®.)