Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar 4;14(3):e0213295.
doi: 10.1371/journal.pone.0213295. eCollection 2019.

Multiplex PCR methods for detection of several viruses associated with canine respiratory and enteric diseases

Xiangqi Hao  1   2   3 , Ruohan Liu  1   2   3 , Yuwei He  1   2   3 , Xiangyu Xiao  1   2   3 , Weiqi Xiao  1   2   3 , Qingxu Zheng  1   2   3 , Xi Lin  1   2   3 , Pan Tao  1   2   3 , Pei Zhou  1   2   3 , Shoujun Li  1   2   3
Affiliations

Multiplex PCR methods for detection of several viruses associated with canine respiratory and enteric diseases

Xiangqi Hao et al. PLoS One. .

Abstract

Viral respiratory and intestinal infections are the most common causes of canine viral illness. Infection with multiple pathogens occurs in many cases. Rapid diagnosis of these multiple infections is important for providing timely and effective treatment. To improve diagnosis, in this study, two new multiplex polymerase chain reactions (mPCRs) were developed for simultaneous detection of canine respiratory viruses (CRV) and canine enteric viruses (CEV) using two separate primer mixes. The viruses included canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine circovirus (CanineCV), canine coronavirus (CCoV) and canine parvovirus (CPV). The sensitivity of the mPCR results showed that the detection limit of both mPCR methods was 1×104 viral copies. Twenty nasal swabs (NS) and 20 anal swabs (AS) collected from dogs with symptoms of respiratory disease or enteric disease were evaluated using the novel mPCR methods as a clinical test. The mPCR protocols, when applied to these respiratory specimens and intestinal samples, could detect 7 viruses simultaneously, allowing rapid investigation of CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV) status and prompt evaluation of coinfection. Our study provides an effective and accurate tool for rapid differential diagnosis and epidemiological surveillance in dogs.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Optimal annealing temperatures in mPCRs.
(A) Lane M, 100 bp DNA ladder; Lane NC, negative control for detection of CRV; Lanes 1~12, gradient annealing temperatures were 55.0°C, 55.5°C, 56.0°C, 57.0°C, 58.2°C, 59.4°C, 60.6°C, 61.8°C, 63.0°C, 64.0°C, 64.5°C, and 65.0°C, respectively. (B) Lane M, 100 bp DNA ladder; Lane NC, negative control for detection of CEV; Lanes 1~12, gradient annealing temperatures were 55.0°C, 55.5°C, 56.0°C, 57.0°C, 58.2°C, 59.4°C, 60.6°C, 61.8°C, 63.0°C, 64.0°C, 64.5°C, and 65.0°C, respectively.
Fig 2
Fig 2. Specificity of the mPCR methods.
Agarose gel electrophoresis (1.5%) of specific fragments amplified by mPCRs from the proviral DNAs and cDNA of MDCK cells, E. coli, Salmonella enterica and RABV. (A) CRV mPCR: Lane M, 100 bp DNA ladder; Lane NC, negative control; Lane 1, E. coli; Lane 2, Salmonella enterica; Lane 3, RABV; Lane 4, MDCK cells; Lanes 5~8, pMD-CPIV, pMD-CIV, pMD-CDV, and pMD-CAV-2; Lane 9, mixed standard of pMD-CPIV/pMD-CIV/pMD-CDV/pMD-CAV-2 plasmids. (B) CEV mPCR: Lane M, 100 bp DNA ladder; Lane NC, negative control; Lane 1, E. coli; Lane 2, Salmonella enterica; Lane 3, RABV; Lane 4, MDCK cells; Lanes 5~8, pMD-CPV, pMD-CCoV, pMD-CanineCV, and pMD-CAV-2; Lane 9, mixed standard of pMD-CPV/pMD-CCoV/pMD-CanineCV/pMD-CAV-2 plasmids.
Fig 3
Fig 3. Sensitivity of the mPCR method for CRV detection.
The four single plasmids (pMD-CAV-2, pMD-CDV, pMD-CIV and pMD-CPIV), diluted from 1×1010 to 1×101 copies/μl, and the mixed plasmids (pMD-CAV-2/pMD-CDV/pMD-CIV/pMD-CPIV), diluted from 1×109 to 1×101 copies/μl, were used to determine the minimum detection limit of the CRV mPCR method. (A) The sensitivity of pMD-CAV-2; (B) the sensitivity of pMD-CDV; (C) the sensitivity of pMD-CIV; (D) the sensitivity of pMD-CPIV; (E) the sensitivity of pMD-CAV-2/pMD-CDV/pMD-CIV/pMD-CPIV; Lane M, 100 bp DNA ladder; Lane NC, negative control.
Fig 4
Fig 4. Sensitivity of the mPCR method for CEV detection.
The four single plasmids (pMD-CAV-2, pMD-CanineCV, pMD-CCoV and pMD-CPV), diluted from 1×1010 to 1×101 copies/μl, and the mixed plasmids (pMD-CAV-2/pMD-CanineCV/pMD-CCoV/pMD-CPV), diluted from 1×109 to 1×101 copies/μl, were used to determine the minimum detection limit of the CEV mPCR method. (A) The sensitivity of pMD-CAV-2; (B) the sensitivity of pMD-CanineCV; (C) the sensitivity of pMD-CCoV; (D) the sensitivity of pMD-CPV; (E) the sensitivity of pMD-CAV-2/pMD-CDV/pMD-CIV/pMD-CPIV; Lane M, 100 bp DNA ladder; Lane NC, negative control.
Fig 5
Fig 5. Reproducibility of the mPCR methods.
Mixed CRV and CEV plasmids were diluted as templates from 1×105 to 1×103 copies/μl to amplify specific fragments by using three different PCR instruments at different times. (A) CRV: Lane M, 100 bp DNA ladder; Lanes 1, 5, and 9, negative controls for the three tests; Lanes 2~4, mPCR amplifying 1×105 copies/μl, 1×104 copies/μl, and 1×103 copies/μl CRV plasmids; Lanes 6~8, mPCR amplifying different dilutions of mixed plasmids a second time; Lanes 10~12, mPCR amplifying different dilutions of mixed plasmids a third time. (B) CEV: Lane M, 100 bp DNA ladder; Lanes 1, 5, and 9, negative controls for the three tests; Lanes 2~4, mPCR amplifying 1×105 copies/μl, 1×104 copies/μl, and 1×103 copies/μl CEV plasmids; Lanes 6~8, mPCR amplifying different dilutions of mixed plasmids a second time; Lanes 10~12, mPCR amplifying different dilutions of mixed plasmids a third time.
Fig 6
Fig 6. Evaluation of clinical samples.
20 NS and 20 AS clinical samples were tested by established mPCRs respectively. (A) CRV: Lane M, 100 bp DNA ladder; Lanes PC, positive controls; Lanes NC, negative controls; Lanes 1~20, multiplex PCR tested on NS samples. (B) CEV: Lane M, 100 bp DNA ladder; Lanes PC, positive controls; Lanes NC, negative controls; Lanes 1~20, multiplex PCR tested on AS samples.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pesavento PA, Murphy BG. Common and emerging infectious diseases in the animal shelter. Veterinary pathology. 2014;51(2):478–91. 10.1177/0300985813511129 . - DOI - PubMed
    1. Buonavoglia C, Martella V. Canine respiratory viruses. Veterinary research. 2007;38(2):355–73. 10.1051/vetres:2006058 . - DOI - PubMed
    1. Erles K, Dubovi EJ, Brooks HW, Brownlie J. Longitudinal study of viruses associated with canine infectious respiratory disease. J Clin Microbiol. 2004;42(10):4524–9. 10.1128/JCM.42.10.4524-4529.2004 - DOI - PMC - PubMed
    1. Jeoung HY, Song DS, Jeong WS, Lee WH, Song JY, An DJ. Simultaneous detection of canine respiratory disease associated viruses by a multiplex reverse transcription-polymerase chain reaction assay. The Journal of veterinary medical science. 2013;75(1):103–6. . - PubMed
    1. Posuwan N, Payungporn S, Thontiravong A, Kitikoon P, Amonsin A, Poovorawan Y. Prevalence of respiratory viruses isolated from dogs in Thailand during 2008–2009. Asian Biomedicine. 2010;4(4):563–9.

Publication types

MeSH terms

Grants and funding

This work was supported in part by the National Natural Science Foundation of China (31672563, 31802204), The National Key Research and Development Program of China (2016YFD0501004), The Natural Science Foundation Guangdong province (2018B030311037, 2018A030313633), The Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases (2017B030314142).