Rapid sample preparation and low-resource molecular detection of hepatopancreatic parvoviruses (HPV) by recombinase polymerase amplification lateral flow detection assay in shrimps (Fenneropenaeus merguiensis)

Abstract

Background Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments. Methods We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity. Results The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F. merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86–100%) and 100% specificity (95% CI: 66–100%) for detection of HPV. Conclusion The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.

Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay

“… 47 , 48 In addition, point-of-care or field-deployable MPXV diagnostics will require rapid and easy sample preparation. A few methods of sample preparations have been reported for point-of care or field-deployable diagnostics, including one-step sample extraction with TNA-Cifer Reagent, 49 using dipstick extraction technology, 50 and disposable pen-like sensor systems, 51 which can be tested along with our mpox detection assay. Our RPA-Cas12a system can be optimized by incorporating the visual readout component and one-step sample preparations with the aim for field and home use.…”

Rapid low-resource detection of Plasmodium falciparum in infected Anopheles mosquitoes

“…Here we report the development of a test for the detection of P. falciparum (Rapid Pf test) in mosquitoes that is suitable for lowresource implementation and offers equivalent sensitivity to PCR. The test uses a novel 10-minute sample preparation method that requires only tube, pestle, and a liquid reagent (23)(24)(25)(26)(27)(28). Sensitive 10minute isothermal amplification of the Plasmodium 18S rRNA gene is performed using recombinase polymerase amplification (RPA) followed by lateral flow detection, as described for other viruses and bacteria (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), thus requiring only a single temperature heating block for operation.…”

“…The test uses a novel 10-minute sample preparation method that requires only tube, pestle, and a liquid reagent (23)(24)(25)(26)(27)(28). Sensitive 10minute isothermal amplification of the Plasmodium 18S rRNA gene is performed using recombinase polymerase amplification (RPA) followed by lateral flow detection, as described for other viruses and bacteria (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), thus requiring only a single temperature heating block for operation. In this study, we report analytical sensitivity of the test, and demonstrate detection of P. falciparum in experimentally infected Anopheles stephensi mosquitoes.…”

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings

“…Comparable to RT-qPCR, reverse transcriptase-recombinase aided amplification (RT-RAA) has been shown to be both rapid ( Xue et al, 2020 ) and clinically sensitive ( Wang et al, 2020 ). However, NAAT test uptake for low-resource detection of disease is hampered by the lack of field-friendly sample preparation techniques, instead requiring purification of RNA using magnetic beads or column-based laboratory technologies ( Pollak et al, 2022 ).…”