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Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus

  • Qian Zhang
  • Jiangting Niu
  • Shushuai Yi
  • Guoying Dong
  • Dejing Yu
  • Yanbing Guo
  • Hailong Huang
  • Guixue HuEmail author
Original Article

Abstract

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per μl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.

Notes

Funding

This study was supported by the Research Project of the National Key Research and Development Plan of China (Grant no. 2016YFD0501002). This work was also supported by the National Key R&D Program of China (Grant no. 2017YFD0501703).

Compliance with ethical standards

Conflict of interest

All the authors declare that they have no conflict of interest.

Ethical approval

All animal experiments conformed to the guidelines and regulatory requirements established by the Animal Care and Use Committee of Jilin Agricultural University, Jilin Province, China. Owners’ consent was obtained to collect samples from healthy and diarrheic cats.

Supplementary material

705_2019_4394_MOESM1_ESM.tif (4.5 mb)
Supplementary Fig. 1 Optimization of the annealing temperature (A) and the number of cycles (B) for multiplex PCR for the detection of FPV, FBoV and FeAstV using mixed primers. (a) Lanes 1-10: 1, 50°C; 2, 51°C; 3, 52°C; 4, 53°C; 5, 55°C; 6, 57°C; 7, 59°C; 8, 60°C; 9, 61°C; 10, 62°C; M, DL2000 DNA marker. (b) Lanes 1-5: 1, 30 cycles; 2, blank; 3, 35 cycles; 4, blank; 5, 40 cycles; M, DL2000 DNA marker (TIFF 4563 kb)
705_2019_4394_MOESM2_ESM.tif (6.4 mb)
Supplementary Fig. 2 Optimization of the reagent concentration for multiplex PCR for the detection of FPV, FBoV and FeAstV using mixed primers. (a) Optimization of the concentration of primers. Lanes 1-6: 1, 0.25 μl; 2, 0.5 μl; 3, 0.75 μl; 4, 1 μl; 5, 1.5 μl; 6, 2 μl; M, DL2000 DNA marker. (b) Optimization of the concentration of the dNTP mixture. Lanes 1-7: 1, 1 μl; 2, 1.5 μl; 3, 2 μl; 4, 2.5 μl; 5, 3 μl; 6, 3.5 μl; 7, 4 μl; M, DL2000 DNA marker. (c) Optimization of the concentration of Ex Taq DNA polymerase. Lanes 1-6: 1, 0.3 μl; 2, 0.4 μl; 3, 0.5 μl; 4, 0.6 μl; 5, 0.8 μl; 6, 1 μl; M, DL2000 DNA marker (TIFF 6527 kb)
705_2019_4394_MOESM3_ESM.tif (3.3 mb)
Supplementary Fig. 3 Ability of multiplex PCR to detect different genotypes of FPV, FBoV and FeAstV. Lanes 1-10: 1, FPV; 2, CPV-2; 3, CPV-2a; 4, CPV-2b; 5, FBoV-1; 6, FBoV-2; 7, FBoV-3; 8, FeAstV-1; 9, FeAstV-2; 10, Negative control; M, DL2000 DNA marker (TIFF 3397 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Qian Zhang
    • 1
  • Jiangting Niu
    • 1
  • Shushuai Yi
    • 1
  • Guoying Dong
    • 2
  • Dejing Yu
    • 1
  • Yanbing Guo
    • 1
    • 3
  • Hailong Huang
    • 1
  • Guixue Hu
    • 1
    Email author
  1. 1.College of Animal Science and TechnologyJilin Agricultural UniversityChangchunChina
  2. 2.College of Global Change and Earth System ScienceBeijing Normal UniversityBeijingChina
  3. 3.Jilin Institute of Animal Husbandry and Veterinary ScienceChangchunChina

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