Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

Hao Li; Kai Li; Zhen Bi; Jun Gu; Deping Song; Dan Lei; Suoxian Luo; Dongyan Huang; Qiong Wu; Zhen Ding; Leyi Wang; Yu Ye; Yuxin Tang

doi:10.1016/j.jviromet.2019.04.019

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

Hao Li
, Kai Li
, Zhen Bi
, Jun Gu
, Deping Song
, Dan Lei
, Suoxian Luo
, Dongyan Huang
, Qiong Wu
, Zhen Ding
, Leyi Wang
, Yu Ye
, Yuxin Tang

Research output: Contribution to journal › Article › peer-review

Abstract

A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.

Original language	English (US)
Pages (from-to)	59-65
Number of pages	7
Journal	Journal of Virological Methods
Volume	270
DOIs	https://doi.org/10.1016/j.jviromet.2019.04.019
State	Published - Aug 2019

Keywords

Nested RT-PCR
Porcine pegivirus
Reverse transcription-loop-mediated isothermal amplification
qRT-PCR

ASJC Scopus subject areas

Virology

Online availability

10.1016/j.jviromet.2019.04.019

Library availability

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@article{04bc1f1d8ff84c72a9c923979ea7f1d6,

title = "Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus",

abstract = "A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.",

keywords = "Nested RT-PCR, Porcine pegivirus, Reverse transcription-loop-mediated isothermal amplification, qRT-PCR",

author = "Hao Li and Kai Li and Zhen Bi and Jun Gu and Deping Song and Dan Lei and Suoxian Luo and Dongyan Huang and Qiong Wu and Zhen Ding and Leyi Wang and Yu Ye and Yuxin Tang",

note = "This work was supported by the National Key Research and Development Program (grant number 2017YFD0500600 ), the Natural Science Foundation of Jiangxi Province (grant number 2018ACB21027 and 20161BAB214169), the Graduate Research \& Innovation Projects in Jiangxi (grant number YC2017-B036 ), and Science and Technology Project of Education Department of Jiangxi Province (grant number GJJ150388 and GJJ160399 ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",

year = "2019",

month = aug,

doi = "10.1016/j.jviromet.2019.04.019",

language = "English (US)",

volume = "270",

pages = "59--65",

journal = "Journal of Virological Methods",

issn = "0166-0934",

publisher = "Elsevier B.V.",

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T1 - Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

AU - Li, Hao

AU - Li, Kai

AU - Bi, Zhen

AU - Gu, Jun

AU - Song, Deping

AU - Lei, Dan

AU - Luo, Suoxian

AU - Huang, Dongyan

AU - Wu, Qiong

AU - Ding, Zhen

AU - Wang, Leyi

AU - Ye, Yu

AU - Tang, Yuxin

N1 - This work was supported by the National Key Research and Development Program (grant number 2017YFD0500600 ), the Natural Science Foundation of Jiangxi Province (grant number 2018ACB21027 and 20161BAB214169), the Graduate Research & Innovation Projects in Jiangxi (grant number YC2017-B036 ), and Science and Technology Project of Education Department of Jiangxi Province (grant number GJJ150388 and GJJ160399 ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

PY - 2019/8

Y1 - 2019/8

N2 - A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.

AB - A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.

KW - Nested RT-PCR

KW - Porcine pegivirus

KW - Reverse transcription-loop-mediated isothermal amplification

KW - qRT-PCR

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Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

Abstract

Keywords

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