Establishment and Application of Real-time Quantitative PCR Method for Poliovirus Sabin Strain
Objective To establish a real-time quantitative PCR method for poliovirus and detection the viral load of different tissues in Transgenic mice susceptible to poliovirus.Method Poliovirus includes three distinct serotypes named types 1,2 and 3.First,Primers were designed targeting the VP1 coding regions of three serotypes,and then construct plasmids after PCR identification and sequencing analysis.Making a standard curve by diluting the plasmid copy concentration ten-time series,and evaluated for the specificity,sensitivity,and repeatability of the standard curve.Meanwhile,detection of viral load in infected mouse tissues using established real-time quantitative PCR method method.Result PCR identification and sequencing analysis showed that the sequence of the standard plasmid was complete and correct.The standard curve established has a good linear relationship between copy number and Ct value,with a R value>0.99.The amplification efficiency is between 99%and 110%,and the minimum detection limit was 10 copy per μL,which is 100 times higher than that of regular PCR.The amplification curve of fluorescence quantitative PCR reaction is S-shaped and the main peak of the dissolution curve is single,indicating good specificity and the amplification conditions were reliable.Applying this method to detect the viral load in various tissues of the infected mouse model,it was found that viral RNA was exclusively detected in the spinal cord and brain tissues,revealing of poliovirus corresponding to the neurotoxicity in monkeys.Conclusion The established real-time quantitative PCR method for poliovirus has good specificity,sensitivity,and repeatability,and can provide a reliable experimental method for quantification of viral load in different tissues in the polio virus permissive mouse model.
万方数据
维普
