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Front Microbiol. 2016 Apr 20;7:554. doi: 10.3389/fmicb.2016.00554. eCollection 2016.

Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip.

Author information

1
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical Sciences Changchun, China.
2
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical SciencesChangchun, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and ZoonosesYangzhou, China.
3
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical SciencesChangchun, China; Animal Science and Technology College, Jilin Agricultural UniversityChangchun, China.
4
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical SciencesChangchun, China; College of Veterinary Medicine, Jilin UniversityChangchun, China.
5
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical SciencesChangchun, China; Changchun SR Biological Technology Co., Ltd., ChangchunChina.
6
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical SciencesChangchun, China; Key Laboratory of Emergency Detection for Public Health of Zhejiang Province, Zhejiang Provincial Center for Disease Control and PreventionHangzhou, China.

Abstract

West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 10(2) copies/μl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 10(1.5) TCID50/ml and 10(1.33) TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

KEYWORDS:

RT-LAMP-VF; West Nile virus; reverse transcription loop-mediated isothermal amplification; visual detection; visualization strip

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