Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus.
Detection
Differentiation
Genotype
PHEV
Real time RT-PCR
Journal
Journal of virological methods
ISSN: 1879-0984
Titre abrégé: J Virol Methods
Pays: Netherlands
ID NLM: 8005839
Informations de publication
Date de publication:
07 2019
07 2019
Historique:
received:
11
02
2019
revised:
04
04
2019
accepted:
05
04
2019
pubmed:
9
4
2019
medline:
4
6
2020
entrez:
9
4
2019
Statut:
ppublish
Résumé
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded, positive-sense RNA virus. PHEV mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. However, our recent findings provide strong evidence that PHEV can also cause respiratory disease in older pigs. Genomic analysis of new PHEV strains identified in our former study further classifies PHEV into three genotypes. Detection and differentiation of these new mutants are critical in monitoring PHEV evolution in the field. In the present study, we report the development of a triplex real-time RT-PCR assay for detection and differentiation of three PHEV genotypes, 1, 2, and 3. Three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the 3' end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of NS2 with different patterns of deletions for detection of both genotypes 1 and 3, or genotype 3 only. Genotype 1 was positive when two probe dyes showed signals, genotype 2 was positive when only one probe dye showed a signal, and genotype 3 was positive when all three probes showed signals. The detection limit of the developed triplex real-time RT-PCR was as low as 8 or 9 DNA copies for three sets of primers and probes. The specificity test showed no cross reaction with other porcine viruses. Positive field-samples were correctly typed by this new assay, which was further confirmed by DNA sequencing. The triplex real-time RT-PCR provides a rapid and sensitive method to detect and differentiate all three US genotypes of PHEV from clinical samples.
Identifiants
pubmed: 30959064
pii: S0166-0934(19)30064-3
doi: 10.1016/j.jviromet.2019.04.008
pmc: PMC7113741
pii:
doi:
Types de publication
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
13-17Subventions
Organisme : NIAID NIH HHS
ID : HHSN272201400006C
Pays : United States
Informations de copyright
Copyright © 2019 Elsevier B.V. All rights reserved.
Références
Arch Virol. 2014 Sep;159(9):2329-37
pubmed: 24756345
Aust Vet J. 1979 Oct;55(10):503-4
pubmed: 231961
Emerg Infect Dis. 2017 Sep;23(9):
pubmed: 28654418
Virus Genes. 2011 Feb;42(1):90-6
pubmed: 21103919
J Virol. 2006 Jul;80(14):7270-4
pubmed: 16809333
Arch Gesamte Virusforsch. 1974;44(1):35-50
pubmed: 4823866
Am J Vet Res. 1972 Oct;33(10):2003-9
pubmed: 5074702
Can J Comp Med Vet Sci. 1962 Mar;26(3):49-56
pubmed: 17649356
Vet Rec. 1969 Feb 15;84(7):175-6
pubmed: 5813276
Emerg Infect Dis. 2008 Mar;14(3):484-6
pubmed: 18325268
Emerg Infect Dis. 2017 Jul;23(7):1168-1171
pubmed: 28628449
Nihon Juigaku Zasshi. 1987 Feb;49(1):85-93
pubmed: 3033381
J Am Vet Med Assoc. 1976 Feb 1;168(3):236-9
pubmed: 1248957
Am J Vet Res. 1975 Jun;36(6):821-3
pubmed: 807134