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Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus

文献类型： 外文期刊

作者： Wang, Songqi 1 ; Shen, Haiyan 2 ; Lin, Qijie 1 ; Huang, Jun 3 ; Zhang, Chunhong 2 ; Liu, Zhicheng 2 ; Sun, Minhua 2 ; Zhang, Jianfeng 2 ; Liao, Ming 2 ; Li, Yugu 1 ; Zhang, Jianmin 1 ;

作者机构： 1.South China Agr Univ, Coll Vet Med, Res Ctr African Swine Fever Prevent & Control, Key Lab Zoonoses Prevent & Control Guangdong Prov, Guangzhou, Peoples R China

2.Guangdong Acad Agr Sci, Inst Anim Hlth, Guangdong Lab LingNan Modern Agr Sci & Technol, Key Lab Livestock Dis Prevent Guangdong Prov,Maomi, Foshan, Peoples R China

3.Foshan Univ, Coll Life Sci & Engn, Guangzhou, Peoples R China

关键词： African swine fever virus; rapid detection; RNase H2; CP-LAMP; point-of-care testing; smartphone quantitation

期刊名称：FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY （ 影响因子：6.073； 五年影响因子：6.34 ）

ISSN： 2235-2988

年卷期： 2022 年 12 卷

页码：

收录情况： SCI

摘要： African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/mu L within 40 min at constant temperature (62 degrees C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV.

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