Variability and cost implications of three generations of the Roche LightCycler® 480

Real time PCR has become a dominant method for the highly sensitive detection of pathogens in clinical material. Real time PCR can generate a fluorescence signal by using fluorescence labelled probes, allowing us to detect and semi quantify the amount of amplified DNA. Here we test the variability of the detection system and cost implications of three different versions of the LightCycler® 480 (LC480), focusing on the intensity of fluorescence and Cq in monoplex and multiplex rtPCRs.
For gastro-intestinal pathogens there was no correlation between the intensity of fluorescence and the Cq value in the different LC480 types. For probes with the dyes FAMTM, HEXTM, Cy5 and Red610 a higher fluorescence intensity was seen in LC480 type II and III compared to LC480 type I. After lowering the probe concentration for the Cy5 dye three-fold (from 0.3μM to 0.1μM) the Cq value remains the same and the intensity of fluorescence decreases. For the LC480 type II and III the difference in fluorescence intensity was much more extreme. The concentration of the different labelled probes can be lowered at least six-fold in LC480 type II and III cyclers while maintaining a fluorescence intensity as high as achieved in the LC480 type I with undiluted probe. In conclusion, the strength of the fluorescence signal of the LightCycler® 480 type III is superior to that of LightCycler® 480 types I and II, allowing the use of lower probe concentrations for all dyes, particularly for the dyes Red610 and Cy5. This results in a two thirds reduction in PCR probe costs. Switching to these newer machines for real-time PCR can reduce dye labelled probe consumption and thus reduce costs significantly.

实时聚合酶链反应（real-time PCR）已成为临床样本中病原体高灵敏度检测的主流检测方法。该技术可通过荧光标记探针（fluorescence labelled probes）产生荧光信号，实现扩增DNA的检测及半定量分析。本研究针对罗氏LightCycler® 480（LC480）的三款不同型号，评估其检测系统的变异性与对应成本影响，重点关注单重与多重实时PCR中的荧光强度与Cq值。针对胃肠道病原体，不同LC480型号的荧光强度与Cq值之间无显著相关性。针对标记有FAM™、HEX™、Cy5及Red610染料的探针，LC480 II型与III型的荧光强度显著高于I型。将Cy5染料标记的探针浓度降低至原浓度的1/3（从0.3μM降至0.1μM）后，Cq值保持不变，但荧光强度出现下降。LC480 II型与III型的荧光强度差异更为显著。在LC480 II型与III型扩增仪中，各类标记探针的浓度可至少降低至原浓度的1/6，仍可获得与未稀释探针的LC480 I型相当的荧光强度。综上，LightCycler® 480 III型的荧光信号强度优于I型与II型，可针对所有染料（尤其是Red610与Cy5）使用更低的探针浓度。此举可使PCR探针成本降低三分之二。将实时PCR实验升级至该新型扩增仪，可减少荧光标记探针的消耗量，从而显著降低实验成本。