A real time high-resolution melting PCR assay for detection and differentiation among sheep pox virus, goat pox virus, field and vaccine strains of lumpy skin disease virus
| Autor: | Alexander Kononov, A.V. Sprygin, Olga Byadovskaya, Yana Pestova |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
040301 veterinary sciences Nucleic Acid Denaturation Real-Time Polymerase Chain Reaction Melting curve analysis Capripoxvirus High Resolution Melt 0403 veterinary science 03 medical and health sciences chemistry.chemical_compound Species Specificity Lumpy skin disease medicine Animals Molecular Biology biology Base Sequence Viral Vaccines 04 agricultural and veterinary sciences Cell Biology Assay sensitivity Amplicon medicine.disease biology.organism_classification Virology Lumpy skin disease virus 030104 developmental biology chemistry DNA |
| Zdroj: | Molecular and cellular probes. 41 |
| ISSN: | 1096-1194 |
| Popis: | In this paper, we report on the development of a real time high-resolution melting (HRM) PCR assay for detection and differentiation among sheep pox virus (SPPV), goat pox virus (GTPV), field isolates and vaccine strains of lumpy skin disease virus (LSDV) based on high-resolution melting curve analysis of their target PCR amplicons. A 111 bp region of LSDV010 ORF, which harbors unique genetic differences for each of these viral species, was selected as the PCR target in this study. During the validation of this assay using DNA from clinical isolates originated from naturally infected animals from the different geographic locations and reference strains, the obtained PCR amplicons demonstrated that the melting temperature picks were specific for each tested viral species, i.e., 74.56 ± 0.04 °C for field LSDV, 74.95 ± 0.08 °C for vaccine LSDV, 74.24 ± 0.06 °C for SPPV and 73.61 ± 0.04 °C for GTPV. The assessment of the assay sensitivity utilizing a LSDV field strain as a PCR template revealed the assay detection limit as low as 0.1 TCD50 lg/ml. Overall, this assay based on Rotor-Gene Q (QIAGEN) platform was shown to be reproducible across replicates and operators and can be recommended as an additional diagnostic tool to the currently available molecular assays for detection and differentiation of the genus Capripoxvirus species, including the differentiation of vaccine strains of LSDV from field isolates. The assay can be used for detection of these viruses in animal- and insect-derived field specimens. |
| Databáze: | OpenAIRE |
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