Premium
Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples
Author(s) -
PinheirodeOliveira T. F.,
FonsecaJúnior A. A.,
Camargos M. F.,
LaguardiaNascimento M.,
GiannattasioFerraz S.,
Cottorello A. C. P.,
de Oliveira A. M.,
GóesNeto A.,
BarbosaStancioli E. F.
Publication year - 2019
Publication title -
transboundary and emerging diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.392
H-Index - 63
eISSN - 1865-1682
pISSN - 1865-1674
DOI - 10.1111/tbed.13168
Subject(s) - digital polymerase chain reaction , virology , reverse transcriptase , outbreak , real time polymerase chain reaction , biology , concordance , virus , disease , polymerase chain reaction , medicine , bioinformatics , genetics , gene
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 ‐dd PCR ) 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 ‐dd PCR , 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 ‐dd PCR and RT ‐ qPCR using the one‐step assay approach and biological samples from Brazilian outbreaks of Senecavirus A . However, dd RT ‐ PCR had a better performance than RT ‐ PCR when swine serum pools were tested. According to the results, the one‐step RT ‐dd PCR 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 ‐dd PCR ), being a useful tool for vesicular diseases control programs.