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Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

Published on Dec 29, 2018in Veterinary Sciences
· DOI :10.3390/vetsci6010002
David De la Torre2
Estimated H-index: 2
,
Claudete S. Astolfi-Ferreira7
Estimated H-index: 7
+ 1 AuthorsAntonio José Piantino Ferreira16
Estimated H-index: 16
Cite
Abstract
Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.
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Published on Jan 1, 2018in MethodsX
Lee S. Toni1
Estimated H-index: 1
(Anschutz Medical Campus),
Anastacia M. Garcia1
Estimated H-index: 1
(Anschutz Medical Campus)
+ 4 AuthorsCarmen C. Sucharov19
Estimated H-index: 19
(Anschutz Medical Campus)
Abstract Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse trans...
2 Citations Source Cite
Published on Nov 1, 2016in Avian Pathology 1.96
Ryan Devaney1
Estimated H-index: 1
('QUB': Queen's University Belfast),
James Trudgett5
Estimated H-index: 5
+ 2 AuthorsVictoria Smyth1
Estimated H-index: 1
ABSTRACTRunting-stunting syndrome (RSS) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. Of specific interest are the viral agents associated with RSS which have been difficult to fully characterize to date. Past research into the aetiology of RSS has implicated a wide variety of RNA and DNA viruses however, to date, no individual vir...
11 Citations Source Cite
Published on Aug 1, 2016in Archives of Virology 2.26
Joana D'Arc Pereira Mascarenhas23
Estimated H-index: 23
,
Delana Andreza Melo Bezerra4
Estimated H-index: 4
+ 3 AuthorsLuana da Silva Soares6
Estimated H-index: 6
The aim of this study was to detect rotavirus F (RVF) and rotavirus G (RVG) in fecal specimens of broiler chickens in Brazil. During 2008 and 2011, a total of 85 fecal samples were collected. The viral genome was extracted, followed by polyacrylamide gel electrophoresis (PAGE), reverse transcription polymerase chain reaction (RT-PCR), and nucleotide sequencing. Samples were screened for rotaviruses by PAGE, and RVF and RVG genome banding patterns were not seen. Using RT-PCR, it was found that 9....
3 Citations Source Cite
Published on Jul 1, 2016in Pesquisa Veterinaria Brasileira 0.30
Luis Fabian N Nuñez5
Estimated H-index: 5
(USP: University of São Paulo),
Silvana Hipatia Santander Parra5
Estimated H-index: 5
(USP: University of São Paulo)
+ 4 AuthorsAntonio José Piantino Ferreira16
Estimated H-index: 16
(USP: University of São Paulo)
Enteric disease is a multifactorial problem in chickens, which causes gastrointestinal alterations, elevated feed conversions and impairment. In the last years, several enteric viruses were implicated in enteric disease; case reports have shown their presence alone or in concomitant infections during outbreaks and have suggested that they might be determining factors in the aetiology of enteric disease. This study shows high detection rates of enteric viruses in the pancreas and spleen in sample...
5 Citations Source Cite
Published on Jun 1, 2016in Poultry Science 2.03
A. M. Lobani1
Estimated H-index: 1
(JUST: Jordan University of Science and Technology),
Saad Gharaibeh11
Estimated H-index: 11
(JUST: Jordan University of Science and Technology),
Ahmad M. Al-Majali15
Estimated H-index: 15
(JUST: Jordan University of Science and Technology)
The aim of this study is to determine if enteric viruses are the cause of diarrhea in broiler flocks in Jordan. Intestinal content samples were collected from 101 broiler flocks from several regions of Jordan to detect the presence of astrovirus, coronavirus, reovirus, and rotavirus, by using reverse transcriptase polymerase chain reaction (RT-PCR). Forty-six of these flocks were clinically healthy with no enteric disease, and the other 55 flocks were clinically suffering from diarrhea. The samp...
2 Citations Source Cite
Published on Nov 2, 2015in Avian Pathology 1.96
Emiliana Falcone15
Estimated H-index: 15
(ISS: Istituto Superiore di Sanità),
Chiara Busi2
Estimated H-index: 2
+ 6 AuthorsMaria Beatrice Boniotti11
Estimated H-index: 11
Avian rotaviruses are still largely undefined despite being widespread in several avian species and despite the economic impact of rotavirus (RV) enteritis in poultry flocks. In this study, the presence of different avian RV groups was investigated in commercial poultry flocks reared in Northern and Central Italy and with a history of enteric diseases. Faeces or intestinal contents from different avian species previously found to contain RV particles by electron microscopy (EM) were analysed by ...
3 Citations Source Cite
Published on Mar 1, 2015in Journal of Virological Methods 1.75
Qiu-Hua Mo3
Estimated H-index: 3
,
Hai-Bo Wang4
Estimated H-index: 4
+ 5 AuthorsZe Yang3
Estimated H-index: 3
Abstract Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a comm...
8 Citations Source Cite
Published on Jan 1, 2015in Pesquisa Veterinaria Brasileira 0.30
Elizabeth C.M. Marconi1
Estimated H-index: 1
(USP: University of São Paulo),
Nara T.C.G. Bernardes2
Estimated H-index: 2
(USP: University of São Paulo)
+ 2 AuthorsFabio Gregori10
Estimated H-index: 10
(USP: University of São Paulo)
Group A Rotavirus (RVA) is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR) was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5) gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing). The overall agreement (kap...
2 Citations Source Cite
Published on Mar 1, 2014in Avian Diseases 1.31
Laila Andreia Rodrigues Beserra2
Estimated H-index: 2
,
B. R. P. Barbosa3
Estimated H-index: 3
+ 2 AuthorsFabio Gregori10
Estimated H-index: 10
SUMMARY Rotaviruses are a major cause of diarrhea in humans and animals, including several mammalian and avian species. Using different PCR protocols, we report the occurrence of rotavirus A in 21 (53.84%; 21/39) from 39 fecal pool samples of broilers, layers, and broiler breeders from Brazilian avian farms. We typed the G5, G8, G11, G19, and P[31] genotypes.
6 Citations Source Cite
Published on Jan 1, 2014in The Scientific World Journal
Elena Mettifogo9
Estimated H-index: 9
,
Luis Fabian N Nuñez5
Estimated H-index: 5
+ 5 AuthorsAntonio José Piantino Ferreira16
Estimated H-index: 16
(USP: University of São Paulo)
Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. C...
20 Citations Source Cite
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