Based On Multi-droplet PCR Coupled Fluorescence Spectrophotometry, Qualitative, Quantitative And High-throughput Detection Techniques For Biological Genes

Detection of genetically modified components and food-borne pathogens in food and feed is particularly critical in raw material control,food industry,and food sales.Traditional PCR technology can usually only detect one target gene in one reaction.When dealing with complex nucleic acid samples,multiple reactions need to be separately performed,which is time consuming and costly.Although conventional multiplex PCR were always used to amplify nucleic acid samples,many problems exist against its wide use,such as preferential amplification of shorter DNA templates,interference of multiple primer pairs and limited substrates,making it incapable in quantitative and high-throughput research.Real-time quantitative PCR has been widely used to quantify target genes with high sensitivity,specificity and a wide dynamic range,the throughput in such a scheme is low because of the limited number of channels in the real-time system.DNA microarray is an approach to analyze complex nucleic samples with high throughput,the complicated procedure and expensive consumption,however,limit its feasible availability for most laboratories.Poor linearity is another weakness related with DNA microarray which limits its application in quantitative analysis.Therefore,developing a novel qualitative,quantitative and high-throughput method for detecting multiple biological genes is on demand.The multiplex emulsion PCR provides a possibility.Emulsion PCR has been developed for high-throughput simultaneous amplification of several DNA targets,either used alone or combined with other methods.In emulsion PCR,the different target DNA molecules are amplified in parallel in millions of compartmentalized micro-reservoirs,which alleviates the drawbacks in conventional multiplex PCR while increasing the throughput and reducing the regent and sample consumption.Here we first report a microdroplet PCR method combined with fluorescence spectrophotometry(MPFS),which allows for qualitative,quantitative and high-throughput detection of multiple DNA targets.In MPFS,each of the fluorophores has its own intrinsic excitation and emission wavelength that can be identified and measured by the Infinite M1000 PRO.Each pair of primers specific to a target sequence was labeled with a specific fluorophore that does not interfere with any other fluorophores in the same reaction set for detection purpose.Through the emulsion PCR,a target DNA was amplified and labeled with the same fluorophore.After PCR reaction,the products were purified by PCR cleanup kit to remove the free primers and other disruptive substances.Finally,the DNA products tagged with different fluorophores could be used for qualitative analysis by the fluorescent intensity determination.The relative fluorescence unit(RFU)was also measured to construct the standard curves and then the quantitative analysis could be achieved.Since a variety of different target samples were simultaneously amplified in a single PCR tube,the co-amplified products with different fluorophores could be simultaneously analyzed to achieve high-throughput detection.GM maize was selected as a model for this purpose.By means of labeling the different gene-specific primer with different fluorophores without detection interference,multiple target DNAs could be co-amplified in a single PCR reaction,and amplicons could be analyzed qualitatively and quantitatively with high throughput.The MPFS method was proved to be free of cross interference from different fluorophores,and of sensitivity of 0.5%(w/w)GM content,lower than 0.9%of EU criterion.In addition,this method generates the data with excellent linearity relationship between the logarithm of RFU values and gene copy number and with a LOD of 10~3 gene copy number.The 4-plex method increased the throughput by microdroplet PCR reaction from single MPFS assay but not sacrifice the quality of resulting data.The small signal strength variation within batches or between batches further confirmed the precision and accuracy of this MPFS method.Finally,the results from foodborne pathogenic bacteria showed the applicability of MPFS method to other biological samples.To our knowledge,the MPFS system described here is the first analytical technology which allows qualitative,quantitative and high-throughput analysis of multiple biological genes simultaneously.The primer fluorescent labeling method used in this study does not require the size and sequence of the PCR product.It is based on the labeled fluorophore.If the specificity of the primer is good,the target gene can be specifically amplified,and the microdroplet PCR product can be identified.This is especially true for simultaneous detection of multiple genes with high homology in DNA sequences and very short conserved regions.Fluorescence microplate readers were used to detect the fluorescently-labeled PCR product quickly.It took only one second to detect one gene.The Thermo VarioSkan Flash multi-function microplate reader has an excitation wavelength range of 200-1000nm.The excitation and emission wavelengths can be appropriately adjusted to detect 20fluorescent markers simultaneously.Using the 384-well microplate reader,more than6000 genes can be detected at a time.The method improves the throughput of target amplification,reduces the consumption of valuable reagents and samples,has the advantages of rapidity,convenience,low cost,easy promotion,and less environmental pollution,and can be used for qualitative,quantitative,and high-level analysis of all biological genes.