Taqman openarray master mix

Open array was performed using the TaqMan^® assay (Applied Biosystems; Thermo Fisher Scientific, Inc.), and genotypes were captured using the OpenArray^® NT Genotyping system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Non-template control (process control) was used in each plate assay. DNA samples (50 ng/µl each sample) and TaqMan^® OpenArray Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) were mixed in a 384 well plate and transferred to the OpenArray plate using an autoloader. The OpenArray plate was subsequently filled with immersion fluid, and sealed with glue. Multiplex TaqMan^® assay reactions were performed in a Dual Flat Block GeneAmp PCR system 9700 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR cycling conditions were as follows: Initial denaturation at 93˚C for 10 min; followed by 50 cycles at 95˚C for 45 sec, 94˚C for 13 sec and 53˚C for 14 sec; and a final step at 25˚C for 2 min. Samples were stored at 4˚C until further use (11 (link),12 (link)).

After total RNA was extracted from respiratory samples, RNA was reverse transcribed to cDNA in the thermocycler. The Influenza A/B and RSV were determined by multiplex RT-PCR using open array technology method and validated at Patients Choice Laboratories using TaqMan open array master mix and customized respiratory panel open array chips (Applied Biosystems, Thermo Fisher Scientific). The open array chip is a microscope slide–sized plate with 48 sub arrays, each with 64 through-holes. It has multiple targets of respiratory viruses and bacteria including influenza and RSV. The readymade open array master mix is mixed with the sample in the 1:1 ratio in the 384 well plates, and samples are transferred into open array chips with the help of Accufill. Each through hole receives 33 nl sample. The open array is loaded into QuantStudio 12K flex instrument for RT-PCR.