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.

中文翻译：

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一种新型通用实时 PCR 系统，使用连接的通用双链探针进行核酸定量分析。

背景技术实时PCR技术被广泛用于核酸分析，但是当前经常使用的实时PCR的一个限制是每个目标分子的标记探针的高成本。结果 我们描述了一种采用附加通用双链探针 (AUDP) 的实时 PCR 技术，与当前的实时 PCR 方法相比，该技术具有通过探针水解和链置换产生荧光的优势。AUDP 涉及一组通用双链探针，其中荧光探针 (FP) 的 5' 端和互补淬灭探针 (QP) 靠得很近，以便可以淬灭荧光。带有通用模板 (UT) 和 FP 的 PCR 引物对与 UT 序列相同。我们已经证明，AUDP 技术可用于在单链和双链实时 PCR 检测中检测多个目标 DNA 序列，用于基因表达分析、基因型鉴定和转基因生物 (GMO) 定量，具有相当的灵敏度、再现性和可重复性与其他实时 PCR 方法。结论 AUDP 实时荧光定量 PCR 技术对 GMO 定量、基因表达分析、基因型鉴定和 GMO 定量结果表明 AUDP 实时 PCR 技术已成功应用于核酸分析，开发的 AUDP 实时 PCR技术将为核酸分析提供一种低成本、高效、可靠和灵活的替代方法。基因型鉴定和转基因生物 (GMO) 定量，具有与其他实时 PCR 方法相当的灵敏度、重现性和可重复性。结论 AUDP 实时荧光定量 PCR 技术对 GMO 定量、基因表达分析、基因型鉴定和 GMO 定量结果表明 AUDP 实时 PCR 技术已成功应用于核酸分析，开发的 AUDP 实时 PCR技术将为核酸分析提供一种低成本、高效、可靠和灵活的替代方法。基因型鉴定和转基因生物 (GMO) 定量，具有与其他实时 PCR 方法相当的灵敏度、重现性和可重复性。结论 AUDP 实时荧光定量 PCR 技术对 GMO 定量、基因表达分析、基因型鉴定和 GMO 定量结果表明 AUDP 实时 PCR 技术已成功应用于核酸分析，开发的 AUDP 实时 PCR技术将为核酸分析提供一种低成本、高效、可靠和灵活的替代方法。