›› 2010, Vol. 37 ›› Issue (4): 43-46.

• 生理生化 • Previous Articles     Next Articles

Preparation of Standard Positive Template with Transcription in Vitro for Real-time Quantitative Reverse Transcription PCR (qRT-PCR) for the Detection of Newcastle Disease Virus

SUN Jun-feng1，2，LIU Hua-lei2，ZHANG Wei2，WANG Zhi-liang2   

  1. (1.Qingdao Agricultural University，Qingdao 266109,China; 2.China Animal Health and Epidemiology Center，Qingdao 266032,China）
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-04-20 Published:2010-04-20
  • Contact: WANG Zhi-liang

Abstract: Primers and probe for qRT-PCR were designed based on the matrix protein gene sequence of Newcastle disease virus(NDV)，and T7 promoter was added to the 5’ end of the forward primer. RT-PCR was used to get the template for transcription in vitro，and the concentration and the D value of the RNA were determined. The results of linear and specific test indicated that the template showed good linearity and specificity.The correlation coefficient was 0.998 when the template was diluted to 2.95×104 to 2.95×108 copies/μL. Storing at -80 ℃,the template still worked well after 30 days. And the coefficient of variation value of 2.95×104 to 2.95×108 copies/μL standard positive template were 0.20%, 0.28%, 1.00%, 0.54% and 0.52% respectively，showing an excellent repetitiveness of the template. tative reverse transcriptase PCR

Key words: Newcastle disease virus; in vitro transcription; one-step real-time quantitative reverse transcriptase PCR

CLC Number: