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Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

Published on Jul 25, 2018in Veterinary Sciences
· DOI :10.3390/vetsci5030069
Luis Fabian N Nuñez5
Estimated H-index: 5
,
Silvana H. Santander-Parra + 6 AuthorsAntonio José Piantino Ferreira16
Estimated H-index: 16
Abstract
Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR® Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR® Green was employed, and the Fast mode of a thermocycler was used. The assay detects 109 to 101 copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 °C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 × 106 CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 × 106 CG/uL DNA were detected in chickens with RSS.
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Published on Mar 29, 2018in Veterinary Sciences
David De la Torre2
Estimated H-index: 2
,
Luis Fabian N Nuñez5
Estimated H-index: 5
+ 1 AuthorsAntonio José Piantino Ferreira16
Estimated H-index: 16
Enteric viruses play an important role in the Brazilian poultry industry due to the economic impact of resulting low yields of broilers, layers, and breeders. The most common enteric viruses affecting commercial flocks in Brazil include Fowl Adenovirus of group I (FAdV-I), Chicken Parvovirus (ChPV), Chicken Astrovirus (CAstV), Avian Nephritis Virus (ANV), Infectious Bronchitis Virus (IBV), Avian Reovirus (AReo), and Avian Rotavirus (ARtV). The aim of this study was to identify single and multipl...
4 Citations Source Cite
Published on Jan 1, 2018in Journal of Virological Methods 1.76
Evelyne Lemaitre5
Estimated H-index: 5
(ANSES),
C. Allée6
Estimated H-index: 6
(ANSES)
+ 2 AuthorsPa. Brown1
Estimated H-index: 1
(ANSES)
Abstract Current molecular methods for the detection of avian and human metapneumovirus (AMPV, HMPV) are specifically targeted towards each virus species or individual subgroups of these. Here a broad range SYBR Green I real time RT-PCR was developed which amplified a highly conserved fragment of sequence in the N open reading frame. This method was sufficiently efficient and specific in detecting all MPVs. Its validation according to the NF U47-600 norm for the four AMPV subgroups estimated low...
3 Citations Source Cite
Published on Sep 1, 2017in Journal of Microbiological Methods 1.70
Libera Maria Dalla-Costa12
Estimated H-index: 12
(UFPR: Federal University of Paraná),
Luis Gustavo Morello1
Estimated H-index: 1
(FIOCRUZ: Oswaldo Cruz Foundation)
+ 6 AuthorsSonia Mara Raboni15
Estimated H-index: 15
(UFPR: Federal University of Paraná)
Abstract Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and its diagnosis remains a challenge. Blood culturing is the gold standard technique for blood stream infection (BSI) identification. Molecular tests to detect pathogens in whole blood enable early use of antimicrobials and affect clinical outcomes. Here, using real-time PCR, we evaluated DNA extraction using seven manual and three automated commercially available systems with whole blood samples artificially...
2 Citations Source Cite
Published on Jan 1, 2017in Current Issues in Molecular Biology 2.27
Jeanette Klenner1
Estimated H-index: 1
,
Claudia Kohl9
Estimated H-index: 9
+ 1 AuthorsAndreas Nitsche37
Estimated H-index: 37
4 Citations Source Cite
Published on Dec 1, 2016in Tropical Animal Health and Production 0.97
Fabrine Finkler2
Estimated H-index: 2
(UFRGS: Universidade Federal do Rio Grande do Sul),
Diane Alves de Lima3
Estimated H-index: 3
+ 6 AuthorsAna Cláudia Franco16
Estimated H-index: 16
(UFRGS: Universidade Federal do Rio Grande do Sul)
Chicken parvovirus (ChPV) has been associated with malabsorption syndrome (MAS) in broilers. However, the participation of this virus in such syndrome is unclear, since it may be detected in diseased and healthy chickens. In the course of these studies, it was argued whether ChPV genome loads might be correlated to the occurrence of MAS. To check such a hypothesis, a SYBR green-based quantitative polymerase chain reaction was developed to detect and quantify ChPV genomes. Cloacal swabs from 68 b...
4 Citations Source Cite
Published on Nov 1, 2016in Archives of Virology 2.16
Bin Feng1
Estimated H-index: 1
,
Zhixun Xie14
Estimated H-index: 14
+ 10 AuthorsYanfang Zhang5
Estimated H-index: 5
A previously unidentified chicken parvovirus (ChPV) strain, associated with runting-stunting syndrome (RSS), is now endemic among chickens in China. To explore the genetic diversity of ChPV strains, we determined the first complete genome sequence of a novel ChPV isolate (GX-CH-PV-7) identified in chickens in Guang Xi, China, and showed moderate genome sequence similarity to reference strains. Analysis showed that the viral genome sequence is 86.4 %–93.9 % identical to those of other ChPVs. Gene...
2 Citations Source Cite
Published on Nov 1, 2016in Avian Pathology 2.05
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 Jul 1, 2016in Pesquisa Veterinaria Brasileira 0.39
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 Jul 1, 2016in Molecular Biology and Evolution 10.22
Sudhir Kumar63
Estimated H-index: 63
(TU: Temple University),
Glen Stecher6
Estimated H-index: 6
(TU: Temple University),
Koichiro Tamura28
Estimated H-index: 28
(Tokyo Metropolitan University)
Abstract We present the latest version of the Molecular Evolutionary Genetics Analysis (Mega) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, Mega has been optimized for use on 64-bit computing systems for analyzing larger datasets. Researchers can now explore and analyze tens of thousands of sequences in Mega The new version also provides an advanced wizard for building timetrees and includes a new functionality to automa...
8,817 Citations Source Cite
Published on Apr 1, 2016in Poultry Science 2.22
L. F. N. Nuñez4
Estimated H-index: 4
(USP: University of São Paulo),
Lilian Rose Marques de Sá2
Estimated H-index: 2
(USP: University of São Paulo)
+ 3 AuthorsA. J. P. Ferreira5
Estimated H-index: 5
(USP: University of São Paulo)
Enteric disorders are an important cause of economic losses in broiler chickens worldwide. Several agents have been associated with enteric problems, such as viruses, bacteria, and parasites. In this study, broiler chickens showing signs of enteric disorders were subjected to molecular diagnosis for several viral agents and also for pathological examination for elucidating this problem. Thus, the chickens were screened for avian nephritis virus (ANV), chicken astrovirus (CAstV), avian rotavirus ...
6 Citations Source Cite
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