Establishment and verification of multiplex fluorescence quantitative PCR method for detection of CHO host cell residual DNA content and fragmentation degree
Objective To establish and verify a multiplex fluorescence quantitative PCR method for detection of CHO host cell residual DNA content and fragmentation degree,so as to provide a reliable method for safety detection of related vaccines.Methods Two sets of primers(Alu-L and Alu)and universal probe were designed for clone250 and clone49c,members of Alu sequence family of CHO cells.The UBI3 gene of capsicum was introduced as external standard gene,and corresponding primers and probe were designed.Multiplex reaction system of Alu sequence and external standard gene was established,and the residual DNA of CHO cells in samples was quantified by the multiplex fluorescence quantitative PCR.The linear range,limit of quantitation(LOQ),specificity,robustness,precision and accuracy of the method were verified.In addition,the DNA fragmentation degree of CHO cells was determined by comparing the △ Ct[Ct(Alu-L)-Ct(Alu)]of the amplification curves of long and short primers of Alu sequences.Results The multiplex reaction standard curve had a good linear relationship in the range of 0.001-0.1 ng/μL,R2 ≥ 0.99.The addition of external standard gene had no effect on the detection ability for CHO target,and the LOQ was 0.5 fg/μL.The DNA of human,E.coli,rats and mice had no effect on the detection,the primers targeting residual DNA of CHO cells had no specific amplification in the genomes of seven animal cell lines,and the frag-mented DNA samples showed no effect on the detection results.The CVs of precision verification were all less than 15％,and the recoveries of simulated samples in normal saline and PBS containing 1％BSA were in the range of 70％-130％.The△ Ct of Alu and Alu-L amplification curves increased with the degree of sample fragmentation,and the fitting curve R2≥ 0.99.Conclusion The multiplex fluorescence quantitative PCR detection method established in this study can rapidly and accu-rately quantify the residual DNA of CHO cells and determine the fragmentation degree of samples according to △ Ct,which can be used for the safety detection of the related vaccines.
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