PCR–ELISA method for Mycoplasma contamination detection in veterinary vaccines

Abstract

AbstractA polymerase chain reaction–enzyme-linked immunosorbent assay (PCR-ELISA) method was set up to detect Mycoplasma in live vaccines and to establish the optimal experimental conditions. According to the 16S rRNA gene sequences published in GenBank, which include Mycoplasma gallisepticum of chicken and M. hyopneumoniae, M. hyosynoviae and M. flocculare of swine (submitted nos: Mg AY744942, Mhp AE017244, Mhs AY973563 and Mf X63377), PCR primers, labelled with digoxin (Dig), and probe, labelled with biotin, were designed using the software DNAstar and Primer Premier 5.0. The system and conditions of PCR–ELISA were optimized using purified genomic DNA extracted from Mycoplasma as a template. Samples of 30 batches of Newcastle disease live vaccines (I strain), 30 batches of Newcastle disease live vaccines (La Sota strain) and 30 batches of swine fever live vaccine were tested by PCR-ELISA. Results showed that the detection rate of Mycoplasma contamination from different vaccines was higher (36.5%) with this technique than with PCR (24.4%). The PCR–ELISA appeared to be a simple, fast and reliable method for qualitative and quantitative analysis of Mycoplasma. The practical use of PCR–ELISA as a kit to detect Mycoplasma contamination in live vaccine seems very promising.