Validation of PCR thermal cycler performance is crucial in order to obtain reliable results. In this study, high resolution melting curve (HRM) analysis is presented as a novel validation method for real-time PCR instruments. By applying HRM analysis using a defined PCR amplicon and EvaGreen dye, information about the temperature accuracy and thermal homogeneity of the heating block was obtained. This pilot study shows the potential of our technique for temperature validation of real-time quantitative PCR thermal cyclers. Our data correlated well with the temperature accuracy data obtained from the Mobile Temperature Acquisition System (MTAS; r2 = 0.93), which conforms to the National Institute of Standards and Technology criteria, and our method was reproducible in independent runs (r2 = 0.95). The advantages of this HRM-based method include: (i) temperature measurement under real world conditions in the reaction liquid in closed reaction tubes; (ii) temperature measurement of all wells; and (iii) applicability to all real-time PCR instruments capable of HRM analysis.
Real-time PCR (1) is becoming more prevalent than conventional PCR and has become an essential technique in molecular biology (2). Much effort has been expended in improving real-time quantitative PCR (qPCR) assays and controls (3,4). A common and unaccounted for source of error in qPCR data is the qPCR instrument itself. PCR instruments are subjected to vast and sudden changes in temperature that may lead to material fatig ue caused by continuous cycles of expansion and contraction or by condensation. The accuracy of qPCR instruments is a crucial requirement for any assay to obtain reliable results. This concerns ramping rates and peak temperatures, but much more important is the homogeneity of temperature in all well positions in the qPCR instrument, especially when results from different wells are to be compared.
With qPCR becoming the method of choice for various diagnostic applications, qPCR instrument performance has been subjected to increased scrutiny by the scientific community. Discrepancies in temperature performance bet ween different brands and t ypes of therma l c yclers have been observed and shown to have a negative influence on PCR performance and results (5-7). Furthermore, the lack of proper methods to validate thermal cycler performance is a major factor limiting the widespread adoption of diagnostic qPCR by end users (8). Va lidation becomes particularly important when performing an assay using different thermal cycler models, since only validation will ensure proper and reproducible results (9).
The mo st com mon com merc i a l validation methods are based on thermistor probes on printed circuit boards that have been developed for the validation of conventional PCR instruments. These usually consist of 15 thermistors that are positioned at predetermined locations in the heating block. For some of these, the lid has to be left open (e.g., Driftcon, Mobile Temperature Acquisition System [MTA S; CyclerTest, Landg raaf, The Netherlands]), while others fit into closed qPCR systems with drawers (e.g., Driftcon FFC). However, neither method takes into account the temperature crosstalk from the heated lid.
Other techniques have been suggested as alternatives to physical temperature sensors. For example, Gronlund et al. (10) have suggested the use of a system based on infrared (IR) thermography. IR thermography detects and visualizes IR energ y radiating from the heating block (10). This method, however, also does not take into account the temperature crosstalk from the heating lid to the reaction wells, since the lid has to be left open. A major drawback of all these methods is that they directly measure the temperature of the block and not the sample. However, the temperature in the sample is important information (5).
Here, we describe a qPCR thermal cycler validation method that determines temperature in every well of the heating block using high resolution melting curve (HR M) analysis. This technique determines the melting point (Tm) of double-stranded DNA by monitoring the loss of fluorescence from a DNA intercalating dye bound to the double-stranded DNA as it is melted into single-stranded DNA (see Supplementary Figure S1). A defined amplicon with a known Tm is combined with the HR M-compatible dye EvaGreen and distributed into every well of a PCR plate. A melting curve analysis leads to Tm values for every well, which will be scattered around the reference Tm depending on the performance of the heating block at every individual well position. Tm values below this nominal temperature indicate that the well was too hot, while Tm values above the nominal temperature indicate that the well was too cold. Our study demonstrates that HR M analysis can be applied as an alternative method to thermistor probes for the validation of qPCR instruments.
Material and methods
Preparation of the reference solution
The reference solution consists of a buffer containing the 256-bp reference PCR fragment (Tm ~90°C) and the intercalating, high resolution dye EvaGreen. The reference PCR fragment is derived from the Homo sapiens glutathione S-transferase theta 1 gene (GSTT1; NM_000853) and was amplified from human genomic DNA isolated from a cervical pap smear as follows: a single PCR was set up in 1× multiplex PCR master mix (Qiagen, Hilden, Germany) containing 1× EvaGreen dye (Jena Bioscience, Jena, Germany), 1 µL human genomic DNA (10 ng), 300 nM forward primer 5′-TTCCTTACTGGTCCTCACATCTC-3′ (positions 522–545), and 300 nM reverse primer 5′-TCACCGGATCATGGCCAGCA-3′ (positions 758–777). The reaction was carried out in a total volume of 50 µL using standard 200-µL PCR tubes. The PCR was set up in a Chromo4 real-time PCR instrument (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 57°C for 20 s, and elongation at 72°C for 45 s. A melting curve analysis was carried out between 50° and 95°C with a plate read every 0.1°C after holding the temperature for 5 s. The resulting amplicon was used as template in an upscaled PCR: 2100 µL PCR mixture were prepared in the sa me ma nner, containing 21 µ L template amplicon. After thorough homogenization, 43 µL PCR mixture were distributed into each of 48 PCR tubes. A PCR without a melting curve analysis was carried out with the same temperature program as described above. After the PCR, the 48 tubes were pooled in a 10-mL tube and vortex mixed thoroughly.