All real time PCR reactions were performed on a Stepone System Real Time PCR cycler (Applied Biosystems, U.S.A).
Fig. 1 LAMP detection of the ATCC 49926 porA gene using an automatic PCR cycler Fig. 2 Specificity of LAMP for porA detection.
The tubes were incubated at 20, 30, 40, 50, 60, 70 and 80 °C in a PCR Cycler and the residual activity was measured with the standard ABTS activity assay.
A TransStart Green qPCR SuperMix UDG kit (TransGen Biotech) was applied to prepare the qPCR samples, which were run in triplicate on a Rotorgen Q real-time PCR cycler (QIAGEN).
Following the foodproof® Alicyclobacillus Detection Kit protocol, the real-time PCR amplification parameters used in the Applied Biosystems 7900HT real-time PCR cycler were as follows: 37°C for 4 min (1 cycle) and 95°C for 5 min (1 cycle), and 95°C for 5 s followed by 60°C for 1 min (50 cycles).
PCR was carried out using a Bio-Rad IQ5 PCR cycler.
cDNA thus generated was used for quantitative RT-PCR (BioRad iCycler iQ™ Real-Time PCR cycler, BioRad, Hercules, CA).
Real-time PCR reactions were performed using iQ SYBR Green (Biorad) and a BioRad real-time PCR cycler as previously described [63].
The resulting cDNA was labeled with the QuantiTect SYBR Green PCR Kit (Qiagen 204143) in a DNA Engine Opticon PCR cycler (MJ Research) following the kit protocol.
Relative level of mRNA transcripts for target genes was measured by quantitative real-time PCR (qPCR) using the Rotor Gene RG 3000 PCR Cycler (Corbett Research, Australia) and Hot Start version of TaKARa ExTaq DNA polymerase.
Cycling was done in a Fast System 7500 Real Time PCR Cycler (Applied Biosystems) according to the following program: step 1: 10 minutes at 95°C; step 2: 15 seconds at 95°C; step 3: 30 seconds at 55°C; step 4: 45 seconds at 60°C (data collection was performed at this step); steps 2 to 4 where repeated for 40 cycles.
