常见乳酸菌PCR检测定性标准样品研制

Development of Qualitative Reference Materials for PCR Detection of Common Lactobacillus

本研究针对食品中常见的乳酸菌研制5种乳酸菌菌种实时荧光PCR检测的核酸标准样品。利用重组质粒技术分别制备5种乳酸菌重组质粒,并培养纯化,经均匀性初检后,稀释至10^(-4)分装,制备标准样品。各标准样品通过核酸测序确定插入目的片段的同源性后,用实时荧光PCR方法进行均匀性、稳定性研究,特性值采用联合定值获得,并计算不确定度。在研制的5种标准样品中,重组质粒插入片段与即将实施的GB 4789.35—2023中特异性序列同源性均为100%。各标准样品组内偏差与组间偏差无明显差异(P>0.05),可在室温(20~25℃)、4℃和-20℃中稳定保存14 d,并且在-80℃中保存12个月且无显著变化,特性值均为阳性。5种标准样品参考Ct值分别为21.62、21.63、18.70、24.58、19.32。研制的5种乳酸菌定性标准样品均匀性、稳定性符合GB/T 15000系列技术规范,可作为乳酸菌PCR鉴定的DNA阳性对照。

In this study,we developed 5 kinds of qualitative reference materials for real-time fluorescent PCR detection of common lactobacillus strains in foods.Recombinant plasmids were prepared by applying gene recombinant technology,and were cultured and purified.After pre-homogeneity test,those recombinant plasmids were all diluted to 10^(-4)fold and sub packaged with equal volume to make 5 types of candidate reference materials.Nucleic sequencing was used to conferring the genetic homology of the inserted target segments.Homogeneity and stability study was conducted by real-time fluorescent PCR.The combined measurement was executed and the uncertainty value was counted at the same time.The inserted target segments in recombinant plasmids were 100%homogeneity with specific sequences in the pending GB 4789.35—2023.There were no significant differences between intra-variance and inter-variance of five reference materials(P>0.05).The reference materials were stable at room temperature(20-25℃),4℃,-20℃for 14 days,and had no significant changes at-80℃for 12 months.The characteristic values were positive for all five reference materials.The reference Ct values for the five reference materials were 21.62,21.63,18.70,24.58 and 19.32,respectively.The homogeneity and stability of the five kinds of lactobacillus reference materials were conformed with GB/T 15000 technical series,which could be used as positive control for real-time fluorescent PCR identification of common lactobacillus strains.