Gotaq probe qpcr kit

TaqMan PCRs were performed in duplicate with a final volume of 10 μL per reaction using the GoTaq Probe qPCR kit from Promega. Each reaction contained the following: 5 μL of 2× GoTaq Probe qPCR master mix, 1 μL of PCR-Pan assay primer/probe mix, 1 μL of PCR-Pf assay primer/probe mix, 1 μL of sample, and enough nuclease-free water to bring the volume to 10 μL. Final concentrations per reaction of the primer mix were forward primer 0.4 μM, reverse primer 0.4 μM, and probe 0.2 μM. Thermal cycling conditions were adapted from Kamau et al. (12 (link)) as follows: 1 cycle of 2 min at 95°C, 45 cycles of 15 s at 95°C, and 1 min at 60°C.

The following RT–PCR kits were tested to determine the best performing assay: (1) OneStep RT–PCR Kit (QIAGEN); (2) TaqPath 1-Step Multiplex Master Mix (Thermo Fisher Scientific); (3) One-Step PrimeScript RT–PCR Kit (Takara Bio); (4) GoTaq Probe qPCR Kit (Promega Corporation); (5) UltraPlex 1-Step ToughMix (4X) (Quanta BioSciences); and (6) iTaq Universal One-Step Kit for RT–PCR (Bio-Rad Laboratories). Of the kits tested, the OneStep RT–PCR Kit (QIAGEN) was chosen for the final mCARMEN protocol.

The FDp status of each sample was determined by triplex qPCR, including a Vitis host gene as the extraction control, according to Pelletier et al. [23 (link)], using the GoTaq Probe qPCR kit (Promega). The presence of “Bois Noir” phytoplasma (BNp) was also tested since BNp causes the same set of external symptoms on grapevines as FDp and is only distinguishable through PCR analysis [3 (link)]. Cycling conditions were 5 min at 95 °C followed by 42 cycles of 15 sec at 95 °C and 30 sec at 60 °C, using a CFX96 real-time PCR instrument (Bio-Rad).
Samples with a Cq value below the limit of repeatability (LR) were considered positive (FDp+). LR is determined using dilution series. It is set at the lowest concentration of the pathogen that can consistently be detected in the grapevine matrix using at least six technical replicates [25 ].
Samples positive to BNp or of dubious status, i.e., with Cq values above the LR, were excluded from further analyses.