Establishment And Application Of Mycoplasma Contaminant Detection Method In Cell Culture

Mycoplasma is a common source of contamination in cell cultures.Mycoplasma is able to coexist with cells for a long time due to its non-lethality.Moreover,cells contaminated with mycoplasmas did not show significant morphological changes,thus is imperceptible.However,mycoplasma contaminated cells showed diminished metabolism and slower growth,which brings great trouble to the majority of researchers.In order to prevent and control mycoplasma contamination and improve the detection technology of mycoplasma contamination in cell culture,143 mycoplasmas genomes sequences that have been published in the NCBI database were compared and analyzed in this study.Total 27 pairs of primers were designed using the regions with higher homology.The primers were tested with 16 different mycoplasmas species preserved in our laboratory,including the 9 main contamination sources specified in the European Pharmacopoeia(EP10edition)and Japanese Pharmacopoeia(JP16 edition):Acholeplasma laidlawii(A.laidlawii),Mycoplasma fermentans(Mfe),Mycoplasma hyorhinis(M.hyorhinis),Mycoplasma orale(M.orale),Mycoplasma arginini(M.arginini),M.pneumonia(Mp),M.gallisepticum(MG),Mycoplasma synoviae(MS),and Spiroplasma citri(S.citri).A pair of primers with excellent sensitivity and specificity was obtained and a polymerase chain reaction(PCR)method for the detection of mycoplasma contamination in cell culture and a Basic Enzymatic Recombinase Amplification(ERA)method were then established based on the filtered primers.The main test results are as follows:1:Establishment of PCR detection method for mycoplasma contamination in cell culture.According to the results of genome alignment,total 27 pairs of primers were designed and synthesized.A pair of broad-spectrum primers that can detect all the 16 mycoplasmas stored in our laboratory were found.A PCR assay for mycoplasma contamination in cell culture was then established which can detect mycoplasmas in cell culture and sera.This method has no cross reaction with common animal origin bacteria and viruses,such as Salmonella,Escherichia coli,Streptococcus suis,Staphylococcus aureus,Haemophilus parasuis,Rtemerella anatipestifer,Actinobacillus pleuropneumoniae,Pasteurella,PCV-2,PRV,PEDV,CSFV and PRRSV,thus demonstrating high specificity.The PCR assay shows an excellent sensitivity with a minimum sensitivity of 10~2genomic equivalents per reaction;In addition to the single isolate of 16 different mycoplasms,the universality of the PCR method were further revealed with different clinical isolates of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.It was found that the method could not only detect different mycoplasma species,but also had a detection rate of 100%in different strains of the same mycoplasma species,which demonstrated an excellent broad-spectrum detection method.2:Application of PCR detection method for mycoplasma contamination in cell culture.Thirty-eight random cell samples from different laboratories in different regions were collected for mycoplasma detection;11 positive samples were detected by the PCR assay established in this test and the method of microbiological culture and DNA fluorescence staining specified in the Chinese Pharmacopoeia;while only 10 positive samples were detected by a commercial nested PCR assay,and 8 positive samples were detected by the Japanese Pharmacopoeia recommended nested PCR assay.Taking the pharmacopoeia culture method as the“gold standard”,it was determined that the contamination rate in the cell samples reached 28.9%,the positive rate detected by the self-built PCR method was 28.9%,and its coincidence rate with the culture method was 100%,which had a higher sensitivity compared with the commercial nested PCR kit and the nested PCR method in the Japanese Pharmacopoeia.A total of 102 serum products from different brands and batches were also collected for mycoplasma detection:40 samples contaminated with mycoplasma were detected by the PCR assay developed by this study,accounting for 39.2%of the total number of samples.Thirty-one positive samples(30.4%)were detected by a commercial nested PCR assay and13 positive samples for mycoplasma contamination(12.7%)were detected by Japanese Pharmacopoeia nested PCR assay.The results show that the PCR assay developed in this study has broad spectrum and better sensitivity.3:Establishment of Basic ERA detection method for mycoplasma contamination in cell culture.Using the screened mycoplasma universal primers,a universal mycoplasma detection method for Basic ERA was established.After three specificity tests,it was demonstrated that the established Basic ERA did not cross-react with a variety of bacteria and viruses.After optimizing the conditions,the assay can detect 10~1genomic equivalents per reaction at 39℃for 20 min,which proves that it has the advantages of convenience,rapidity,specificity and high sensitivity.