Estabishment and evaluation of a new method for rapid detection of CA16 hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer technique / 吉林大学学报(医学版)

ABSTRACT

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 1128 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 X 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 I525 ntu= 15. 452 IgC-9. 746, R2 = 0.993 2, the detection limit was as 1 X 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.