Development Of Real-time PCR Genotyping Methods Based On Probe Coding Technology

This dissertation consists of four chapters. The first chapter describes the development and principle of probe coding technology (PCT), the following three chapters deal with the application of PCT genotyping in Bacillus cereus, human papillomavirus, and hepatitis C virus.In chapter one, a novel real-time PCR genotyping approach named probe coding technology (PCT) was described. The basic principle of PCT is that each genotype to be analyzed is coded by from one to four fluorophores -labeled displacing probes,which could discriminate sequences difference at single nucleotide level. In such a way, using n fluorophores, totally 2n-1 targets can be coded. The genotyping results can be obtained directly after real-time PCR using these probes based on the appearance of fluorescence corresponding to the probes. PCT can be regarded as a multiplex real-time PCR genotyping strategy concerning the targets number to be analyzed. PCT actually combines the merits of high-throughout and simplicity of real-time PCR, the extremely high specificity of displacement probes, and the legible mathematics rule, and has thus greatly improved the genotyping capacity of real-time PCR.In chapter two, the feasibility of PCT was tested by detecting eight different genotypes of a food-born pathogen Bacillus cereus. A four-step protocol and asymmetric PCR was adopted to facilitate the hybridization between the probes and targets. The results showed that using totally nine probes labeled with four different colors, i.e., FAM, HEX, ROX and Cy5, the eight genotypes Bacillus cereus could be unequivocally discriminated within 2 hr. The robustness of PCT was further validated by genotyping of seven strains isolated from food samples and the results were confirmed with bi-directional sequencing.In chapter three, a multiple real-time PCR genotyping of fifteen human papillomavirus in a single-tube was developed. The design reached the theoretical upper limit of PCT genotyping, i.e., 15 genotypes were discriminated using 4 fluorophores-labeled probes. The results showed that using thirty-two probes labeled with different combination of FAM, HEX, ROX, and Cy5, 15 HPV genotypes could be discriminated within 2 hr. In addition, differentiation of co-infection was also tested by changing the ratio the fluorescence of two probes. The established method was applied to two clinical samples. The results showed that one sample was HPV6, the other was HPV16. These results were concordant with the sequencing method.In the last chapter, PCT was applied to reverse-transcription PCR genotyping of HCV. First, 6 armored RNA of HCV fragments were prepared standing for the 6 different genotypes mostly popular among Chinese population. Using 9 probes labeled with three different colors, i.e., HEX, ROX and Cy5, the 6 HCV genotypes could be discriminated within 3 hr. Finally, 10 clinical samples were genotyped and confirmed by sequencing.In comparison with conventional genotyping methods, PCT is more accurate, reliable, and high-throughput. The analytical capacity of PCT is close to low density microarray or chip of the same aim, but PCT has obvious advantages in manipulation and readout interpretation. It is more suitable for routine clinical testing and large-scale genetic screening.