1.5-fold Discrimination for qPCR Quantification Applications

A valid real-time PCR assay is characterized by four key properties:

  • PCR efficiency
  • Sensitivity
  • Reproducibility
  • Specificity

Multiple factors can affect these critical points; beside plastic ware, primer design, and master mix, the performance of the real-time PCR cycler itself influences the results. It is mandatory, for this reason, to guarantee the best properties of the real-time PCR cycler in order to rely on the outcome of the applied real-time PCR.

Discriminating between small dilution steps, even down to 1:1.5 is one of the most challenging requirements. This proves the accuracy and dependability of the performance of the real-time PCR cycler and so, of all data generated using the device.

Methods and materials

Primers that are specific for the 16S rRNA gene of E. coli have been employed for this experiment for the amplification of a 120 bp fragment in seven dilution steps with six replicates á 20 µl reaction volume for each concentration.

The master mix employed was Analytik Jena’s innuMIX qPCR SyGreen Sensitive. The time-temperature-profile begins with 2 minutes at 95 °C, followed by 45 cycles with 5 sec at 95 °C, 5 sec at 58 °C and 15 sec at 72 °C and finishes with a melting curve (60 – 95 °C, 15-sec equilibration and 1 °C increment).

Instrumentation

Data were acquired in color module 1 (470 nm/520 nm, gain = 5) of qTOWER³ G touch for SyGreen.

Results

The linear regression of the 1.5-fold dilution series yields a PCR efficiency of 97 % (slope = -3.41) and R² = 0.998.

Amplification plot for 6-time 1.5-fold dilution of E. coli genomic DNA, amplified using primers specific for 16S rRNA gene. With automated threshold settings results are as follows for Ct (SD): 106 copies, dark red = 16.11 (0.04); 6,67·105 copies, light green = 16.83 (0.05); 4.44·105 copies, dark blue = 17.31 (0.04); 2.96·105 copies, green = 18.05 (0.03); 1.98·105 copies, red = 18.59 (0.03); 1.32·105 copies, yellow = 19.18 (0.03); 8.78·104, blue = 19.72 (0.03).

Figure 1. Amplification plot for 6-time 1.5-fold dilution of E. coli genomic DNA, amplified using primers specific for 16S rRNA gene. With automated threshold settings results are as follows for Ct (SD): 106 copies, dark red = 16.11 (0.04); 6,67·105 copies, light green = 16.83 (0.05); 4.44·105 copies, dark blue = 17.31 (0.04); 2.96·105 copies, green = 18.05 (0.03); 1.98·105 copies, red = 18.59 (0.03); 1.32·105 copies, yellow = 19.18 (0.03); 8.78·104, blue = 19.72 (0.03).

Melting peaks of the PCR amplicons result in a melting temperature of 78.80 °C with a SD of 0.13 °C.

Figure 2. Melting peaks of the PCR amplicons result in a melting temperature of 78.80 °C with an SD of 0.13 °C.

Conclusion

The Analytik Jena qTOWER³ G touch can detect and clearly differentiate between minute concentration alterations. In the 1.5-fold dilution series that was carried out, high precision discrimination was seen in a reaction with a R² of 0.998 and a PCR efficiency of 97 %.

About Analytik Jena US

Analytik Jena is a provider of instruments and products in the areas of analytical measuring technology and life science. Its portfolio includes the most modern analytical technology and complete systems for bioanalytical applications in the life science area.

Comprehensive laboratory software management and information systems (LIMS), service offerings, as well as device-specific consumables and disposables, such as reagents or plastic articles, complete the Group’s extensive range of products.

About life science

The Life Science product area demonstrates the biotechnological competence of Analytik Jena AG. We provide a wide product spectrum for automated total, as well as individual solutions for molecular diagnostics. Our products are focused to offer you a quality and the reproducibility of your laboratory results.

This will surely ease your daily work and speed up your work processes in a certain way. All together we support you through the complete process of the lab work. Besides we offer customized solutions and are able to adapt our products to your needs. Automated high-throughput screening systems for the pharmaceutical sector are also part of this segment’s extensive portfolio.

About analytical instrumentation

Analytik Jena has a long tradition in developing high-performance precision analytical systems which dates back to the inventions made by Ernst Abbe and Carl Zeiss. We have grown to become one of the most innovative manufacturers of analytical measuring technology worldwide.

Our business unit Analytical Instrumentation offers excellent competencies in the fields of optical spectroscopy, sum parameters and elemental analysis. Being proud of our core competency we grant all our customers a long-term warranty of 10 years for our high-performance optics.

About lab automation

With more than 25 years of market experience, Analytik Jena with its CyBio® Product Line is a leading provider for high quality liquid handling and automation technologies. In the pharmaceutical and life science industries, our products enjoy the highest reputation for precision, reliability, robustness and simplicity.

Moreover, the Automation Team designs, produces and installs fully automated systems tailored to our clients' application, throughput and capacity requirements. From stand-alone CyBio® Well up to fully customized robotic systems we handle your compounds, biomolecules and cells with great care.


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Last updated: Sep 30, 2020 at 3:02 AM

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