Detection of Classical swine fever virus in various types of clinical samples by RT-PCR

Classical swine fever (CSF) is an economically important Office International des Epizooties (OIE) list A disease of swine which is characterized by high fever and multiple hemorrhages. A rapid method of reverse transcription-polymerase chain reaction (RT-PCR), for detection of classical swine fever virus in clinical samples, was developed using E2 and 5’NTR region specific primers. Further, the nested PCR was standardized using primary amplicon as template to enhance the sensitivity and specificity of PCR. The nested PCR resulted in a band of 271 bp with both E2 and 5’NTR specific primers. The PCR was confirmed by electrophoresis in 1.5% agarose gel and sequencing of nested PCR product. Later, the test was applied to screen CSFV in suspected clinical materials, which included spleen, mesenteric lymph nodes, tonsils, part of intestine, blood, etc. These samples were comprised of clinical tissues collected on ice, preserved in formalin or buffered glycerol, decomposed tissues, tissue homogenate kept at room temperature for more than 20 days with out any preservative and PK-15 adapted cell culture virus. Over 202 samples collected from different states of country including Assam, Mizoram, Bihar, Haryana, Punjab, Uttar Pradesh and Maharashtra were processed. Out of which 92 samples were found positive by RT-PCR. From this study, it was concluded that for CSFV-RT-PCR analysis, clinical samples can be transported or stored at room temperature without any preservatives, even in tropical countries and also it is mandatory to clean-up RNA isolated from decomposed tissues with either RNeasy or any other commercially available kits.