QIAGEN 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.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

For the confirmation of serological results, the Microbiological Diagnostic Laboratory at DKFZ applied conventional PCR for minute virus of mice immunosuppressive variant (MVMi) and MAdV-1 using the Taq PCR core Kit from Qiagen as recently described with some modifications [34], [35]. Amplicon detection was done by gel-electrophoresis.

Briefly, the parvovirus singleplex PCR (MVMi) comprised a mastermix volume of 46 µL with two family-specific primers in a final concentration of 0.2 µM, 5 µL of 5×PCR buffer, 1 µL of dNTP (10 mM) and 2.5 µL of a fast start high fidelity enzyme (1 U/µL) (Roche Applied Science, Germany) per reaction. Four µL of cell culture supernatant containing MVMi was added. A 30 s denaturation step at 95°C was followed by 35 cycles of amplification in a Mastercycler (Eppendorf, Hamburg, Germany). Each cycle included a denaturation step for 30 s at 95°C, an annealing step for 30 s at 57°C and an extension step for 1 min at 72°C. The final extension was prolonged for further 7 min and reactions were stored at 4°C. The amplified PCR fragment was approximately 310 bp long.

The MAdV-1 singleplex PCR setting comprised a mastermix in a volume of 20 µL with a final primer concentration of 0.2 µM, 2 µL of 10×PCR buffer, 0.5 µL of dNTP (10 mM) and 1.5 µL of Taq polymerase (1 U/µL) (Qiagen, Germany) per reaction. A 5 min activation step at 95°C was followed by 31 cycles of amplification in a Mastercycler (Eppendorf, Hamburg, Germany). Each cycle included a denaturation step for 1 min at 94°C, an annealing step for 1 min at 52°C and an extension step for 1 min at 72°C. The final extension was prolonged for further 7 min and reactions were stored at 4°C. The PCR fragment was 281 bp long.

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