Mic qpcr instrument

In October 2019, suspected strobilurin-resistant wheat powdery mildew samples (n = 12) collected from different farms and brought by growers attending an industry field day at Northern Yorke Peninsula (South Australia), were processed in-field (Table 1). The tissue was not excised using the coring tool, rather cut into small fragments using scissors and forceps disinfected with 70% v/v ethanol between samples. Each sample was added up to the 100 µl graduation in 1.5 ml microfuge tubes. This was followed by a quick DNA extraction and ASqPCR assay on the MIC qPCR instrument (Bio Molecular Systems) as detailed above, and mutant frequency results communicated to the growers upon assay completion.

Viral RNA was extracted from approximately 10 mg of spleen using the NucleoMag^® Vet kit (MACHEREY-NAGEL, Germany) on a KingFisher^™ Flex Purification System, according to the manufacturer’s instructions, and eluted with 100 μL of the supplied elution buffer. Complementary DNA (cDNA) was made using 2 μL qScript^™ Supermix (Quanta Biosciences, USA) and 8 μL RNA in a 10 μL reaction, according to the manufacturer’s instructions. Spleen samples (n = 191) were tested in duplicate on a Mic qPCR instrument (Bio Molecular Systems), using 1 μL template and PowerUp^™ SYBR^® Green Master Mix (Applied Biosystems, USA), with the primer concentration and cycling conditions as described previously [13 (link)]. Primers targeted the RNA dependent RNA polymerase gene within ORF1b [13 (link)]. Samples were considered positive if the amplification curve crossed the automatically defined threshold and the melting peak was between 85 °C and 86.5 °C. Samples were considered equivocal if only one of the duplicates was positive with the Cq value >33 or if both duplicates showed the correct melting peak, but the Cq was >37. Samples with equivocal results were retested in duplicate with 2 μL and 5 μL template, using a conventional PCR targeting a 321 bp conserved region in ORF1b [14 (link)].

Primers (HRM-2826-F/R) were designed to bind flanking regions of sgRNA binding site. The HRM curve analysis was performed on a magnetic induction cycler (MIC) qPCR instrument (Bio Molecular Systems, Australia) using Precision Melt Supermix (BioRad, #1,725,112, USA) in a 20-µL reaction with 1 × Precision Melt Supermix, each primer at concentration of 300 nM and 5 ng of genomic DNA. At least two technical replicates were performed for each sample. The qPCR amplification was performed with the following program: initial denaturation of 3 min at 95 °C, followed by 40 cycles of 95 °C for 10 s, 61 °C for 30 s, with fluorescence reading at the end of each extension step. The qPCR was followed by a melting program where the amplicon was heated 65 to 98 °C by ramping up the temperature at 0. 2 °C/s with a continuous signal acquisition. The HRM curve data (Additional file 1: Fig. S2) were analyzed using the in-built HRM analysis software (Bio Molecular Systems, Australia). To confirm mutants identified by HRM, Pf2826 gene was amplified with Q5 High-Fidelity DNA Polymerase (NEB MA, USA) using primers Pf2826-F and Pf2826-R, resulting amplicons were purified and sequenced.