Shanmuganatham, K. | Search bioRxiv

bioRxiv2024

Rapid identification of African swine fever virus in diagnostic samples using CRISPR-Cas

African Swine Fever Virus (ASFV) is a high consequence, highly transmissible pathogen affecting swine causing African Swine Fever (ASF), a devastating disease, with high mortality rates in naive populations. Due to the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States (U.S.) to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten U.S. swine due to their proximity and involvement in movement to and from North America. While surveillance programs are ongoing, there are limited point-of-care (POC) tests available during outbreaks that maintain the sensitivity and specificity standards of laboratory testing (e.g., qPCR). However, the recently developed CRISPR-Cas testing systems may provide comparable high-quality results. In a CRISPR-based diagnostic assay, CRISPR effectors can be programmed with CRISPR-RNA (crRNA) to target specific DNA or RNA. Upon target binding, the Cas enzyme undergoes collateral cleavage of nearby fluorescently quenched reporter molecules (ssDNA or ssRNA), which can be detected under blue light or a fluorescence microplate reader. Furthermore, this tool is rapid, simple, cost-effective and can be performed with inexpensive equipment. For these reasons, we sought to develop a low-cost visual detection method for ASFV by employing the recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a point-of-care-assay. Our CRISPR-Cas12a assay demonstrated comparable sensitivity and specificity to qPCR, both visually and when quantified using a fluorescent reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, the CRISPR assay achieved a sensitivity of 98.3% (102 DNA copies) and a specificity of 100%. Finally, an assessment of the reaction time constraints indicated that results can be visualized in as little as seven minutes with a peak fluorescence at 40 min (RPA and CRISPR steps). The results of this feasibility assay validation allow for the rapid development of sensitive and specific POC tests that may be used for outbreak response in the future.

microbiology↗

bioRxiv2023

Inactivation of Highly Transmissible Livestock and Avian Viruses Including Influenza A and Newcastle Disease Virus for Molecular Diagnostics

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection but maintains the nucleic acid quality required for diagnostic PCR testing. This inactivation method is needed to alleviate concerns about transmission potential, reduce shipping complications and cost, and allow testing in lower containment laboratories to improve disease diagnostics by improving turn-around time. This study evaluated a panel of ten surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial (PrimeStore(R)) molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99% as determined by infectivity and serial passage assays. However, detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and shipment of infectious substances. Contribution to the fieldActive surveillance and confirmatory testing efforts are in place to protect animals in the United States from certain highly contagious diseases and to limit financial impacts to consumers and producers when the food supply is disrupted. Confirmatory testing typically utilizes nucleic acid detection to identify active infection. Testing is required to be completed in high containment facilities due to the elevated pathogenicity and impact potential of animal diseases. The requirement for testing in high containment facilities limits the ability for regional and state laboratories to test for Tier 1 select agents. Shipment of diagnostic samples is costly, as well as time and temperature sensitive to avoid deterioration of sample quality needed for testing. These constraints lengthen response time and testing turn-around time. Here, we showed that a commercial (PrimeStore(R)) molecular transport media (PSMTM) completely inactivated all viruses tested without affecting nucleic acid detection/integrity. These data suggest that highly contagious agents are effectively inactivated by PSMTM without compromising the nucleic acid needed for diagnostic testing. These data provide support that this inactivation method can be utilized during sample collection to reduce constraints in disease diagnostics and in reagent sharing among international laboratories.

microbiology↗