High Throughput Two-Dimensional PCR Technology

Aim:In this research,we devoted our effort to the development of a novel two-dimensional(2D)PCR technology on the basis of base quenched technology,which can identify target genes by both fluorescence and melting temperature(Tm)at closed tube condition.Methods:We add different tag sequence to the 5' end of one primer of each target gene detected in the same fluorescence detection channel.These tag sequences can be the same as the detecting probe or only contain several bases different form the base-quenched probe.During the Melting curve analysis after the PCR amplification,the detecting probe targets at the tags'reverse complemented sequences.In the same fluorescence detection channel,we only need one detecting probe and various target genes can all be distinguished by different melting temperature(Tm)values.Meanwhile,we use different fluorophores to label different detecting probes.By regarding the emission wavelength as the Y-axis and the Tm as X-axis,all target genes detected in multiple channels will be distinguished in a two-dimensional way.In this way,2D PCR can identify multiple target genes in one closed tube.We implemented 2D PCR in detecting 10 genes including 9 human papillomavirus(HPV)subtypes and human hemoglobin gene by using both two fluorescence detection channel mode and three channel mode.To test the functionality of 2D PCR,we conducted sensitivity tests,clinical sample detection tests and agreement analysis.Results:The library for 3 channels' probes and tags was built.And 2D PCR was successfully applied to detecting 10 genes.In two channel mode,5 genes were detected in the FAM channel,including HPV16,HPV 18,HPV 31,hemoglobin gene and HPV 35.The approximate Tms were 41,48,52,60 and 65?,respectively.In the HEX channel,5 genes,including HPV 45,HPV 51,HPV 52,HPV 58 and HPV 68,were detected.The approximate Tms were 48,54,60,63 and 71?,respectively.In three channel mode,only hemoglobin gene was transferred to Cy5 channel while the others remained the same.The melting temperature for hemoglobin gene in Cy5 channel was about 72?.In sensitivity tests,the lowest concentration 2D PCR can detect was 2×101 copies per reaction in single gene detection and 4×101 copies per reaction in double infection test.2D PCR also successfully showed the 5 genes in multi-infection test.Comparing 2D PCR results of clinical sample detection with the other two methods,the agreement ratios were 90.58%,92.14%,and the kappa coefficient were 0.8228,0.8403,respectively.Conclusion:2D PCR have the capability of identifying multiple genes in one closed tube.This method is particularly suitable for distinguishing microorganisms,single-nucleotide polymorphisms and methylation of genes.