title: Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay.
abstract: BACKGROUND
  From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus.
  
  
  RESULTS
  The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation.
  
  
  CONCLUSIONS
  The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, PR China.
authors.forename: Youling
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Yuan
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Li
authors.forename: Kexiang
authors.initials: K
authors.lastname: Yu
authors.forename: Jinxing
authors.initials: J
authors.lastname: Yang
authors.forename: Huaiying
authors.initials: H
authors.lastname: Xu
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kangzhen
authors.initials: K
authors.lastname: Yu
authors.forename: Ming
authors.initials: M
authors.lastname: Liao
authors.forename: Zhuoming
authors.initials: Z
authors.lastname: Qin
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D001715:Bird Diseases / Q000175:diagnosis* / Q000821:virology; D002681:China; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D004372:Ducks; D005416:Flavivirus / Q000235:genetics* / Q000302:isolation & purification; D018177:Flavivirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I