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Comparative Study
. 2017 Aug 31;13(1):278.
doi: 10.1186/s12917-017-1179-0.

Development of duplex PCR for differential detection of goatpox and sheeppox viruses

Zhixun Zhao  1 , Guohua Wu  1 , Xinmin Yan  1 , Xueliang Zhu  1 , Jian Li  1 , Haixia Zhu  1 , Zhidong Zhang  2 , Qiang Zhang  3
Affiliations
Comparative Study

Development of duplex PCR for differential detection of goatpox and sheeppox viruses

Zhixun Zhao et al. BMC Vet Res. .

Abstract

Background: Clinically, sheeppox and goatpox have the same symptoms and cannot be distinguished serologically. A cheaper and easy method for differential diagnosis is important in control of this disease in endemic region.

Methods: A duplex PCR assay was developed for the specific differential detection of Goatpox virus (GTPV) and Sheeppox virus (SPPV), using two sets of primers based on viral E10R gene and RPO132 gene.

Results: Nucleic acid electrophoresis results showed that SPPV-positive samples appear two bands, and GTPV-positive samples only one stripe. There were no cross-reactions with nucleic acids extracted from other pathogens including foot-and-mouth disease virus, Orf virus. The duplex PCR assay developed can specially detect SPPV or GTPV present in samples (n = 135) collected from suspected cases of Capripox.

Conclusions: The duplex PCR assay developed is a specific and sensitive method for the differential diagnosis of GTPV and SPPV infection, with the potential to be standardized as a detection method for Capripox in endemic areas.

Keywords: Differential diagnosis; Duplex PCR assay; Goatpox virus; Sheeppox virus.

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Conflict of interest statement

Ethics approval

This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval number LVRIAEC 2012–018). Goats and sheep, from which tissues samples were collected, were handled with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China (AEPGPRC). The collection of tissues samples was performed as part of routine process of disease monitoring and surveillance for these goats and sheep. The owners of goats and sheep had given permission for the collection of tissues samples.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Target gene sequences and primers. Nucleotide sequences of the E10R and RPO132 amplicon (E10R, RPO132 GenBank accession no. AY077832.1) and locations of the primers along the sequence
Fig. 2
Fig. 2
Optimization of annealing temperature (Tm) for duplex PCR reaction in the detection of GTPV or SPPV using mix primers. Agarose gel electrophoresis showing the effect of Tm on duplex PCR. a Duplex PCR amplicated products using 100 ng SPPV gDNA as template. b Duplex PCR amplicated products using 100 ng GTPV gDNA as template. Lane 1–6 is 46 °C, 48 °C, 50 °C, 52 °C, 54 °C and 56 °C, respectively; Lane M: 2000 bp DNA Ladder Marker (TaKaRa, Dalian) and Lane C: No template control
Fig. 3
Fig. 3
Optimization of primers rate for duplex PCR reaction. Lane 1–5 and 1′-5′: E10R primers is 0.5 μM, RPO132 primers is 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM, respectively; Lane 6–10 and 6′-10′: RPO132 primers is 0.5 μM, E10R primers is 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM, respectively. Lane1–5 and 6–10: 100 ng SPPV genome as templates; Lane1’-5’and 6′-10′: 100 ng GTPV genome as templates, respectively. Lane C and C′: No template control. Lane M:2000 bp DNA Ladder Marker (TaKaRa, Dalian). The strip in red grid showed the result is better using 1 μM RPO132 primers and 0.2 μM E10R primers in the reaction
Fig. 4
Fig. 4
Optimization of reaction cycles for duplex PCR reaction in the detection of GTPV or SPPV using mix primers. Agarose gel electrophoresis showing the effect of cycles on duplex PCR. a Duplex PCR amplicated products using 100 ng SPPV gDNA as template. b Duplex PCR amplicated products using 100 ng GTPV gDNA as template. Lane 1–5: Reaction annealing temperature is 50 °C for 20 cycles, 25 cycles, 30 cycles, 35 cycles and 40 cycles; Lane C: No template control and Lane M: 2000 bp DNA Ladder Marker (TaKaRa, Dalian)
Fig. 5
Fig. 5
Duplex PCR sensitivity. Amplification using different concentration gradient of the gene as template, nucleic acid electrophoresis test results. a Lane 1–10: SPPV gDNA concentration gradient for 1.011 × 1010–1.011 × 100 copies as template, (b) Lane 1–10: GTPV gDNA concentration gradient for 1.043 × 1010–1.043 × 100 copies as template, respectively. Lane M: 2000 bp DNA Ladder Marker (TaKaRa, Dalian) and Lane C: No template control
Fig. 6
Fig. 6
Specificity of duplex PCR for detection different pathogen nucleic acid. Aboat 100 ng DNA or cDNA template of ten different sheep or goat pathogens were used in LAMP reaction. Agarose gel electrophoresis (1%) of PCR products stained with Ethidium bromide and visualized under UV transilluminator. Lane 1: SPPV; Lane 2: GTPV; Lane 3: Orf virus; Lane 4: FMDV O/China 99; Lane 5: M. ovippneumoniae; Lane 6: Chlamydia psittaci; Lane 7: L.interrogans; Lane 8: Toxoplasma gondii; Lane 9: Babesia; C: No template control and Lane M: 2000 bp DNA Ladder Marker (TaKaRa, Dalian)
Fig. 7
Fig. 7
Single PCR and duplex PCR for detection different GTPV and SPPV nucleic acid using E10R primers and RPO132 primers or mix primers. Lane 1–3: single PCR detect 100 ng SPPV gDNA, 100 ng GTPV gDNA and no template using RPO132 primers; Lane 4–6: single PCR detect 100 ng SPPV gDNA, 100 ng GTPV gDNA and no template using E10R primers; Lane 7–9: duplex PCR detect 100 ng SPPV gDNA, 100 ng GTPV gDNA and no template using mixture of RPO132 primers and E10R primers. Lane M: 2000 bp DNA Ladder Marker (TaKaRa, Dalian)
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