Development of quantification kit for bovine viral diarrhea virus

Introduction and Objectives: Bovine viral diarrhea virus (BVDV), one of the most bovine pathogens, is belong to the genus Pestivirus and family of Flaviviridae. Infection with this virus is associated with a wide range of clinical or subclinical manifestations, including transient fever, diarrhea, respiratory problems, abortion and teratogenesis. The aim of this research was development of quantification kit for detection of bovine viral diarrhea virus. Materials and Methods: Total RNA was extracted from BVDV-infected cell culture and reversed to cDNA by TAKARA cDNA synthesized kit. The 5′ untranslated region of BVDV was amplified using PCR method and cloned into T/A cloning plasmid. The recombinant plasmid was propagated and purified from competent cells Escherichia coli DH5a. Ten-fold serial dilutions of the recombinant plasmid was prepared and standard curves were generated by TaqMan Real-Time PCR. Results: The detection limit of cDNA BVDV copy numbers per μl was 1-10 copy and was applied for infected cell cultures and clinical samples. Conclusion: This method can detect and quantify BVDV RNA in cell culture or samples with higher specificity and sensitivity than conventional RT-PCR.

کلیدواژه ها:

TaqMan Real-Time RT-PCR ، Bovine viral Diarrhea virus ، quantification kit