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Abstract In this experiment, we hope to establish a method to detect Mycoplasma in th live vaccine by PCR-ELISA, and to inquire the optimum experiment conditions and superiority combination of PCR and ELISA. According to the 16s rRNA gene sequence published in GenBank, 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 probe labled by Dig and Biotin was designed by 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 priactice. This method was applied to detect the samples of 30 batches Newcastle Disease live vaccines(I strain), 30 batches Newcastle Disease live vaccines (La Sota Strain) and 30 batches Swine Fever live vaccine which were bought from market randomly. The results showed that the rate of Mycoplsma contamination from different vaccines by PCR-ELISA was 36.5%. It was 11.2% higher than the result detected by PCR.The method of PCR-ELISA has the character of simlification, speediness and reliability for qualitive and quantifictional analysis of Mycoplasmas. It is hopeful to be used in practice as a detection kit for Mycoplasmas contamination in live vaccine.
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Received: 01 June 2006
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