反转录酶活性检测实时荧光定量PCR方法的建立

目的在传统产物增强性反转录酶（PERT）活性检测方法基础上建立检测反转录酶活性的实时荧光定量PCR方法。方法以噬菌体MS2RNA为模板,设计、合成引物和探针,并分别以连续10倍倍比稀释的AMV反转录酶（1×10-1～1×10-9U/μl）标准品催化体外反转录反应合成相应cDNA。采用实时荧光定量PCR法检测底物cDNA模板量,并获得相应的PCR荧光值曲线,分析AMV反转录酶的相对活性。同时应用传统PERT方法对cDNA进行扩增,测定该方法的灵敏度。结果将AMV反转录酶标准品连续10倍倍比稀释后催化反转录反应,实时荧光定量PCR分析所得的反转录产物cDNA,结果得到与理论值完全相符合的PCR标准荧光值曲线;定量分析结果显示,浓度为1×10-2～1×10-8U/μl的AMV反转录酶均可得到相应的荧光值。不同稀释度的AMV反转录酶,经传统PERT方法扩增后,结果显示浓度为1×10-3～1×10-7U/μl的AMV反转录酶均可见大小为112bp的阳性条带,检测灵敏度达到1×10-7U/μl。结论成功建立了基于实时荧光定量PCR技术的检测反转录酶活性的新方法,实验操作简单,具有高精确性和无污染性,为生物制品外来污染尤其是反转录病毒污染的检测提供了参考指标。

Development of a real-time fluorescence quantitative PCR method for detection of reverse transcriptase activity

Objective To develop a real-time fluorescence quantitative PCR method for detection of reverse transcriptase activity, based on the traditional product enhanced reverse transcriptase (PERT) activity detection method. Methods Based on phage MS2 RNA as a template to design and synthesize primers and probe, AMV reverse transcriptase standard in a continuous of 10-fold serial dilution (1 × 10-1 - 1 × 10-9 U/μl) were used to catalyze reverse transcription reaction in vitro and synthesize cDNA. Using real-time fluorescence quantitative PCR method to detect the amount of substrate cDNA template, obtaining the real-time fluorescence quantitative PCR curves and analyzing the relative activity of the AMV reverse transcriptase. The traditional PERT method was used to amplify cDNA, and determining the sensitivity of the method. Results Using AMV reverse transcriptase standard in a continuous of 10-fold serial dilution to catalyze the reverse transcriptase reaction, and the real-time fluorescence quantitative PCR method to analyze the reverse transcriptase products cDNA, the standard real-time fluorescence quantitative PCR curves fully consistent with the theoretical value were obtained; the quantitative analysis showed that the concentrations of 1 × 10-2 - 1 × 10-8 U/μl of AMV reverse transcriptase could get the corresponding fluorescence values. After different dilutions AMV reverse transcriptase were amplified by the traditional PERT method, the results showed the concentrations of 1 × 10-3 - 1 × 10-7 U/μl of AMV reverse transcriptase appeared the size of 112 bp positive band, the detection sensitivity was 1 × 10-7 U/μl. Conclusion The new real-time fluorescence quantitative PCR method for detection of reverse transcriptase activity was established successfully, the operation process was simple, and with high accuracy and non-polluting, it could provide a reference for detection of biological products external contamination, and especially retrovirus contamination.