PDF(Pubmed)
Abstract：
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.
摘要：
近年来，禽流感病毒（AIV）亚型H9N2已成为鸡群中的主要亚型，对家禽业务构成了重大威胁，鸭子,还有鸽子.此外,H9N2AIV的公共卫生方面对人类构成重大威胁。因此,H9N2AIV的早期和快速诊断非常重要。在这项研究中,利用CRISPR/Cas13a技术成功建立了基于荧光强度检测H9N2AIV的新方法。Cas13a蛋白首先在原核系统中表达，并使用镍离子亲和层析纯化，产生高纯度的Cas13a蛋白。设计并筛选出最佳的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H9N2AIV检测系统。确定Cas13a和crRNA的最佳浓度以优化构建的测定。优化后的检测系统灵敏度优异，最低检测限为10°拷贝/μL，不与其他禽类易感病毒反应，具有优异的特异性。该检测方法为H9N2一AIV的现场检测提供了依据。
