Selected article for: "Applied Biosystems real Time pcr system and GAPDH gene"
Author: Park, Donghyun; Huh, Hee Jae; Kim, Yeon Jeong; Son, Dae-Soon; Jeon, Hyo-Jeong; Im, Eu-Hyun; Kim, Jong-Won; Lee, Nam Yong; Kang, Eun-Suk; Kang, Cheol In; Chung, Doo Ryeon; Ahn, Jin-Hyun; Peck, Kyong Ran; Choi, Sun Shim; Kim, Yae-Jean; Ki, Chang-Seok; Park, Woong-Yang
Title: Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus
  • Document date: 2016_11_23
    • ID: xgp2vx6o_29
    Snippet: MERS-CoVs were detected in specimens using real-time reverse transcription (rRT)-PCR. Extracted RNAs were screened by targeting the upE region, and positive results were confirmed by a subsequent amplification of orf1a using a PowerChek MERS Real-Time PCR kit (Kogene Biotech, Seoul, South Korea). All rRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with a total reaction volume of 20 μL (15 μL of PCR.....
    Document: MERS-CoVs were detected in specimens using real-time reverse transcription (rRT)-PCR. Extracted RNAs were screened by targeting the upE region, and positive results were confirmed by a subsequent amplification of orf1a using a PowerChek MERS Real-Time PCR kit (Kogene Biotech, Seoul, South Korea). All rRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with a total reaction volume of 20 μL (15 μL of PCR reaction mixture and 5 μL of template RNA). The thermocycling conditions included a reverse transcription reaction for 30 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 60 sec at 60°C. A positive viral template control and a nontemplate control were included in each run. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified simultaneously as an internal control to monitor PCR inhibition. A positive result was identified by a well-defined exponential fluorescence curve that crossed the defined threshold at ≤35 cycles in both the upE and ORF1a assays.

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