Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (21): 4492-4500.doi: 10.3864/j.issn.0578-1752.2012.21.018

• VETERINARY SCIENCE • Previous Articles     Next Articles

Fluorescence Quantitative RT-PCR Assay for Detection of Tembusu Virus

 YU  Chun-Mei, DIAO  You-Xiang, TANG  Yi, CUI  Jing-Teng, GAO  Xu-Hui, ZHANG  Ying, JU  Xiao-Jun, WU  Li-Li   

  1. 1.山东农业大学动物科技学院  山东泰安 271018
  • Received:2012-03-23 Online:2012-11-01 Published:2012-09-27

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Abstract: 【Objective】The objective of the study is to establish a method for detecting Tembusu virus by SYBR GreenⅠ relative fluorescence quantitative RT-PCR. 【Method】 Special primers based on Tembusu virus NS5 and E gene were designed and a pair of primers of house-keeping gene β-actin was chosen. Then these amplified fragments were cloned into pMD18-T. Using the plasmids NS5-pMD18-T, E-pMD18-T and β-actin-pMD18-T as standard products, a real-time quantitative reverse transcription-polymerase chain reaction ( RT-PCR) was performed to construct the standard curves of NS5, E and β-actin gene and detect the sensitivity, specificity and repeatability. 【Result】 The results showed a precise linear relationship with a correlation coefficient of R2＞0.99. The detection limits was 10 copies of DNA plasmid reaction. The amplification curve showing a single peak could only been detected for Tembusu virus. The variation coefficient was less than 0.5% by within and between the group of repeatability tests. The clinical samples were detected 3 times by this method, and all results were positive.【Conclusion】 The developed real-time PCR assay was highly specific, sensitive, and reproducible and could be an available tool for diagnosis and monitoring of Tembusu virus in duck farms.

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