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

BMC Microbiology, Jan 2014

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.

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

BMC Microbiology Development of loop-mediated isothermal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus Zhixun Zhao 0 Bin Fan 0 Guohua Wu 0 Xinmin Yan 0 Yingguo Li 2 Xiaoli Zhou 1 Hua Yue 4 Xueling Dai 3 Haixia Zhu 0 Bo Tian 0 Jian Li 0 Qiang Zhang 0 0 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 1 Yunnan entry exit inspection and quarantine bureau , Kunming , PR China 2 Chongqing entry exit inspection and quarantine bureau , Chongqing , PR China 3 Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute , CAS, Lanzhou, Gansu , PR China 4 College of Life Science and Technology, Southwest University for Nationalities , Chengdu , PR China 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. Goat pox virus (GTPV); Sheep pox virus (SPPV); Inverted terminal repeat (ITR) regions; Loop-mediated isothermal amplification (LAMP); Differential diagnosis Background Sheep pox and goat pox are economically important diseases in goat and sheep producing areas of the world. Sheep pox and goat pox result from infection by SPPV or GTPV respectively, and are closely related members of the Capripoxvirus genus in the family Poxviridae. Clinically, sheep pox and goat pox have indistinguishable symptoms. Several PCR-based assays have been reported to distinguish SPPV from GTPV including cleaved amplification polymorphism sequence-tagged sites (RFLPPCR) and real-time PCR (qPCR) [ 1-5 ]. In our laboratory, distinction of GTPV from SPPV was established via a Hinf I digest of the p32 gene, followed by a sequence alinment of G-protein-coupled chemokine receptor (GpCR) genes [ 5,6 ]. Some of the advantages of qPCR include speed, sensitivity, and real time monitoring to determine exact concentrations. However, this approach requires expensive high precision instrumentation and specialized training for operation and data analysis, presenting a need for a more convenient alternative that is robust, inexpensive, and easy to operate and maintain. Recently, loop-mediated isothermal amplification (LAMP) has been developed for the diagnosis of a number of diseases [ 1,7,8 ]. The LAMP reaction can be conducted under isothermal conditions ranging 60-65°C by using four or six primers recognizing six or eight distinct regions [ 9 ]. LAMP produces large quantities of amplified product resulting in easy visual detection either via turbidity or fluorescence [ 10 ]. The present study established the ability of LAMP assays to differential detect GTPV and SPPV through the targeting of inverted terminal repeat (ITR) sequences. Compared to conventional PCR techniques, the newly established LAMP assay is simple, efficient, cost-effective and convenient, making it a useful diagnostic tool for clinical samples. Results Primers and gene sequences Several GTPV and SPPV genomic sequences were downloaded from GenBank and aligned using MegAlign, with the most conserved ITR segment (...truncated)


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Zhixun Zhao, Bin Fan, Guohua Wu, Xinmin Yan, Yingguo Li, Xiaoli Zhou, Hua Yue, Xueling Dai, Haixia Zhu, Bo Tian, Jian Li, Qiang Zhang. Development of loop-mediated isothermal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus, BMC Microbiology, 2014, pp. 10, 14, DOI: 10.1186/1471-2180-14-10