Study On The Mycoplasma Contamination In Live Vaccines By PCR-ELISA

Mycoplasma is a kind of prokaryote that is harmful to the health of livestock and poultry ,which can dramaticly affect the economic effect of the husbandry manufacture. It can contaminate biologics,e.g cell culture and vaccine ,and decrease the effect of vaccines to prevent or curl disease.Checking and measuring Mycoplasma infection is very important.In this experiment,we hope to establish a method to detect Mycoplasma in the live vaccine by PCR-ELlSA,and to inquire the optimum experimental conditions and superiority combination of PCR and ELISA. According to the 16s rRNA gene sequence published in GeneBank, which include the sequences of 1 chicken Mycoplasmas (M. gallisepticum) and 3 swine Mycoplasmas (M. hyopneumoniae, M. hyosynoviae and M. flocculare),The PCR primers and the prober labled by Dig and Biotin was designed through the software DNAStar and Primer 5.0. At the same time,the genome DNA of Mycoplasma was extracted and purified.Then the system and condition of PCR-ELISA was optimized.At last,this method was evaluated through comparing with PCR-electrophoretic method in practice.This method was applied to detect the samples got 30 batches Newcastle Disease live vaccines ( I Strain),30 batches Newcastle Disease live vaccines (La Sota Strain) and 30 batches Swine Fever live vaccines from market randomly. The results showed that the rate of Mycoplasma contamination from different vaccines by PCR-ELISA was 35.6%.It was 11.2% higher than the result detected by PCR.The method of PCR-ELISA has the character of simplify,speediness, and reliability for qualitive and quantificational analysis of Mycoplasmas.It is hopeful to be used in practice as a detection kit for Mycoplasma contamination in live vaccines .