基于荧光共振能量转移技术的快速检测CA16型手足口病病原体新方法的建立及其评价

目的：基于荧光共振能量转移（FRET）技术建立一种柯萨奇病毒A组16型（CA16）手足口病病原体快速检测的新方法，并对检测效果进行评价，使其达到疾病爆发流行期间大样本量检测的要求。方法：采用SDS-PAGE凝胶电脉技术和蛋白浓度定量试剂盒测定CA16鸡卵黄抗体（CA16-IgY）纯度及蛋白水平，采用间接ELISA法检测抗体效价及特异性，采用紫外可见光谱（UV-Vis）、红外光谱（FTIR）和透射电子显微镜（TEM）等方法对所制备的金纳米粒子（AuNPs）及其生物探针（IgY-AuNPs）的尺寸、形貌和性能进行表征；基于FRET技术构建CA16检测体系，通过优化检测体系中IgY-AuNPs浓度、氯化钠（NaCl）用量和荧光恢复时间等指标，对检测方法的灵敏度、特异度和临床样本检测进行评价。结果：CA16-IgY纯度较高，抗体平均蛋白水平为12.15 mg·L^-1，抗体效价高达1：128 000，特异性良好；AuNPs及IgY-AuNPs的UV-Vis、FTIR和TEM观察结果，IgY已成功标记至AuNPs表面，提示已通过静电自组装成功制备了可以特异性识别CA16的IgY-AuNPs。基于FRET技术构建CA16检测体系，体系经优化后，确定IgY-AuNPs的最佳浓度为0.52×10^-3g·L^-1，NaCl的最佳用量为40 μL，荧光恢复最佳时间为90 min，建立的检测方法的标准曲线为I_525nm=15.452IgC-9.746，R²=0.993 2，检测限为1×10⁴ PFU·mL^-1，与临床检测金标准实时荧光定量PCR（qRT-PCR）法比较，一致率可达93.75%。结论：成功建立了CA16型手足口病病原体的快速检测新方法，可用于实验室和临床检测。

Establishment and evaluation of a new method for rapid detection of CA16 hand,foot and mouth disease pathogens based on fluorescence resonance energy transfer technique

Objective: To establish a new method for rapid detection of Coxsachie virus A16 (CA16) hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer (FRET) technique, to evaluate the detection effect and to make the method to meet the requirements of large sample size detection during the outbreak of disease. Methods: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and bicinchoninic acid (BCA) protein assay were used to identify the purity of CA16 chicken yolk antibody(CA16-IgY) and the protein level. Indirect enzyme-linked immunosorbent assay (iELISA) was used to detect the titer and specificity of anti-CA16 IgY antibody. The size, morphology and characterization of gold nanoparticles (AuNPs) and their biological probes (IgY-AuNPs) were determined by UV-visible spectroscopy (UV-Vis), infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The CA16 detection system was constructed based on FRET technique. The sensitivity and specificity of the detection method and clinical sample detection were evaluated by optimizing the IgY-AuNPs concentration, sodium chloride (NaCl) dosage, fluorescence recovery time and other indicators. Results: The CA16-IgY had high purity, the titer was 1: 128 000,the average protein level was 12.15 mg·L^-1,and CA16-IgY had good specificity. The results of UV-Vis, FTIR and TEM of AuNPs and IgY-AuNPs showed that IgY was successfully labeled onto the surface of AuNPs, which suggested that IgY-AuNPs could specially recognize CA16 was successfully prepared by electrostatic self-assembly. The CA16 detection system was constructed based on FRET technology, after optimization of the detection system, the optimal dosage of IgY-AuNPs was determined to be 0.52×10^-3 g·L^-1, the optimal dosage of NaCl was 40 μL and the optimal fluorescence recovery time was 90 min. The standard curve of the established detection method was I_{525 nm}=15.452 IgC-9.746, R²=0.993 2, the detection limit was as 1×10⁴ PFU·mL^-1. Compared with qRT-PCR, the agreement rate reached 93.75%. Conclusion: A new rapid detection method for CA16 hand, foot and mouth disease pathogens is successfully established, which can be applied to laboratory and clinical tests.