Establishment Of Real Time Fluorescence Quantitative PCR Diagnostic Method For Porcine Rotatirus

Porcine Rotavirus(PRV) belongs to the family reoviridae,species rotavirus.It is one of the major pathogens that cause life-threatening diarrhea in piglets.The young animals are the most easily infected.Its infectious ratio is over 80%,and its death rate is 7～20% in 1～4 weekage pigs.Rotavirus diarrhea's typically clinical manifestation is serious diarrhea.Partial infected piglets were dead due to severe loss of water,imbalance of acid alkali and secondary infection.accurate diagnosis and effective vaccination are main means for the prevention of porcine diarrhea, so,early diagnosis for the prevention of rotavirus are particularly important.In this study,porcine rotavirus was successfully isolated from diarrheal piglet fecal samples from Gansu province,and PRV was propagated on MA-104 monolayer cell. accordance with the porcine rotavirus strains vp6 gene coding sequence which has been submitted in Genebank, and in accordance with this sequence,using primers premier 5.0 software design Primer. Samples collected on the treatment and viral RNA extraction, take the Oligo (dT)18 as primers in RT-PCR, Reverse transcription of RNA to cDNA, Then to design a pair of primers and suitable conditions for PCR amplification, approximately 1100bp amplified fragment of the gene.To develop a more sensitive and specific diagnostic diagnostic test for rotavirus by applying real-time quantitative RT-PCR technology is our objectives.however,the majority of which reported to date have been used in a quanlitative format.Real-time PCR using SYBR Green I dye as fluorimetry it has proved it self a valuable tool in true quantitation of different taeget nucleic acids at ptesent,and it has engendered wider acceptance of the PCR due to its improved rapidity,sensitivity,reproducibility and the reduced risk of carry-over contamination.We get a table in the end , Negative control sample and the fluorescence curves are the same straight line, and the fluorescence curve of positive samples, there is a visible rise, and rise in the level with the template curve was directly proportional to the concentration. Positive samples at this time of the curve more smooth, and can be seen an initial "S" curve.This study procides the basis evidence and material basis for PRV molecular epidemiology investigation,research of diagnostic reagent and genetic engineering vaccine.