Explorement And Preliminary Validation Of Nested-PCR Method For Detection Of Mycoplasma Contamination

Mycoplasma is common in cell contamination events. The major contaminantsare two bovin mollicutes, Mycoplasma arginini and Acholeplasma laidlawii; a porcinemollicutes, Mycoplasma hyorhinis; and two human Mollicutes, Mycoplasma oraleand Mycoplasma fermentans. The 5 species account for >95% of cell mollicutecontaminations. Comparing to traditional detecting methods, the advantage of PCRmethod for detecting mycoplasma is easy, short cycle, high sensitivity and so on, sothe method is developed in recent years. However, the issue of the method is that itcouldn't detect t five common mycoplasma simultaneously, has cross-reactivity withbacterial genera with close phylogenetic relation to mycoplsma or couldn't identifymycoplasma species according to PCR product. In view of this, we expected toestablish a nested-PCR method, and judgy whether samples were contaminatedaccording to PCR product , identify mycoplasma species according to PCR ampliconsize, futher confirme the result by sequencing the second-stage PCR product tospeculate the contamination source.we targeted 16S-23S rDNA intergenic spacer(ISR) and made bioinformaticsalignments analysis of intraspecies ISR and interspecies ISR for five mycoplasma.According to the analysis result, we designed four pairs of primers to detect fivecommon mycoplasma. We extracted the genomic DNA of M.arginini,M.hyorhinis,M.orale and A.laidlawii(without M.fermentans), and sequenced two stage PCRproducts after nested-PCR amplication. Sequencing results showed that the nucleicacid sequence and fragment length of amplicons were identical to the expectedresults. We also constructed a plasmid containing the ISR of M.orale as positivecontrol. Preliminary validation of nested-PCR: we studied the limit ofdetection(LOD50),specificity and robustness of the method, and evaluated crosscontamination. 50% limit of detection:the LOD50 of M.arginini, M.hyorhinis, M.orale and A.laidlawii were 1.5,1.0,0.6 and 2.7gc/reaction. Specificity: our object wereClostridium, Lactobacillus and Streptococcus which were close phylogenetic relationto mycoplsma, 12 bacteria which were common bacterial contamination inproduction and vero cell DNA. The result showed that there was not crossamplication. Robustness: we evaluated the effect of operators, PCR thermal cyclers,storage condition, host cell DNA and cell contents. The results showed that onlystorage condition of samples had effect on the efficiency of the nested-PCRamplication. Cross contamination: exclusion of cross contamination wasdemonstrated by assaying highly positive specimens alternating with negative ones.The result showed that there was no cross contamination for the assay if weoperated strictly. Preliminary application: We detected 13 samples by the nested-PCRmethod and indicator cell culture method simutanously. The inconsistent results wasfuther tested by culture method. The results showed that the nested-PCR wasreliable.We have established nested-PCR method for detection of mycoplasmacontamination and made Preliminary validation. The method has high sensitivity,good specificity and robustness, which can offer a technical reference for detectionof mycoplasma contamination. Because we can obtain results in 4 hours, we may useit for process control purpose. Same-day results enable multiple in-process samplingpoints which can help us decide next operation immediately and protect from thespread of contamination.