实时荧光定量PCR意外中断后再扩增对检测结果的影响及有效应对措施研究

The Impact of Reamplification after Unexpected Interruption of Real-time Fluorescence Quantitative PCR on Detection Results and the Research on Effective Measures to Address this Issue

目的 研究实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)意外中断后再扩增对检测结果的影响及有效的应对措施。方法 目的收集2023年1—11月佛山市中医院住院患者中丙型肝炎病毒核酸检测阳性的血清样本,并随机抽取30例样本进行丙型肝炎病毒核酸(hepatitis C virus ribonucleic acid, HCVRNA)定量检测。通过点击软件强制中断按钮模拟不同扩增阶段的意外中断,分别采用原程序重新扩增以及用修正程序从断点处再扩增的方法来进行补救,评价两种补救方法对检测结果的影响。再以弱阳性样本检测为研究对象,研究扩增中断后体系的保存温度及时长对检测结果的影响,并探究扩增体系的最佳暂存条件。结果 逆转录中断组和变性中断组的循环阈值(Ct值)及HCV-RNA检测定量值与对照组比较,差异无统计学意义(P均>0.05)。预扩增中断组Ct值低于对照组比较,差异有统计学意义(P<0.05),但两组HCVRNA定量值对比,差异无统计学意义(P>0.05)。逆转录中断组、变性中断组以及预扩增中断组的Ct值和HCV-RNA定量值与对照组对比,差异无统计学意义(P均>0.05)。基线期意外中断组的Ct值低于对照组,差异有统计学意义(P<0.05),但两组HCV-RNA定量值对比,差异无统计学意义(P>0.05)。扩增中断后体系弱阳性样本4℃避光保存0、2、4、6、8、12 h后的Ct值分别为25.06±0.60、24.83±0.39、25.07±0.49、25.28±0.46、25.18±0.60、24.89±0.56,不同保存时间Ct值比较,差异无统计学意义(F=3.179,P>0.05)。结论 出现扩增意外中断时,如断点在扩增循环基线期及其之前各阶段,可采用相应的修正程序,从断点处再扩增的方法来补救。但对于已经进入指数扩增期及其以后各阶段的意外中断,则建议直接重建扩增体系进行检测。若因各种原因导致无法立即进行再扩增的体系,应放置在4℃冰箱避光保存,且最好不超过12 h。

Objective To study the effect of reamplification after unexpected interruption of real-time quantitative polymerase chain reaction(PCR)on the detection results and effective countermeasures.Methods Objectively collect positive serum samples of hepatitis C virus nucleic acid from patients in Foshan Hospital of Traditional Chinese Medi-cine from January to November 2023,and randomly select thirty samples for quantitative detection of hepatitis C virus ribonucleic acid(HCV-RNA).By clicking the software forced interrupt button to simulate the unexpected interruption in different amplification stages,the original program is re-amplified and the correction program is re-amplified from the breakpoint to remedy the method,and the influence of the two remedial methods on the detection results is evalu-ated.Then taking weak positive samples as the research object,the influence of storage temperature and time on the detection results of the system after the interruption of amplification was studied,and the best temporary storage condi-tions of the amplification system were explored.Results There were no significant differences in Ct value and HCV-RNA quantitative value between the two groups(all P>0.05).The Ct value of the pre-amplification interrupted group was lower than that of the control group,the difference was statistically significant(P<0.05),but the quantitative value of HCV-RNA between the two groups was not statistically significant(P>0.05).There were no significant differences in Ct value and HCV-RNA quantitative value in reverse transcription interruption group,denateration interruption group and pre-amplification interruption group compared with control group(all P>0.05).The Ct value of the unex-pected interruption group at baseline was lower than that of the control group,the difference was statistically signifi-cant(P<0.05),but the quantitative value of HCV-RNA between the two groups was not statistically significant(P>0.05).After amplification interruption,the Ct values of the weak positive samples of the system were 25.06±0.60,24.83±0.39,25.07±0.49,25.28±0.46,25.18±0.60 and 24.89±0.56 after 0,2,4,6,8 and 12 h at 4℃,respectively.There were no significant differences in Ct values between different preservation times(F=3.179,P>0.05).Conclu-sion When the amplification is interrupted unexpectedly,if the breakpoint is in the baseline period of the amplifica-tion cycle and its previous stages,it can be remedied by the corresponding correction program and re-amplification from the breakpoint.However,for the unexpected interruption that has entered the exponential expansion period and its subsequent stages,it is recommended to directly reconstruct the amplification system for detection.If the system cannot be immediately reamplified for various reasons,it should be stored in a refrigerator at 4℃away from light,and it is best not to exceed 12 hours.