College of Veterinary Medicine, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Provincial Key Laboratory of Animal Microecology and Healthy Breeding, Engineering Research Center of Microecological Vaccines (Drugs) for Major Animal Diseases, Ministry of Education, Jilin Agricultural University, Changchun, 130118, China.
College of Veterinary Medicine, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Provincial Key Laboratory of Animal Microecology and Healthy Breeding, Engineering Research Center of Microecological Vaccines (Drugs) for Major Animal Diseases, Ministry of Education, Jilin Agricultural University, Changchun, 130118, China.
Poult Sci. 2024 Oct;103(10):104068. doi: 10.1016/j.psj.2024.104068. Epub 2024 Jul 9.
Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.
禽流感病毒（AIV）亚型 H9N2 近年来对家禽业构成了重大威胁，已成为鸡、鸭和鸽子群体中的主要亚型。此外，H9N2 AIV 的公共卫生方面对人类构成重大威胁。因此，早期和快速诊断 H9N2 AIV 非常重要。在这项研究中，成功地建立了一种基于荧光强度的新型 H9N2 AIV 检测方法，该方法基于 CRISPR/Cas13a 技术。首先在原核系统中表达 Cas13a 蛋白，并使用镍离子亲和层析进行纯化，得到高纯度的 Cas13a 蛋白。设计和筛选了最佳的 RPA（重组酶聚合酶扩增）引物对和 crRNA，成功构建了基于 CRISPR/Cas13a 技术的 H9N2 AIV 检测方法。确定了 Cas13a 和 crRNA 的最佳浓度，以优化构建的检测系统。优化后的检测系统具有出色的灵敏度，最小检测限为 10°拷贝/μL，与其他禽源易感病毒无反应，具有出色的特异性。该检测方法为 H9N2 AIV 的现场检测提供了基础。
