A combination of recombinase polymerase amplification with CRISPR technology rapidly detects goose parvovirus with high accuracy and sensitivity

TL;DR

A new rapid and sensitive test for detecting goose parvovirus combines CRISPR and RPA technologies, enabling on-site diagnosis with high accuracy.

Contribution

A novel RPA-CRISPR/Cas12a assay for GPV detection with high sensitivity and specificity, suitable for on-site use.

Findings

The assay detects 10 copies/μL of GPV VP3 gene within one hour with high sensitivity.

The assay showed 100% diagnostic sensitivity and 95.5% specificity in field samples.

The method supports visual readouts using portable blue light transilluminators for on-site use.

Abstract

Goose parvovirus (GPV) poses a significant threat to the waterfowl industry, necessitating reliable detection methods. However, conventional techniques are often time-consuming, equipment-dependent, or lack sufficient sensitivity for detecting early-stage infection. In contrast, emerging CRISPR/Cas12a-based systems offer a promising alternative for rapid, sensitive, and on-site diagnostics.

We developed and optimized a recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting the conserved VP3 gene of GPV. The analytical and diagnostic performance of this assay was rigorously validated using plasmid standards and clinical specimens from both experimentally infected and field-collected ducklings.

Our developed assay combines RPA with CRISPR/Cas12a technology for rapid GPV nucleic acids detection. This method achieves a detection limit of 10 copies/μL of the VP3 gene within one hour, demonstrating high sensitivity and rapid turnaround. The assay exhibited exceptional specificity, with no cross-reactivity against other waterfowl viruses, and showed robust reproducibility, with intra- and inter-assay coefficients of variation consistently below 5.0%. Clinical validation using 42 field samples confirmed a diagnostic sensitivity of 100% and 95.5% specificity, showing superior performance to real-time quantitative PCR (qPCR) in both metrics. Furthermore, the assay supports flexible visual readouts using portable blue light transilluminators, facilitating on-site interpretation.

This study established a highly field-deployable RPA-CRISPR/Cas12a assay for rapid, visual detection of GPV with outstanding sensitivity and specificity. Its capability for instrument-free on-site diagnosis via blue light transillumination makes this approach particularly promising for resource-limited settings.

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