5 plex rotorgene q

A species-specific assay was used to confirm P. falciparum infection, using established primers, probe, and conditions15 (link); forward primer was 5′-CTTTTGAGAGGTTTTGTTACTTTGAGTAA-3′, reverse primer 5′-TATTCCATGCTGTAGTATTCAAACACAA-3′, and probe sequence 5′-TGTTCATAACAGACGGGTAGTCATGATTGAGTTCA-3′ labeled with 5′ FAM and 3′ TAMRA as reporter and quencher, respectively. A real-time generic Plasmodium spp. assay was then used to quantify gene copies of 18S rDNA using established primers, probe, and conditions16 (link); forward primer was 5′-GCTCTTTCTTGATTTCTTGGATG-3′, reverse primer 5′-AGCAGGTTAAGATCTCGTTCG-3′, and probe sequence 5′-ATGGCCGTTTTTAGTTCGTG-3′.
Amplification and real-time measurements were performed in the 5-plex Rotorgene Q (Qiagen) with the following thermal profile for quantitative PCR (qPCR): 10 minutes at 95°C, 50 cycles of 15 seconds at 95°C, and 1 minute at 60°C. For the reaction, 2 μL template was added to 8 μL reaction master mix containing 1× QuantiTect Multiplex PCR Master Mix NoROX (Qiagen), 0.4 μM of each primer, and 0.2 μM probe.
Relevant primers derived from the kelch13 gene sequence were used to amplify the full kelch13 open reading frame using a nested PCR protocol.17 (link)

In order to determine the load of calpox virus in various organs and body fluids, a real-time PCR targeting the gene region of the ankyrin repeat-containing protein (GenBank accession number HQ420898) was used. A quantified molecular DNA standard with the target DNA cloned into a TOPO-TA vector (Invitrogen, Karlsruhe, Germany) was applied to calculate the concentration of calpox viral DNA either per gram of tissue or per microliter of blood. The TaqMan^® Universal PCR Master Mix (Thermo Fisher Scientific (formerly Life Technologies), Darmstadt, Germany) was used as follows: 7.5 µL of TaqMan^® Universal PCR Master Mix was mixed with 1 µL of each primer at a concentration of 10 nmol (Calp1for: 5′-CCggCATgCgTgACTgAATT-3′ and Calp1rev 5′-TAAgATgCgAgCCgAgAAgC-3′), 0.5 µL of the 10 nmol of TaqMan probe TIB-1 (FAM -TgCTCCgTgTTCTACCATCgTgCg-TAMRA), 4 µL molecular biology grade water and 1 µL of the extracted DNA. Oligonucleotides were synthesized by TIB Molbiol (Berlin, Germany). The real-time PCR was performed using the 5-Plex Rotor-Gene Q (Qiagen, Hilden, Germany) and the following cycling conditions were applied: initial activation at 95 °C for 10 min, and 45 cycles of 94 °C for 15 s, and 58 °C for 45 s. Absolute quantification was conducted via Rotor-Gene Q Series Software 2.1.0 (Qiagen, Hilden, Germany).