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Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System

Bovine rotavirus (BRV), bovine parvovirus (BPV), and bovine viral diarrhea virus (BVDV) are the pathogens that cause diarrhea primarily in newborn calves. A mixed infection of BRV, BPV, and BVDV makes clinical diagnosis difficult. In this study, we designed dual-priming oligonucleotide (DPO) primers...

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Autores principales: Wang, Mengmeng, Yan, Yue, Wang, Ruichong, Wang, Li, Zhou, Han, Li, Yijing, Tang, Lijie, Xu, Yigang, Jiang, Yanping, Cui, Wen, Qiao, Xinyuan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920155/
https://www.ncbi.nlm.nih.gov/pubmed/31921061
http://dx.doi.org/10.3389/fmicb.2019.02884
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author Wang, Mengmeng
Yan, Yue
Wang, Ruichong
Wang, Li
Zhou, Han
Li, Yijing
Tang, Lijie
Xu, Yigang
Jiang, Yanping
Cui, Wen
Qiao, Xinyuan
author_facet Wang, Mengmeng
Yan, Yue
Wang, Ruichong
Wang, Li
Zhou, Han
Li, Yijing
Tang, Lijie
Xu, Yigang
Jiang, Yanping
Cui, Wen
Qiao, Xinyuan
author_sort Wang, Mengmeng
collection PubMed
description Bovine rotavirus (BRV), bovine parvovirus (BPV), and bovine viral diarrhea virus (BVDV) are the pathogens that cause diarrhea primarily in newborn calves. A mixed infection of BRV, BPV, and BVDV makes clinical diagnosis difficult. In this study, we designed dual-priming oligonucleotide (DPO) primers the VP6 gene of BRV, VP2 gene of BPV, and 5′UTR gene of BVDV and synthesized gold nanoparticles (GNPs) with an average diameter of 10 nm. We combined the DPOs with the GNPs to develop a DPO-nanoPCR assay for detecting BRV, BPV, and BVDV. The annealing temperature, primer concentration, and GNP concentration were optimized for this assay. Compared to a conventional PCR assay, the DPO-nanoPCR assay allowed the use of a wider range of annealing temperatures (41–65°C) to effectively amplify target genes. PCR amplification was the most efficient at 56.2°C using conventional primers. The optimal volume of all the primers (10 μM) was 1.0 μL. The optimal volume of GNPs (10 nM) for all the reactions was 0.5 μL. The detection limits of DPO-nanoPCR for pMD19-T-VP6, pMD19-T-VP2, and pMD19-T-5′UTR were 9.40 × 10(2) copies/μL, 5.14 × 10(3) copies/μL, and 4.09 × 10(1) copies/μL, respectively; and those using conventional PCR were 9.40 × 10(4) copies/μL, 5.14 × 10(5) copies/μL, and 4.09 × 10(4) copies/μL, respectively. The sensitivity of DPO-nanoPCR was at least 100-fold higher than that of conventional PCR. The specificity detection showed that the DPO-nanoPCR was able to specifically detect BRV, BPV, and BVDV. Use of clinical samples indicated that target viruses can be detected accurately. Thus, DPO-nanoPCR is a new powerful, simple, specific, and sensitive tool for detecting mixed infections of BRV, BPV, and BVDV.
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spelling pubmed-69201552020-01-09 Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System Wang, Mengmeng Yan, Yue Wang, Ruichong Wang, Li Zhou, Han Li, Yijing Tang, Lijie Xu, Yigang Jiang, Yanping Cui, Wen Qiao, Xinyuan Front Microbiol Microbiology Bovine rotavirus (BRV), bovine parvovirus (BPV), and bovine viral diarrhea virus (BVDV) are the pathogens that cause diarrhea primarily in newborn calves. A mixed infection of BRV, BPV, and BVDV makes clinical diagnosis difficult. In this study, we designed dual-priming oligonucleotide (DPO) primers the VP6 gene of BRV, VP2 gene of BPV, and 5′UTR gene of BVDV and synthesized gold nanoparticles (GNPs) with an average diameter of 10 nm. We combined the DPOs with the GNPs to develop a DPO-nanoPCR assay for detecting BRV, BPV, and BVDV. The annealing temperature, primer concentration, and GNP concentration were optimized for this assay. Compared to a conventional PCR assay, the DPO-nanoPCR assay allowed the use of a wider range of annealing temperatures (41–65°C) to effectively amplify target genes. PCR amplification was the most efficient at 56.2°C using conventional primers. The optimal volume of all the primers (10 μM) was 1.0 μL. The optimal volume of GNPs (10 nM) for all the reactions was 0.5 μL. The detection limits of DPO-nanoPCR for pMD19-T-VP6, pMD19-T-VP2, and pMD19-T-5′UTR were 9.40 × 10(2) copies/μL, 5.14 × 10(3) copies/μL, and 4.09 × 10(1) copies/μL, respectively; and those using conventional PCR were 9.40 × 10(4) copies/μL, 5.14 × 10(5) copies/μL, and 4.09 × 10(4) copies/μL, respectively. The sensitivity of DPO-nanoPCR was at least 100-fold higher than that of conventional PCR. The specificity detection showed that the DPO-nanoPCR was able to specifically detect BRV, BPV, and BVDV. Use of clinical samples indicated that target viruses can be detected accurately. Thus, DPO-nanoPCR is a new powerful, simple, specific, and sensitive tool for detecting mixed infections of BRV, BPV, and BVDV. Frontiers Media S.A. 2019-12-12 /pmc/articles/PMC6920155/ /pubmed/31921061 http://dx.doi.org/10.3389/fmicb.2019.02884 Text en Copyright © 2019 Wang, Yan, Wang, Wang, Zhou, Li, Tang, Xu, Jiang, Cui and Qiao. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Wang, Mengmeng
Yan, Yue
Wang, Ruichong
Wang, Li
Zhou, Han
Li, Yijing
Tang, Lijie
Xu, Yigang
Jiang, Yanping
Cui, Wen
Qiao, Xinyuan
Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System
title Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System
title_full Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System
title_fullStr Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System
title_full_unstemmed Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System
title_short Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System
title_sort simultaneous detection of bovine rotavirus, bovine parvovirus, and bovine viral diarrhea virus using a gold nanoparticle-assisted pcr assay with a dual-priming oligonucleotide system
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920155/
https://www.ncbi.nlm.nih.gov/pubmed/31921061
http://dx.doi.org/10.3389/fmicb.2019.02884
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