Rapid and Easy-Read Porcine Circovirus Type 4 Detection with CRISPR-Cas13a-Based Lateral Flow Strip
文献类型： 外文期刊
作者： Wang, Jieru 1 ; Zhu, Xiaojie 2 ; Yin, Dongdong 1 ; Cai, Chang 3 ; Liu, Hailong 5 ; Yang, Yuqing 5 ; Guo, Zishi 5 ; Yin, Lei 1 ; Shen, Xuehuai 1 ; Dai, Yin 1 ; Pan, Xiaocheng 1 ;
作者机构： 1.Anhui Acad Agr Sci, Inst Anim Husb & Vet Sci, Key Lab Pig Mol Quantitat Genet Anhui Acad Agr Sci, Livestock & Poultry Epidem Dis Res Ctr Anhui Prov,, Hefei 230031, Peoples R China
2.China Inst Vet Drug Control, Beijing 100000, Peoples R China
3.Zhejiang A&F Univ, Coll Anim Sci & Technol, China Australian Joint Lab Anim Hlth Big Data Anal, Hangzhou 311300, Peoples R China
4.Zhejiang A&F Univ, Coll Vet Med, Hangzhou 311300, Peoples R China
5.Huazhong Agr Univ, Minist Educ, Key Lab Agr Anim Genet Breeding & Reprod, Wuhan 430070, Peoples R China
6.Huazhong Agr Univ, Minist Agr & Rural Affairs, Key Lab Swine Genet & Breeding, Wuhan 430070, Peoples R China
关键词： porcine circovirus 4; CRISPR-Cas13a; RPA; LFD; rapid detection
期刊名称：MICROORGANISMS （ 影响因子：4.5； 五年影响因子：4.8 ）
ISSN：
年卷期： 2023 年 11 卷 2 期
页码：
收录情况： SCI
摘要： First identified as a new circovirus in Hunan Province in China in 2019, porcine circovirus (PCV4) is now widely detected in other Chinese provinces and South Korea. In recent years, the virus has threatened pig health and operations in the pig industry. Hence, early PCV4 detection and regular surveillance are required to control the spread of infection and prevent collateral damage to the industry. Due to PCV4 being difficult to isolate in vitro, molecular detection methods, such as conventional PCR and real-time PCR, and serological assays are currently the main methods used for the detection of PCV4 infection. However, they are time-consuming, labor-intensive, and complex and require professional personnel. To facilitate rapid pen-side PCV4 diagnoses, we used clustered regularly interspaced short palindromic repeats (CRISPR) and Cas13a technology to develop a quick testing kit. Five recombinase-aided amplification (RPA) primer sets were designed based on the conserved PCV4-Cap gene nucleotide region, which were used to determine several key lateral flow strip (LFD) characteristics (sensitivity, specificity, and accuracy). The results showed that the RPA-Cas13a-LFD reaction could detect PCV4 within 1.5 h in genomic DNA harboring a minimum of a single copy. Furthermore, the assay showed good specificity and absence of cross-reactivity with PCV2, PCV3, or other porcine viruses. When we tested 15 clinical samples, a high accuracy was also recorded. Therefore, we successfully developed a detection assay that was simple, fast, accurate, and suitable for on-site PCV4 testing.
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