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

Sep 20, 2021ACS synthetic biology

Sensitive CRISPR-Based Fluorescent Test for Detecting Porcine Reproductive and Respiratory Syndrome Virus

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Abstract

The developed assay can detect PRRS virus (PRRSV) with a sensitivity of a single copy in just 25 minutes.

Detection of PRRSV is achieved using a fluorescence assay that combines reverse transcription-recombinase polymerase amplification with a CRISPR/Cas12a system.
The assay allows for isothermal and visual detection, simplifying the process to a single tube reaction.
No cross-reactivity with other porcine viruses was observed, indicating high specificity.
A strong correlation was found between PRRSV detection results from clinical samples using the new assay and the quantitative reverse transcription polymerase chain reaction method, suggesting high accuracy.

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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.

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

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