Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples

Abstract

Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot‐and‐mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT‐ddPCR) using one‐step and two‐step approaches. Analytical sensitivity and specificity were done in parallel with real‐time PCR, RT‐qPCR (one‐step and two‐step) for comparison of sensitivity and specificity of both methods. In the standardization of RT‐ddPCR, the double‐quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one‐step techniques showed superior performance than two‐step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT‐ddPCR and RT‐qPCR using the one‐step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT‐PCR had a better performance than RT‐PCR when swine serum pools were tested. According to the results, the one‐step RT‐ddPCR and RT‐qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT‐ddPCR), being a useful tool for vesicular diseases control programs.

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

“…These include a conventional two-step RT-PCR assay targeting the VP3/VP1 region [6], a conventional nested-PCR assay targeting the VP1 region [28], a SYBR Green-based real-time RT-PCR assay targeting the VP1 region [29], two TaqMan probe-based real-time RT-PCR assays respectively targeting the 3D region [30] and the VP1 region [31], two reverse transcription droplet digital PCR assays both targeting the 3D region [32,33], and several reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays targeting the VP1, VP2, 5′ UTR, or VP3/VP1 region [34,35]. We did not perform a head-to-head comparison of our SVV rRT-PCR and RT-iiPCR to these previously published assays.…”

“…A number of SVV specific gel-based (conventional) RT-PCR, nested RT-PCR, real-time RT-PCR (rRT-PCR), reverse transcription droplet digital PCR, and loop-mediated isothermal amplification assays have been described in the literatures although not all of them have been fully validated [6,[28][29][30][31][32][33][34][35]. Compared to the conventional RT-PCR assays, rRT-PCR is generally more sensitive and suitable for high throughput testing with shorter turnaround time.…”

Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR detects B.1.1.7 lineage in Washington State

“…lineage or other variants of concern (42)(43)(44). The S982A SNP, for instance, is specific for B.1.1.7 lineages, whereas the N501Y mutation is not (40).…”

Possible intrauterine SARS-CoV-2 infection: Positive nucleic acid testing results and consecutive positive SARS-CoV-2-specific antibody levels within 50 days after birth

“…However, one-step RT-ddPCR tests performed on throat and anal swabs were positive (750 and 892 copies/ml) on day 7 of life, while RT-PCR was still negative at the same time ( Figure 1 A). In fact, the sensitivity of the one-step RT-ddPCR is much higher than that of RT-PCR for virus nucleic acid testing ( Pinheiro-de-Oliveira et al, 2019 ). However, the one-step RT-ddPCR technology was not available for the detection of SARS-CoV-2 during the early stage of the pandemic and not until day 7 of the neonate's life.…”