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Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method

文献类型： 外文期刊

作者： Li, Chunhua 1 ; Liang, Jieling 3 ; Yang, Dan 3 ; Zhang, Qi 4 ; Miao, Denian 1 ; He, Xizhong 1 ; Du, Yanan 4 ; Zhang, Wanjing 4 ; Ni, Jianping 1 ; Zhao, Kai 2 ;

作者机构： 1.Shanghai Acad Agr Sci, Inst Anim Husb & Vet Sci, Shanghai 201106, Peoples R China

2.Shanghai Acad Agr Sci, Key Lab Agr Genet & Breeding, Shanghai 201106, Peoples R China

3.Taizhou Univ, Sch Life Sci, Taizhou 318000, Peoples R China

4.Shanghai Normal Univ, Coll Life Sci, Shanghai 200234, Peoples R China

5.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China

关键词： porcine epidemic diarrhea virus; RT-LAMP; visual detection

期刊名称：ANIMALS （ 影响因子：3.231； 五年影响因子：3.312 ）

ISSN： 2076-2615

年卷期： 2022 年 12 卷 19 期

页码：

收录情况： SCI

摘要： Simple Summary Porcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic test for PEDV-the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. This new assay proved to be specific and sensitive when applied to clinical specimens. Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 degrees C and 3 min at 80 degrees C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/mu L. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis.

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