Establishment Of Real-time PCR Detection Method For Bovine And Ovine Derived Materials In Feedstuff

The first bovine spongiform encephalopathy(BSE) was diagnosed in British cattle in 1986, then it spreaded extensively in the country,and even spreaded to the whole Europe.The epidemioiogic survey has proved that BSE and scrapie caused by prions can be transmitted through food chain and is related to Creutzfeldt-Jakob Disease.The spread of Bovine spongiform encephalopathy(BSE) has brought strong influence and losing in cattle raising,feedstuff industry,beef and its products,at the same time,it deeply threatened the life and health of the mankind.The epidemiologic survey has proved that the feedstuffs contaminated by bovine or ovine/goat derived materals is the main cause of transmission of BSE.So it is necessary to detect the bovine and ovine/goat derived materals in feedstuffs to avoid the spread of BSE.Now the analytical methods currently applied in the world for the detection and identification of animal tissues in feedstuff are microscopic analysis,polymerase chain reaction,immunoassay analysis and near infrared spectroscopy.There are some disadvantages in these methods,e.g.the small number of samples,false positive results and much experience of operators.The method of Real-time PCR is a much better analytical method which is more quick,accurate and economical than them.In this work,primers/probe BRTF/BRTR/ProbeB and ORTF/ORTR/ProbeO were designed according to 12SrRNA special region on mtDNA of bovine and ovine.;common primers and probe 16SRTF/16SRTR/Probe16 were designed according to bovine and ovine conserved 16SrRNA region.Besides,universal primer and probe for animal and plant designed according tol 8SrRNA were used to verify the absence of inhibition in real-timePCR.The length of target amplicons for bovine and ovine specific detection and common detection is 90bp,106bp,and 90bp respectively.Three overlapping primer pairs were designed to amplify three longer fragments that could cover shorter target sequences.The three longer fragments were ligated together,then cloned into pGEM-T Easy vector and sequenced.The standard preparations were prepared by diluting the right recombinant plasmid,then three standard curves were established.The coefficient correlation(R~2)of the three standard curves are all above 0.99.The specificity of the three primer pairs and probes designed was tested using genomic DNA of 21 species(15 animals and 6 plants) commonly used in feedstuffs,and no cross-species amplification was observed.The reproducibility of intra-assay and inter-assay is that the CV%is all below 5%。Real time PCR assay developed here allowed the detection of as few as 0.08 ng and 0.06 ng of bovine and ovine genomic DNA, respectively.The detection limit for MBM in contaminated feedstuffs processed according to EU legislation(133℃,3bar,20 min) was0.1%.Therefore real-time PCR method developed here was specific,reproductive and sensitive.It will play an important role in preventing China from BSE,keeping national economic satety and people healthy and promoting the development of food and feedstuff industry.