Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling

Yang, Inchul; Kim, Young‐Ho; Byun, Ji-Young; Park, Sang-Ryoul

doi:10.1016/j.ab.2004.09.035

Cited by 35 publications

(32 citation statements)

References 18 publications

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“…These in-tube methods use temperature sensors in water or oil filled PCR tubes, which are positioned in the wells of the thermal cycler [6,11,14,15,16].…”

Section: In-tube Methodsmentioning

confidence: 99%

“…manufacturer or method based specifications) [5]. In sensitive PCR reactions as little as 0.5 °C deviation in temperature can already result in different, incorrect or failing PCR results [6]. Therefore, the expanded combined measurement uncertainty of thermal cycler calibration systems shall be 0.15 °C (k=2) to enable meaningful compliancy reporting.…”

Section: Evaluation Of Existing Methodsmentioning

confidence: 99%

“…The biochemistry or chemistry based methods, like thermographic liquid crystals based optical temperature verification discs [7], PCR based temperature verification kits [8,9,10], temperature sensitive PCR methods [6,11,12] and melt curve based qPCR methods [13], heavily reply on the use of biochemicals or chemicals. Biochemicals and chemicals can seldomly be obtained as 100% pure substance.…”

Section: Biochemical Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Measurement uncertainty in calibration and compliancy testing of PCR and qPCR thermal cyclers

Span¹,

Verblakt²,

Hendrikx³

2017

18th International Congress of Metrology

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Abstract. Both testing and medical laboratories use thermal cycler and real-time thermal cycler equipment for Polymerase Chain Reaction (PCR) based DNA testing. These thermal cyclers require regular calibration to ensure proper functioning. Currently, a variety of thermal cycler calibration and verification methods exits. Most of these methods mimic the PCR reaction, are not traceable to the International System of Units (SI) and connected to substantial measurement uncertainties, impeding compliancy reporting. Therefore these methods are inapt for application in metrology. This paper discusses a universal temperature calibration method for thermal cyclers that can be applied to any brand and model PCR and qPCR # thermal cycler, irrespective of the heating and cooling technology used. The calibration method and low associated measurement uncertainties do allow meaningful compliancy reporting to meet the requirements of ISO/IEC 17025, ISO 15189, ILAC P10 and ILAC G8. In addition, the paper offers effective practical solutions to determine PCR method based specifications, rather than thermal cycler manufacturer specifications, to link the PCR method to the thermal cycler, in order to qualify if the instrument is suitable for the intended use.

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“…These in-tube methods use temperature sensors in water or oil filled PCR tubes, which are positioned in the wells of the thermal cycler [6,11,14,15,16].…”

Section: In-tube Methodsmentioning

confidence: 99%

Section: Evaluation Of Existing Methodsmentioning

confidence: 99%

Section: Biochemical Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Measurement uncertainty in calibration and compliancy testing of PCR and qPCR thermal cyclers

Span¹,

Verblakt²,

Hendrikx³

2017

18th International Congress of Metrology

View full text Add to dashboard Cite

show abstract

“…Denaturation is a very robust step, but a too high temperature may have a nasty effect on the Taq polymerase. Annealing seems to be the more critical step for PCR yield and specificity [4]. Elongation is dependent on the polymerase activity range.…”

Section: Technical Aspects Of Pcrmentioning

confidence: 99%

Metrology in Thermal cyclers: a new approach

Cherpin¹,

Bozonnet²

2017

18th International Congress of Metrology

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Abstract.Metrological laboratories propose different protocols to qualify thermal cyclers, each depending on a different approach. But all these protocols may not match the laboratory need. Some questions must be answered before any metrological test. Has the qualification protocol be performed with a fixed temperature program? What must be done if the metrological qualification is out of specification? Can specifications be set already? With reference to standardised publications [1-3], we are going to try to focus on the current state of art about metrological qualification. It seems to be necessary to confirm any metrological test by a biological one. And to conclude that a difference between measured value and fixed value does not necessary lead to an end-oflife your thermal cycler even a false genetic analysis.

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“…Two main approaches has been proposed in the ISO/TS 20836 standard (Anonymous when experiencing problems it can be hard to identify the source of the problem and therefore it is feasible to supplement the biochemical validation procedure with a physical one where the actual temperature within each well during temperature cycling is measured (Anonymous 2005;Schoder et al 2003;Schoder et al 2005;Yang et al 2005). Probes for measuring temperatures in-tube have been developed (Schoder et al 2003;Yang et al 2005). Alternatively, the temperature distribution in the heating block (not in the sample) can be recorded using temperature sensitive probes.…”

mentioning

confidence: 99%

The Use of Infrared Thermography as a Novel Approach for Real-Time Validation of PCR Thermocyclers

Grønlund

Löfström

Helleskov³

et al. 2009

Food Anal. Methods

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Validation of PCR thermocycler performance is crucial to obtain reliable results. In this study, infrared (IR) thermography was evaluated as a novel validation tool. After stabilisation, no significant difference in the temperatures recorded using thermography and a reference block-based system was found. By employing IR thermography information about the length of the time until temperature stabilisation in the sample could be obtained. This study shows the potential of using IR thermography for validation of thermocyclers.Keywords: PCR, thermocycler, thermal cycler, infrared thermography, validation 3 Accurate thermocyclers is a prerequisite for all PCR based applications including diagnostic PCR for foodborne pathogens. Differences in temperature performance, between brands and types of cyclers has been demonstrated to influence PCR performance (Kim et al. 2008;Saunders et al. 2001;Yang et al. 2005). Proper validation of the thermocycler performance is one factor limiting the spread of diagnostic PCR to end-users (Malorny et al. 2003). Validation is important to ensure proper and reproducible results from PCR analysis and when transferring an assay between different thermocycler models (Schoder et al. 2005). It can also reveal problems caused by poor thermocycler performance (Kim et al. 2008;Schoder et al. 2005).Ideally, a validation procedure should be able to measure the temperature in-sample simultaneously in all wells. Two main approaches has been proposed in the ISO/TS 20836 standard (Anonymous when experiencing problems it can be hard to identify the source of the problem and therefore it is feasible to supplement the biochemical validation procedure with a physical one where the actual temperature within each well during temperature cycling is measured (Anonymous 2005;Schoder et al. 2003;Schoder et al. 2005;Yang et al. 2005). Probes for measuring temperatures in-tube have been developed (Schoder et al. 2003;Yang et al. 2005). Alternatively, the temperature distribution in the heating block (not in the sample) can be recorded using temperature sensitive probes. Several commercial systems are available for this purpose (e.g. TAS, Quanta Biotech, Somerset, UK and DRIFTCON, GENO-tronics, Landgraaf, The Netherlands). However, heat transfer and distribution through the sample itself, not the block alone, is crucial. Substantial variation in the effective length of a temperature step after the sample has reached the set temperature has been reported (Kim et al. 4 2008). For sensitive assays, this might affect the outcome of PCR and there is a need to calibrate the block temperature with the performance of PCR.Infrared (IR) thermography is a technology to detect and visualize IR energy radiating from an object for displaying images of temperature measurement or temperature distribution (for a review see, (Meola and Carlomagno 2004). The equipment used to perform IR thermography is often referred to as an IR camera or thermocamera. IR thermography has been employed as a tool for non-destructive evalua...

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Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling

Cited by 35 publications

References 18 publications

Measurement uncertainty in calibration and compliancy testing of PCR and qPCR thermal cyclers

Measurement uncertainty in calibration and compliancy testing of PCR and qPCR thermal cyclers

Metrology in Thermal cyclers: a new approach

The Use of Infrared Thermography as a Novel Approach for Real-Time Validation of PCR Thermocyclers

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