Study On The Diagnosis Method Of Early Pregnancy In Dairy Cattle Based On EPF Detection

At present,the early diagnosis of dairy cows in our country continues to develop at the same time,foreign early pregnancy diagnosis products are also emerging.With the increase of the scale of dairy farming,the simple and effective and economical method of pregnancy diagnosis is more and more important to adapt to the situation of industrial development.Early pregnancy factor(EPF)in dairy cows is one of the earliest known biochemical markers to confirm pregnancy,and it can be used as an important indicator of early pregnancy diagnosis.EPF generated by pregnancy from different sources,may be related to the uterus,ovaries,fallopian tubes,fertilized eggs,placenta,pituitary and so on.Real-time quantitative PCR was used to detect the gene copy number of the organism,at the gene level to quantitatively analyze the expression of EPF gene in cow's uterus,placenta,ovary,fallopian tube,vulva skin and vaginal mucous tissue.It is of great significance.Colloidal gold technology has the advantages of easy preparation,low cost,intuitive and reliable results.The use of cow early pregnancy factor combined with colloidal gold technology to prepare immunochromatographic colloidal gold rapid test strip,the rapid diagnosis of cows in the field of pregnancy status is important.In this study,we first used Real-time PCR to detect the difference of EPF gene expression in pregnant and non-pregnant uterus,placenta,ovary,fallopian tube,vulva skin and vaginal mucosa from 4 to 6 years old.Real-time quantitative PCR EPF expression as a cows early pregnancy feasibility.At the same time,the double antibody sandwich ELISA method was used to screen out the matched monoclonal antibodies,and the method of immunochromatographic colloidal gold for rapid detection of early pregnancy test strips was discussed.The specific research results are as follows:1.Real-time fluorescence quantitative PCR found that the expression of EPF in uterine and vulvar skin was not significantly different from the negative control group;EPF expression in ovary and placenta was significantly different from that in negative control group(P <0.05);The expression of EPF in tubal and vaginal mucus was significantly higher than that in the negative control group(P <0.01).Bovine EPF is present in the uterus,ovaries,placenta,fallopian tubes,vaginal mucus and vulva skin.Vaginal mucus collection is relatively convenient,and does not hurt domestic animals,the real-time fluorescence quantitative PCR detection of EPF gene expression that can be used as an effective method to diagnose cows early pregnancy.2.The double antibody sandwich ELISA method was successfully used to screen six pairs of monoclonal antibodies.The linear range of detection of artificial recombinant bovine EPF protein was234~937ng/mL.The detection sensitivity was about 60ng/mL,but no EPF was detected in the bovine serum.3.The minimum detectable rate of artificial recombinant bovine EPF protein detected by colloidal gold immunochromatographic test strip(GICA)prepared by paired monoclonal antibody was 1.5?g/mL.However,EPF in pregnant calf serum and vaginal mucus was not detected clinically.