Absolute Quantification Study Of Influenza A Viruses Based On Digital PCR Technology

Influenza is an important infectious disease,which can cause a serious disease burden,resulting in high morbidity and mortality.Effective antiviral therapy can prevent and treat influenza.It is helpful to evaluate the effect of antiviral therapy by quantitative detection of influenza virus load,which can be used to monitor the treatment and prognosis of the disease.With the development of molecular biology technology,nucleic acid quantitative detection methods are constantly updated.At present,the nucleic acid quantitative detection methods included nucleic acid sequence-based amplification,brunched DNA and quantitative PCR.In recent years,the new nucleic acid quantitative method,digital PCR technology,has been widely used.It can be used to absolute quantify,which provide a new method of quantitative nucleic acid.Digital PCR(dPCR)is a new method for absolute quantification of nucleic acids.dPCR,which originated from the target quantification using limiting dilutions,divides the standard PCR reaction mixture into hundreds to millions of partitions(such as droplet,chip).After PCR amplification,positive and negative partitions are counted and the absolute concentration of target copies in the original sample is calculated by Poisson distribution.Unlike qPCR,dPCR can directly obtain the copy number of samples without standard curve.Nowadays,there are many studies on quantitative analysis of adenovirus,hepatitis B virus and HIV virus by using digital PCR.There is no systematic establishment and application of digital PCR absolute quantification method for influenza A virus.Then in this study we setup droplet digital polymerase chain reaction method for absolute quantification of influenza A virus.In this study,we used digital PCR method to quantify the reference standard of influenza A virus nucleic acid to ensure the optimization of the annealing temperature.We found that the optimal annealing temperature was determined to be 64.4?.It was determined that the detection range of the droplet digital PCR was at least four orders of magnitude,and the limit of quantification was 23 copies/?l.The liner correlation coefficient(R2)was evaluated as 0.9988,indicating the high reliability of our digital PCR method.Additionally,the dPCR method could be applied to detect the influenza A viruses clinically.In summary,we have developed a dPCR method for absolute quantification of Influenza A viruses.It could be effectively used to quantitate influenza A viruses in clinical samples,and therefore we provided a new tool for the absolute quantification of viral load in clinically.In this thesis,by establishing droplet digital PCR method,we compared the accuracy,sensitivity and agreement of the digital PCR and real-time quantitative PCR.Two methods were used to quantify the reference standard of influenza A virus nucleic acid.Quantitative PCR method was calculated the copy number of samples by establishing standard curve.From the quantitative results,it can be seen that the quantitative copy number of digital PCR was closer to the reference standard sample,and the quantitative concentration was higher than that of quantitative PCR.For the low concentration reference standard,when quantitative PCR(Ct)was 35(Ct=35),the digital PCR method can be quantified successfully,and its sensitivity was slightly higher than that of quantitative PCR.After Bland-Altman analysis,the two methods had a good quantitative agreement.The limit of agreement was from-0.2814 to 0.1686.For the samples with high concentration,the droplet digital PCR can't be quantified,and it was beyond the detection range.In conclusion,we have developed a digital PCR method for absolute quantification of Influenza A viruses,ensured the detection range and the limit of quantification.Additionally,we compared the two methods agreement of detection.The results showed that the digital PCR can be used to quantitatively detect the clinical samples.The droplet digital PCR can be used as an effective alternative to quantitative PCR method for quantitative detection of clinical samples.Therefore,we provided a new method to quantity clinical sample.It can monitor the changes of viral load accurately,to guide the clinical drug treatment,to monitor the effect of treatment and the prognosis of the disease.