The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.

] Kist Europe Saarbrücken Saarland 66123 Germany [2] Central European Institute of Technology Brno University of Technology Technická 3058 10 CZ 616 00 Brno Czech Republic [3] Northwestern Polytechnical University School of Mechanical Engineering 127 West Youyi Road Xi'an Shaanxi 710072 P R China

Kist Europe Saarbrücken Saarland 66123 Germany

Zobrazit více v PubMed

Saiki R. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985). PubMed

Higuchi R., Fockler C., Dollinger G. & Watson R. Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nat Biotech 11, 1026–1030 (1993). PubMed

Witham P. K., Yamashiro C. T., Livak K. J. & Batt C. A. A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Applied and environmental microbiology 62, 1347–1353 (1996). PubMed PMC

Holland P. M., Abramson R. D., Watson R. & Gelfand D. H. Detection of Specific Polymerase Chain-Reaction Product by Utilizing the 5′-]3′ Exonuclease Activity of Thermus-Aquaticus DNA-Polymerase. P Natl Acad Sci USA 88, 7276–7280 (1991). PubMed PMC

Andrus A. et al. High-throughput synthesis of functionalized oligonucleotides. Nucleic acids symposium series, 317–318 (1997). PubMed

Safdar M. & Abasiyanik M. F. Simultaneous identification of pork and poultry origins in pet foods by a quick multiplex real-time PCR assay using EvaGreen florescence dye. Applied biochemistry and biotechnology 171, 1855–1864 (2013). PubMed

Abd-Elmagid A. et al. Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia. Journal of Microbiological Methods 92, 293–300 (2013). PubMed

Vandesompele J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome biology 3, research0034 (2002). PubMed PMC

Osman F., Hodzic E., Omanska-Klusek A., Olineka T. & Rowhani A. Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D. Journal of Virological Methods 194, 138–145 (2013). PubMed

Kim Y. et al. A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level. Yonsei Med J 54, 1220–1226 (2013). PubMed PMC

Mehrabadi J. F., Morsali P., Nejad H. R. & Imani Fooladi A. A. Detection of toxigenic Vibrio cholerae with new multiplex PCR. Journal of infection and public health 5, 263–267 (2012). PubMed

Lo C. L. et al. One-step rapid reverse transcription-PCR assay for detecting and typing dengue viruses with GC tail and induced fluorescence resonance energy transfer techniques for melting temperature and color multiplexing. Clinical chemistry 53, 594–599 (2007). PubMed

Bohling S. D., King T. C., Wittwer C. T. & Elenitoba-Johnson K. S. Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis. The American journal of pathology 154, 97–103 (1999). PubMed PMC

Safdar M. & Abasıyanık M. F. Simultaneous Identification of Pork and Poultry Origins in Pet Foods by a Quick Multiplex Real-Time PCR Assay Using EvaGreen Florescence Dye. Applied biochemistry and biotechnology 171, 1855–1864 (2013). PubMed

Gudnason H., Dufva M., Bang D. D. & Wolff A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Research 35, e127 (2007). PubMed PMC

Noorani M. S. et al. Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA. Journal of Virological Methods 193, 103–107 (2013). PubMed

Huang Q. et al. Multicolor Combinatorial Probe Coding for Real-Time PCR. PLoS ONE 6, e16033 (2011). PubMed PMC

Nazarenko I. et al. Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res 30, e37 (2002). PubMed PMC

Rajagopal A., Scherer A., Homyk A. & Kartalov E. Supercolor Coding Methods for Large-Scale Multiplexing of Biochemical Assays. Analytical Chemistry 85, 7629–7636 (2013). PubMed

Lind K., Stahlberg A., Zoric N. & Kubista M. Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis. BioTechniques 40, 315–319 (2006). PubMed

Cheah E. S. et al. A two-tube combined TaqMan/SYBR Green assay to identify mycobacteria and detect single global lineage-defining polymorphisms in Mycobacterium tuberculosis. J Mol Diagn 12, 250–256 (2010). PubMed PMC

Van Poucke M., Van Zeveren A. & Peelman L. J. [Letter to the editor] Combined FAM-labeled TaqMan probe detection and SYBR green I melting curve analysis in multiprobe qPCR genotyping assays. BioTechniques 52, 81–86 (2012). PubMed

Rienzo M. et al. Identification of valid reference housekeeping genes for gene expression analysis in tumor neovascularization studies. Clin Transl Oncol 15, 211–218 (2013). PubMed

Tan S. et al. Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors. Mol Biol Rep 39, 4857–4867 (2012). PubMed PMC

Moniotte S. et al. Real-time RT-PCR for the Detection of Beta-adrenoceptor Messenger RNAs in Small Human Endomyocardial Biopsies. Journal of Molecular and Cellular Cardiology 33, 2121–2133 (2001). PubMed

Ward C. L. et al. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. Journal of Clinical Virology 29, 179–188 (2004). PubMed PMC

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis

Recent advances in lab-on-a-chip technologies for viral diagnosis

IoT PCR for pandemic disease detection and its spread monitoring

Handheld real-time PCR device