Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses

Abstract

Background: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability. Methods: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems. Results: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments. Conclusions: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.

Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae

“…We also found that longer annealing and extension times increased the intensity of the RAPD bands. Although RAPD has been shown to be sensitive to slight fluctuations of annealing temperature and temperature ramp rate of the thermal cycler, temperature-calibrated thermal cyclers are likely to generate repeatable RAPD profiles (MacPherson et al 1993;Saunders et al 2001). Our results showed highly repeatable and reproducible RAPD profiles using three thermal cyclers set at the same ramp temperature.…”

“…Fereshteh Eftekhar, Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, G.C., Tehran, Iran. E-mails: f-eftekhar@cc.sbu.ac.ir or efereshteh@hotmail.com ing concentration of primer, dNTP, DNA polymerase and MgCl 2 , DNA template and purity, thermal cycling profile and type of thermal cycler (Ellsworth et al 1993;MacPherson et al 1993;Saunders et al 2001;Blixt et al 2003;Atienzar and Jha 2006;Peng et al 2007). These factors have usually been optimized one at a time neglecting the interactions between them (Perry et al 2003;Peng et al 2007).…”

Virulence‐associated gene profiling, DNA fingerprinting and multilocus sequence typing of Haemophilus parasuis isolates in Australia

“…The study used the default machine settings, proprietary reagents and other potential variables such as ramp rates and magnesium concentration . A study comparing 33 thermocyclers using the same protocol and ingredients found that variation in PCR results can be attributable to suboptimal thermal cycler performance …”

Conserved flanking microsatellite sequences (ReFS) differentiate between Lepidoptera species, and provide insight into microsatellite evolution

“…Lallias et al , 2007), and is much higher than those often encountered for less predictable random amplified polymorphic DNA (RAPDs; e.g. Saunders et al , 2001). The high repeatability of ReFS presumably arises from the relatively large size of the primers (20 bp versus the 10 bp that is typically used during RAPDs), and the relatively high annealing temperature (61°C).…”