The traditional PCR technology is to amplify the target gene by PCR amplification. A DNA template is duplicated into thousands of offspring and then detected by gel electrophoresis. However, the gel electrophoresis detection can only judge the molecular size of the amplified products, but can not deduce the content of DNA in the initial samples. Therefore, quantitative analysis can not be carried out. Real-time quantitative PCR can be used for absolute quantification and relative quantification, and absolute quantification is made of standard curves with a series of known concentrations. Under the same conditions, the fluorescence signal measured by the target gene was compared with the standard curve to obtain the quantity of the target gene. The standard sample could select purified plasmid DNA or ssDNA synthesized in vitro, and the relative content of DNA in the initial sample could be converted by internal parameters. Digital PCR is the third generation of PCR technology based on traditional PCR and real-time fluorescent quantitative PCR. It does not need standard products, nor does it need to make standard curves, that is, it can achieve more sensitive and accurate absolute quantification.
Another important factor in the high accuracy of digital PCR is the application of Poisson statistics to the detection of digital PCR. Because digital PCR is a terminal detection analysis method, if the target molecule is not well dispersed and there is more than one target DNA in a reaction chamber, then the concentration obtained is not credible. Poisson statistics is a descriptive method of random distribution. When the reaction chamber/droplet volume and the negative ratio are known, the initial concentration can be obtained by Poisson model. Therefore, if the reaction chamber is not saturated, the researchers can also calculate the number of starting molecules of the sample. Beer et al. have shown that the advantage of droplet-based method lies in its scalability. With the increase of the number of reaction containers, the quality of data increases accordingly. That is to say, when the number of reaction vessels is enlarged or increased, the Poisson accuracy will also increase.
At the same time, digital PCR can effectively avoid the influence of reaction inhibitors. As the reaction chamber increases, the reaction is less affected by the inhibitor.
Search the Web of Knowledge Platform Science Citation Index Expanded database for the keywords "digital PCR, dPCR" from 1999 to June 2016 (up to June 25, 2016). The search results show that there are 1 787 related articles, of which 1 405 articles of art type are analyzed by literature statistics. Figure 2 shows that the number of research papers related to digital PCR has increased year by year, and the number of papers has increased rapidly since 2010. Among many countries, the United States and China are the main countries in the research field of digital PCR (Figure 3). According to a study published by Kalorama Information, the global market for digital and qPCR is expected to reach US$3.97 billion in 2019, with China as the main emerging market.
In routine tissues and blood samples, it is difficult to detect single mutation because of its low somatic cell content. However, dPCR can dilute or partition the complex background with limited dilution, which can reduce the background signal of wild genotype, so that the low abundance target sequence can be detected sensitively, especially for the detection of rare mutations. Studies show that the mutation frequency as low as 1/100 000 can be detected by [5-6]. And with the increase of reaction chamber, the analysis of rare mutation is more sensitive.
DPCR has a wide range of applications and research in rare mutation detection, especially in cancer-related detection and quantitative research, such as EGFR , BRAF , KRAS , PIK3CA [10-11], JAK2  and microRNAs . PIK3CA can be activated by growth factors such as IGF-1, HGF and EGF. It acts with growth factors on receptor tyrosine kinase, promoting proliferation, angiogenesis and cell metabolism. Kim et al. used ddPCR to detect the PIK3CA mutation of free DNA in serum . The low abundance PIK3CA mutation in serum was successfully detected. Thirty-eight metastatic cholangiocarcinoma samples were tested. One matched serum sample PIK3CA p.E542K was positive for 28 mutant copies, which was equivalent to the prevalence of 48 copies/mL serum and 0.3% allele. Another serum sample PIK3CA p.H1047R was positive for 10 mutant copies, which was equivalent to 18 copies/mL serum and 0.2% allele. The rate of disease. Miotto et al showed that EvaGreen dye method and TaqMan probe method could be used to detect circulating microRNAs in human plasma and serum by ddPCR.