Study On The Detection Of Mycoplasma Contamination In Live Vaccines By Nested-PCR

Mycoplasma is the smallest and simplest prokaryotes without cell wall, which is one of the widespread contaminants found in cell cultures and vaccines. There is a potential harmfulness to spread mycoplasma disease of animal. So checking and measuring mycoplasmal infection is very important. We searched the published 16s rRNA gene sequences of 1 chicken Mycoplasmas (M. gallisepticum) and 3 swine Mycoplasmas (M. hyopneumoniae, M. hyosynoviae and M. flocculare) from GenBank to design the nested-PCR primers using the software DNAStar and Primer 5.0. The outer primers were the forward primer 1(5′-TGG GGA GCA AAC AGG ATT A-3′) and the reverse primer 2(5′-TTG TAG TAG CCA TTG TAG CAC G-3′). The inner primers for amplifying DNA of chicken Mycoplasma were the forward primer 3(5′-AAC CTT ACC TAG ACT TGA CAT C-3′) and the reverse primer 4(5′-GGC AGT CTC GTT AGA TAA AGT–3′). The inner primers for amplifying DNA of swine Mycoplasmas were the forward primer 5(5′-ATC CGC CTG AGT AGT ATG-3′) and the reverse primer 6(5′-GCA GTA TCT CTA GGG TTC TC-3′). The genome DNA of Mycoplasma is extracted and purified. Then we optimized the system and condition of nested-PCR amplification. The samples got from market randomly-30 Newcastle Disease live vaccines (ⅠStrain), 30 Newcastle Disease live vaccines (La Sota Strain) and 35 Swine Fever live vaccines were examined using these primers. The results showed that this method could examine Mycoplasma contamination from different vaccines. 24.2%(23/95), 21.1%(20/95) and 14.7%(14/95) were positive amplified by outer primers P1 and P2, inner primers P3, P4 and inner primers P5, P6, respectively. This method that we installed is hopeful to be used as a detection kit for Mycoplasma contamination, which must be brought into play in practice.