Abstract
BackgroundFrom 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.ResultsThe 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.ConclusionThe 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.
Highlights
In April 2010, a severe viral disease spread out in most duck-farming regions in China including Zhejiang, Jiangsu, Hebei and Shandong provinces
Design of Duck Encephalitis virus (DEV) specific RT- LAMP primers Based on the sequence of newly isolated Tembusurelated Flavivirus BYD-1 published in GenBank (Genbank accession no.JF312912.1) and other unpublished sequences that we collected, a highly conserved region of the E gene was chosen to design the reverse transcription loop-mediated isothermal amplification (RT-LAMP) primers
Following RT-LAMP amplification, white turbidity was visually observed in the bottom of the tube
Summary
In April 2010, a severe viral disease spread out in most duck-farming regions in China including Zhejiang, Jiangsu, Hebei and Shandong provinces. The disease affected both meat ducks and laying ducks. In some young-duck farms, the disease developed as early as in 10-day-old ducklings, with a peak in 20-40 days old ducklings. From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected laying ducks and 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
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