Hahn-Schickard , Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg , Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Anal Chem. 2016 Mar 1;88(5):2590-5. doi: 10.1021/acs.analchem.5b02960. Epub 2016 Feb 16.
Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2-plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.
聚合酶链反应 (PCR) 中的多重PCR 是一种广泛应用的技术，它可以节省成本和样本材料，并与将样本分配到多个单重反应相比，提高了灵敏度。检测不同扩增产物的最常见方法之一是使用在不同波长（颜色）处发射的荧光探针。为了减少检测通道的数量，已经提出了几种用于单色多重化的方法。然而，它们对可扩增的靶标长度、序列或熔点施加了限制。为了规避这些限制，我们提出了一种新方法，该方法使用具有相同发射最大值的不同荧光团。通过它们在光漂白过程中不同的荧光稳定性来实现区分。atto488（与 6-羧基荧光素、fam 的发射波长相同）和 atto467n（与菁染料 5、cy5 的发射波长相同）被发现比 fam 和 cy5 显著更少地漂白；也就是说，atto 染料的最终荧光比 fam 和 cy5 多三倍以上。我们通过使用仅 2 个颜色通道对金黄色葡萄球菌中的抗生素耐药基因进行 4 重 PCR 来成功应用了这种方法。在高拷贝数初始拷贝数为 100000 拷贝的情况下，对目标物的区分置信度>99.9%。对于 cy5 通道，两个目标物都存在的情况下可以具有相同的区分置信度，而对于 fam 通道，置信度降低（>68%）。此外，还展示了在 1 个颜色通道中的 2 重数字 PCR 反应。在未来，可以通过添加具有其他发射波长的荧光探针对来增加多重化程度。该方法也可以应用于其他探针和检测格式，例如Förster 共振能量转移 (FRET) 探针和免疫测定。
