Azure Cielo™ Real-Time PCR Optical Design Eliminates the Need for Passive Reference Dye

ROX is a widely used inert fluorescent dye, which is usually added to qPCR reaction solution to correct the fluorescence signal. However, with the development of fluorescence technology in instruments, such as Azure Cielo™ Real-Time PCR system, it is no longer necessary to use dye alone for standardization. When the real-time fluorescent quantitative PCR system was first introduced into the market, many products used fixed light source and detection system to image the 96 well plate at the same time, resulting in different light path of each hole in the hot block (Fig. 1). The optical path difference will cause the absolute fluorescence measurement value of each hole to change, depending on their position on the plate. For example, a hole with a shorter optical path may have a higher fluorescence reading than a hole with a longer optical path, resulting in an increase in the deviation of fluorescence values between the two holes. Therefore, many researchers began to use reference dyes (such as ROX) to standardize the signal changes.

Azure Cielo™ Real-Time PCR Optical Design

It can be seen from Figure 1 that in the whole plate imaging of traditional real-time PCR system, the hole in the central area can detect higher fluorescence signal than the hole in the corner. Since the Rox signal is not affected by qPCR reaction, as long as the properties of qPCR mixture remain unchanged, it will remain stable throughout the qPCR process. For this reason and the superior spectral characteristics of ROX, ROX is regarded as a good reference dye, which can be used for the normalization of fluorescence signal to calibrate the fluorescence changes caused by optical path difference. The challenge with using ROX as a reference dye is that it eliminates a channel that could potentially be used for detecting a target of interest. With applications such as detection of Influenza and COVID-19 gene targets (see ANDiS SARS-CoV-2 and Influenza A/B RT-qPCR Detection Kit) becoming more common, multiple reactions are becoming more and more routine, which also shows the importance of multi-channel detection.

Azure Cielo™ Real-Time PCR system uses a new high-performance optical system, and does not need Rox calibration in qPCR experiments. The optical system adopts 16 groups of optical fiber single hole scanning design, one optical fiber is used for high-energy LED excitation, the other is used for optical signal detection, which can collect 16 holes at the same time to ensure the consistency of data acquisition (Fig. 2). In addition, using the "total well detection" technology, about 100,000 pixels can be collected from each hole, and the average value of the readings is taken during the detection to accurately measure the fluorescence intensity of each hole (Fig. 3). This "total well detection" provides more reproducible and reliable data for each acquisition time point than the single hole scanning system, because the single hole scanning system only collects a small number of data points.

Application Example:

1、Methods:

20 μL reaction solution was prepared, and the reaction was carried out in Azure Cielo™ Real-Time PCR system was used for qPCR amplification of 10 repeat wells.

2、Results and Discussion：

Results show that Azure CieloTM Real-Time PCR system can uniformly detect amplifications in every qPCR well. The real-time amplification curves from all ten replicate wells show a uniform increase of signal detected, giving a C q mean value of 17.54 with standard deviation as low as 0.02 from the statistical analysis (Fig. 4). The coefficient of variation (CV) of C q values is 0.13%, reflecting the excellent precision of the data.

The above results further prove the characteristics of Azure CieloTM Real-Time PCR system , which can completely eliminate the optical path difference, and can still obtain high-precision and reliable data without using ROX for standardization.

Azure Cielo™ Real-Time PCR System

Azure Cielo™ Real-Time PCR System is from Azure Biosystems company of the United States, which can provide 3 / 6 detection channels for you, and can be flexibly configured according to the experimental requirements. This product uses high-energy LED as the light source system, which can ensure high light intensity and good light source consistency; high quality Peltier temperature module as the temperature control system, with fast temperature rise and fall speed, which can set 12 columns of 30 °C span temperature gradient; excellent CMOS photography + optical fiber signal transmission as the detection system, with high CMOS detection sensitivity, fast optical fiber transmission speed, no light loss and noise, there is no need for Rox calibration. Azure Cielo™ Real-Time PCR System can provide experimental results with high accuracy, high sensitivity and high reliability for your scientific research.