RT-qPCR genotyping assays for differentiating Rift Valley fever phlebovirus strains.
Genotyping assays
Melt curve analysis
Reassortment
Rift Valley fever phlebovirus
Strain-specific 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:
05 2023
05 2023
Historique:
received:
21
11
2022
revised:
07
02
2023
accepted:
14
02
2023
pmc-release:
01
05
2024
medline:
28
3
2023
pubmed:
22
2
2023
entrez:
21
2
2023
Statut:
ppublish
Résumé
Rift Valley fever phlebovirus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen. Real time RT-qPCR genotyping (GT) assays were developed to differentiate between two RVFV wild-type strains (128B-15 and SA01-1322) and a vaccine strain (MP-12). The GT assay uses a one-step RT-qPCR mix, with two different RVFV strain-specific primers (either forward or reverse) with long or short G/C tags and a common primer (either forward or reverse) for each of the 3 genomic segments. The GT assay produces PCR amplicons with unique melting temperatures that are resolved in a post PCR melt curve analysis for strain identification. Furthermore, a strain specific RT-qPCR (SS-PCR) assay was developed to allow for specific detection of low titer RVFV strains in mixed RVFV samples. Our data shows that the GT assays are capable of differentiating L, M, and S segments of RVFV strains 128B-15 versus MP-12, and 128B-15 versus SA01-1322. The SS-PCR assay results revealed that it can specifically amplify and detect a low titer MP-12 strain in mixed RVFV samples. Overall, these two novel assays are useful as screening tools for determining reassortment of the segmented RVFV genome during co-infections, and could be adapted and applied for other segmented pathogens of interest.
Identifiants
pubmed: 36801236
pii: S0166-0934(23)00018-6
doi: 10.1016/j.jviromet.2023.114693
pmc: PMC10040438
mid: NIHMS1879342
pii:
doi:
Types de publication
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
114693Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM130448
Pays : United States
Informations de copyright
Copyright © 2023 Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Références
Biotechniques. 2005 Dec;39(6):885-93
pubmed: 16382908
Res Virol. 1989 Mar-Apr;140(2):129-38
pubmed: 2756240
PLoS One. 2017 Sep 19;12(9):e0185194
pubmed: 28926632
Virus Genes. 2019 Feb;55(1):1-11
pubmed: 30426314
J Gen Virol. 1990 Oct;71 ( Pt 10):2307-12
pubmed: 2230736
Emerg Infect Dis. 2018 Sep;24(9):1717-1719
pubmed: 30124402
Clin Diagn Lab Immunol. 2002 May;9(3):713-5
pubmed: 11986283
Vaccines (Basel). 2017 Sep 19;5(3):
pubmed: 28925970
Viruses. 2011 May;3(5):493-519
pubmed: 21666766
Sci Rep. 2015 Jun 23;5:11353
pubmed: 26100494
N Am J Med Sci. 2011 Oct;3(10):478-85
pubmed: 22363089
Front Cell Infect Microbiol. 2012 Oct 26;2:131
pubmed: 23112960
Clin Chem. 2010 Aug;56(8):1340-4
pubmed: 20567024
Am J Vet Res. 1997 Oct;58(10):1110-4
pubmed: 9328663
Annu Rev Entomol. 2016;61:395-415
pubmed: 26982443
PLoS Negl Trop Dis. 2022 Jan 24;16(1):e0009852
pubmed: 35073355
J Virol Methods. 2003 Nov;113(2):103-12
pubmed: 14553896
Virol J. 2013 Sep 12;10:284
pubmed: 24028349
Am J Trop Med Hyg. 2010 Aug;83(2 Suppl):28-37
pubmed: 20682903
J Gen Virol. 1985 Oct;66 ( Pt 10):2271-7
pubmed: 4045430
Emerg Infect Dis. 2011 Dec;17(12):2270-6
pubmed: 22172568
Emerg Infect Dis. 2002 Dec;8(12):1492-4
pubmed: 12498669
Zoonoses Public Health. 2015 Aug;62(5):309-25
pubmed: 25256804
J Virol Methods. 2011 Nov;177(2):140-6
pubmed: 21827790
Vaccine. 1991 Jan;9(1):35-41
pubmed: 2008798
J Virol. 1999 Oct;73(10):8196-200
pubmed: 10482570
Vet Microbiol. 2014 Aug 6;172(1-2):44-50
pubmed: 24856133