不同保存温度和反复冻融次数对鸭疫里默氏杆菌DNA的影响

Effects of Different Storage Temperature and Freeze-thaw Times on DNA of Riemerella anatipestifer

为探究不同保存温度和反复冻融次数对鸭疫里默氏杆菌DNA的影响,研究了不同保存温度和反复冻融次数对不同浓度梯度组鸭疫里默氏杆菌DNA样品浓度及荧光定量Ct值的影响。根据OmpA基因保守序列设计了鸭疫里默氏杆菌荧光定量PCR特异性引物,产物大小为112 bp,退火温度为56℃。荧光定量循环阈值Ct值随DNA浓度的下降而升高。常温保存的DNA样品7 d后出现明显降解,保存温度为4、-20、-80℃时则对荧光定量PCR检测效率影响不大。较高浓度的A、B组DNA样品Ct值未受冻融次数的影响,而浓度较低的C、D组则在冻融21次后Ct值明显升高。研究建立了鸭疫里默氏杆菌荧光定量PCR鉴定方法,Ct值随DNA浓度的下降而升高,DNA样品应低于4℃保存,低浓度DNA应避免反复冻融。

To explore the effects of different storage temperature and freeze-thaw times on the DNA of Riemerella anatipestifer,the effects of different storage temperatures and freeze-thaw times on the concentration and fluorescence quantitative CT values of DNA samples of Riemerella anatipestifer in different concentration gradient groups were studied.According to the conserved sequence of OmpA gene,a specific primer for fluorescent quantitative PCR of Riemerella anatipestifer was designed.The product was 112 bp,and the annealing temperature was 56℃.With the decrease of DNA concentration,the Ct values of fluorescence quantitative increased.The DNA samples stored at room temperature showed obvious degradation after 7 days.However,the storage temperature of 4℃,-20℃and-80℃had little effect on the detection efficiency of fluorescent quantitative PCR.The Ct values of DNA samples in group A and group B with higher concentration were not affected by the freeze-thaw times,while the group C and group D with lower concentration increased significantly after 21 freeze-thaw times.The method of fluorescence quantitative PCR for the identification of Riemerella anatipestifer was successfully established in the study.The Ct values increased with the decrease of DNA concentration.The DNA samples should be kept below 4℃,and the low-concentration DNA should not being frozen and thawed repeatedly.