Estudo molecular de pestivirus em amostras de cultura celular e soro de pequenos ruminantes

Abstract

The Ruminants Pestiviruses are often associated with temporary and subclinical or mild clinical signs, with the largest losses related to the infection of pregnant females that can cause fetal loss and birth of persistently infected (PI) animals. The Pestivirus are distributed in many countries, including Brazil, where studies in goats and sheep are scarce. Several studies have reported the use of molecular techniques for detection of Pestivirus, among them, the RT-PCR and the RT-qPCR.They are able to detect PI animals, which are the main source of the virus-Care in the herd. The objective of this study was to determined the lower limit of detection for Pestivirus from supernatant cell culture and serum samples from small ruminants using RT-PCR and qRT-PCR. The extraction of nucleic acids was performed with a commercial kit QIAamp ® MinElute ® Virus Spin, the reverse transcriptase reaction followed the protocol recommended by the manufacturer of the enzyme reverse transcriptase M-MLV. The evaluation of the detection limit of RT-qPCR and RT-PCR was performed using a Pestivirus calibration curve (dilution factor 10) with known and decreasing TCID50/mL called panpestivirus using primers (Vilcek et al., 1994).The detection limit of RT-qPCR in cell culture supernatant was 10-3 TCID50/mL and of RT-PCR was 10.2 TCID50/mL, since the lower limit of detection using sera weeding, sheep and Gibco ® Lamb as a diluent were TCID50/mL. The tests conducted under the conditions described in this study proved to be sensitive and can be used as a strategy for etiologic diagnosis of Pestivirus, especially for the molecular identification of IP animals, which is essential for the control of Pestiviroses.