Methodology article

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

Litao Yang¹ , Wanqi Liang¹ , Lingxi Jiang¹ , Wenquan Li² , Wei Cao² , Zoe A Wilson³ and Dabing Zhang¹

¹GMO detection laboratory, SJTU-Bor Luh Food Safety Center, School of life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China

²Amergen Biomed Co., Ltd., 22B, 3131 Hong Mei Rd., Shanghai 201103, PR China

³Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK

author email corresponding author email

BMC Molecular Biology 2008, 9:54doi:10.1186/1471-2199-9-54

Published:	4 June 2008

Abstract

Background

Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule.

Results

We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods.

Conclusion

The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.