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.
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.
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, #61 Dai Zong Avenue Tai'an, Shandong 271018, China.
Yi
Y
Tang
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, #61 Dai Zong Avenue Tai'an, Shandong 271018, China.
Hao
H
Chen
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, #61 Dai Zong Avenue Tai'an, Shandong 271018, China.
Youxiang
Y
Diao
D000595:Amino Acid Sequence; D000818:Animals; D004372:Ducks / Q000821:virology; D005416:Flavivirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques; D015588:Observer Variation; D016366:Open Reading Frames; D015203:Reproducibility of Results; D048348:Reverse Transcription; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051981:Uracil-DNA Glycosidase / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism
C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins; D051981:Uracil-DNA Glycosidase