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      Evaluation of a multiplex PCR method for the detection of porcine parvovirus types 1 through 7 using various field samples

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          Abstract

          Porcine parvoviruses (PPVs) are small, nonenveloped DNA viruses that are widespread in the global pig population. PPV type 1 (PPV1) is a major causative agent of reproductive failure and has been recognized since the 1960s. In recent decades, novel PPVs have been identified and designated as PPVs 2 through 7 (PPV2~PPV7). Although the epidemiological impacts of these newly recognized parvoviruses on pigs are largely unknown, continuous surveillance of these PPVs is needed. The aim of this study was to develop an improved and efficient detection tool for these PPVs and to assess the developed method with field samples. Using 7 sets of newly designed primers, a multiplex polymerase chain reaction (mPCR) protocol was developed for the simultaneous detection of the seven genotypes of PPV (PPV1~PPV7). The sensitivity of the mPCR assay was analyzed, and the detection limit was determined to be 3×10 3 viral copies. The assay was highly specific in detecting one or more of the viruses in various combinations in specimens. The mPCR method was evaluated with 80 serum samples, 40 lung or lymph node samples and 40 intestine or fecal samples. When applied to these samples, the mPCR method could detect the 7 viruses simultaneously, providing rapid results regarding infection and coinfection status. In conclusion, the developed mPCR assay can be utilized as an effective and accurate diagnostic tool for rapid differential detection and epidemiological surveillance of various PPVs in numerous types of field samples.

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          Rates of evolutionary change in viruses: patterns and determinants.

          Laura A. Shackelton, Siobain Duffy, Edward C Holmes (2008)
          Understanding the factors that determine the rate at which genomes generate and fix mutations provides important insights into key evolutionary mechanisms. We review our current knowledge of the rates of mutation and substitution, as well as their determinants, in RNA viruses, DNA viruses and retroviruses. We show that the high rate of nucleotide substitution in RNA viruses is matched by some DNA viruses, suggesting that evolutionary rates in viruses are explained by diverse aspects of viral biology, such as genomic architecture and replication speed, and not simply by polymerase fidelity.
          Article 0 comments Cited 188 times – based on 0 reviews     Review now
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            Multiplex polymerase chain reaction: a practical approach.

            P Markoulatos, N Siafakas, M Moncany (2002)
            Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features. Copyright 2002 Wiley-Liss, Inc.
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              Discovery of a novel Parvovirinae virus, porcine parvovirus 7, by metagenomic sequencing of porcine rectal swabs.

              Namita Mitra, Ben M. Hause, Rachel Palinski (2016)
              Parvoviruses are a diverse group of viruses containing some of the smallest known species that are capable of infecting a wide range of animals. Metagenomic sequencing of pooled rectal swabs from adult pigs identified a 4103-bp contig consisting of two major open reading frames encoding proteins of 672 and 469 amino acids (aa) in length. BLASTP analysis of the 672-aa protein found 42.4 % identity to fruit bat (Eidolon helvum) parvovirus 2 (EhPV2) and 37.9 % to turkey parvovirus (TuPV) TP1-2012/HUN NS1 proteins. The 469-aa protein had no significant similarity to known proteins. Genetic and phylogenetic analyses suggest that PPV7, EhPV2, and TuPV represent a novel genus in the family Parvoviridae. Quantitative PCR screening of 182 porcine diagnostic samples found a total of 16 positives (8.6 %). Together, these data suggest that PPV7 is a highly divergent novel parvovirus prevalent within the US swine.
              Article 0 comments Cited 53 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Seung-Chai Kim: Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draft
                Chang-Gi Jeong: Role: ValidationRole: Writing – original draft
                Salik Nazki:
                ORCID: https://orcid.org/0000-0002-2837-3365
                Role: ConceptualizationRole: Data curationRole: Methodology
                Sim-In Lee: Role: InvestigationRole: Resources
                Ye-Chan Baek: Role: InvestigationRole: Resources
                Yong-Jin Jung: Role: InvestigationRole: Resources
                Won-Il Kim:
                ORCID: https://orcid.org/0000-0002-0465-0794
                Role: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Simon Clegg: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS One
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 28 January 2021
                Publication date Collection: 2021
                Volume: 16
                Issue: 1
                Electronic Location Identifier: e0245699
                Affiliations
                [1 ] College of Veterinary Medicine, Jeonbuk National University, Iksan, Korea
                [2 ] The Pirbright Institute, Pirbright, United Kingdom
                [3 ] Animal and Plant Quarantine Agency, Gimcheon, Korea
                University of Lincoln, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-2837-3365
                https://orcid.org/0000-0002-0465-0794
                Article
                Publisher ID: PONE-D-20-32226
                DOI: 10.1371/journal.pone.0245699
                PMC ID: 7842984
                PubMed ID: 33508002
                SO-VID: cd43bdad-3a2a-4a96-ba57-6fc3cbcfefdc
                Copyright © © 2021 Kim et al
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 15 October 2020
                Date accepted : 5 January 2021
                Page count
                Figures: 4, Tables: 2, Pages: 13
                Funding
                This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Animal Disease Management Technology Development Program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (118093-03).
                Categories
                Subject: Research Article
                Subject: Biology and Life Sciences
                Subject: Molecular Biology
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Research and Analysis Methods
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Digestive System
                Subject: Gastrointestinal Tract
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Digestive System
                Subject: Gastrointestinal Tract
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Eukaryota
                Subject: Animals
                Subject: Vertebrates
                Subject: Amniotes
                Subject: Mammals
                Subject: Swine
                Subject: Biology and Life Sciences
                Subject: Zoology
                Subject: Animals
                Subject: Vertebrates
                Subject: Amniotes
                Subject: Mammals
                Subject: Swine
                Subject: Biology and life sciences
                Subject: Organisms
                Subject: Viruses
                Subject: DNA viruses
                Subject: Parvoviruses
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Viral Pathogens
                Subject: Parvoviruses
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Pathogens
                Subject: Microbial Pathogens
                Subject: Viral Pathogens
                Subject: Parvoviruses
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Viruses
                Subject: Viral Pathogens
                Subject: Parvoviruses
                Subject: Medicine and Health Sciences
                Subject: Medical Conditions
                Subject: Infectious Diseases
                Subject: Bacterial Diseases
                Subject: Salmonella
                Subject: Salmonella Enterica
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Bacterial Pathogens
                Subject: Salmonella
                Subject: Salmonella Enterica
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Pathogens
                Subject: Microbial Pathogens
                Subject: Bacterial Pathogens
                Subject: Salmonella
                Subject: Salmonella Enterica
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Bacteria
                Subject: Enterobacteriaceae
                Subject: Salmonella
                Subject: Salmonella Enterica
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Lymphatic System
                Subject: Lymph Nodes
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Lymphatic System
                Subject: Lymph Nodes
                Subject: Research and Analysis Methods
                Subject: Electrophoretic Techniques
                Subject: Gel Electrophoresis
                Subject: Agarose Gel Electrophoresis
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Bacterial Pathogens
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Pathogens
                Subject: Microbial Pathogens
                Subject: Bacterial Pathogens
                Custom metadata
                Data Availability All relevant data are within the paper and its Supporting Information files.

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