QuantiFast Multiplex PCR Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	PCR plates, tubes and tips should be UV sterilized for 20-30 mins

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
PCR plates, tubes and tips should be UV sterilized for 20-30 mins

Publication protocol

Real-time PCR
Reactions were carried out on the ABI 7500 Fast instrument (Applied Biosystems, Paisley, UK). mRT-PCR assays were carried out in a total volume of 20 μL, comprising 10 μL of QuantiFast Multiplex PCR mastermix (Qiagen), 2 μL of nuclease-free water (Promega), 2 μL of oligonucleotide mixture (Table 2), and 6 μL of nucleic acid extract. Cycle parameters were 95°C for 5 min, followed by 45 cycles of 95°C for 45 s and 60°C for 45 s. GAPDH real-time PCR was carried out with the same reaction components, but with different cycle parameters: 95°C for 5 min, followed by 45 cycles of 95°C for 30 s and 60°C for 30 s. PhHV PCR was carried out with 10 μL of Express qPCR Universal SuperMix (Invitrogen, Paisley, UK), 1 μL of oligonucleotide mixture (Table 2), and 9 μL of nucleic acid extract. Cycle parameters were 95°C for 20 s, followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. Runs were accepted if negative (no template) controls were negative and positive controls for each amplification target were positive. For quantification, mixed plasmid dilution series ranging from 6 × 101 to 6 × 106 gene copies/reaction were included in each run. ABI 7500 Fast System SDS software v. 1.4 (Applied Biosystems) was used to construct a six-point standard curve and extrapolate a quantitative result. Runs were accepted if standard curves were linear (reaction efficiency of 90–119%, R2 > 0.98). Clinical specimens were classified as positive and quantifiable for a bacterial target if the bacterial load was ≥100 CFUs/reaction. Quantitative results were accepted if the internal control was positive with a quantification cycle (Cq) value within the range ±1 log (Cq ± 3.33) difference from negative extraction controls. CFUs/reaction were assumed to be equivalent to gene copies/reaction, because all of the bacterial target genes are single copy. Conversion of gene copies/reaction to CFUs/mL was based on a 6-μL input per PCR reaction drawn from 100 μL of nucleic acid extract concentrated from 200 μL of specimen.

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Manufacturer protocol

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