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Open Access Methodology article

Development of loop-mediated isothermal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus

Zhixun Zhao1, Bin Fan1, Guohua Wu1, Xinmin Yan1, Yingguo Li2, Xiaoli Zhou3, Hua Yue4, Xueling Dai5, Haixia Zhu1, Bo Tian1, Jian Li1 and Qiang Zhang1*

Author Affiliations

1 Key Laboratory of Animal virology of the Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu, PR China

2 Chongqing entry exit inspection and quarantine bureau, Chongqing, PR China

3 Yunnan entry exit inspection and quarantine bureau, Kunming, PR China

4 College of Life Science and Technology, Southwest University for Nationalities, Chengdu, PR China

5 Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, CAS, Lanzhou, Gansu, PR China

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BMC Microbiology 2014, 14:10  doi:10.1186/1471-2180-14-10

Published: 17 January 2014

Abstract

Background

Capripox viruses are economically important pathogens in goat and sheep producing areas of the world, with specific focus on goat pox virus (GTPV), sheep pox virus (SPPV) and the Lumpy Skin Disease virus (LSDV). Clinically, sheep pox and goat pox have the same symptoms and cannot be distinguished serologically. This presents a real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Capripox outbreaks.

Results

A LAMP method was developed for the specific differential detection of GTPV and SPPV using three sets of LAMP primers designed on the basis of ITR sequences. Reactions were performed at 62°C for either 45 or 60 min, and specificity confirmed by successful differential detection of several GTPV and SPPV isolates. No cross reactivity with Orf virus, foot-and-mouth disease virus (FMDV), A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi or Babesia sp was noted. RFLP-PCR analysis of 135 preserved epidemic materials revealed 48 samples infected with goat pox and 87 infected with sheep pox, with LAMP test results showing a positive detection for all samples. When utilizing GTPV and SPPV genomic DNA, the universal LAMP primers (GSPV) and GTPV LAMP primers displayed a 100% detection rate; while the SPPV LAMP detection rate was 98.8%, consistent with the laboratory tested results.

Conclusions

In summary, the three sets of LAMP primers when combined provide an analytically robust method able to fully distinguish between GTPV and SPPV. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of GTPV and SPPV infections, with the potential to be standardized as a detection method for Capripox viruses in endemic areas.

Keywords:
Goat pox virus (GTPV); Sheep pox virus (SPPV); Inverted terminal repeat (ITR) regions; Loop-mediated isothermal amplification (LAMP); Differential diagnosis