Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

• Published in: Scientific reports Vol. 6; no. 1; p. 27605
• Main Authors: Tang, Yi, Chen, Hao, Diao, Youxiang
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.06.2016; Nature Publishing Group
• Subjects: 38/71; 45; 45/43; 631/326/107; 631/326/596; Amino Acid Sequence; Animals; Contaminants; Deoxyribonucleic acid; DNA; Ducks - virology; Embryos; Epidemics; Flavivirus - classification; Flavivirus - genetics; Flavivirus - isolation & purification; Genes; Genomes; Humanities and Social Sciences; Humans; Limit of Detection; multidisciplinary; Nucleic Acid Amplification Techniques; Observer Variation; Open Reading Frames; Poultry production; Proteins; Public health; Reproducibility of Results; Reverse Transcription; RNA polymerase; Science; Science (multidisciplinary); Sequence Alignment; Sequence Homology, Amino Acid; Uracil-DNA Glycosidase - genetics; Uracil-DNA Glycosidase - metabolism; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - metabolism; Virology; China
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep27605

Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 10 0.89  − 1 × 10 1.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10 −16  g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV.