非洲猪瘟病毒荧光量子点检测试纸条的研制

Development of fluorescent quantum dots test strip for African swine fever virus

根据非洲猪瘟病毒(ASFV)VP72蛋白的氨基酸序列筛选合成多肽,并与载体蛋白BSA进行偶联,将以此得到的多肽作为抗原免疫小鼠,用来制备鼠源抗ASFV VP72蛋白的单克隆抗体。将上述方法制备的针对ASFV VP72蛋白的单抗作为荧光量子点标记的抗体,以与这种标记单抗互相配对的另一单抗包被检测线(T线),采用山羊抗鼠二抗Ig G包被质控线(C线),建立了一种简便、快速、准确的用于ASFV抗原检测的荧光量子点免疫层析试纸条。结果表明,制备的试纸条对于猪瘟等8种猪常见疫病免疫用灭活疫苗检测时无交叉反应;可检测到8μg/m L的VP72重组蛋白;与商品化的ASFV荧光定量PCR检测试剂盒进行比对检测,其对临床样品检测的阳性符合率为82.1%(23/28),阴性符合率为93.8%(75/80)。该试纸条特异性好、灵敏度高以及稳定性好,为ASFV的现场快速初步诊断提供了一种技术支持。

According to the amino acid sequence of African swine fever virus(ASFV)VP72 protein,synthetic peptides was screened and coupled with carrier protein BSA(bovine serum albumin).The obtained polypeptide was used as antigen to immunize mice and prepare mouse monoclonal antibody against ASFV VP72 protein.A simple,rapid,and accurate fluorescent quantum dot immunochromatographic test strip for ASFV VP72 antigen detection was established by using the monoclonal antibody against ASFV VP72 protein prepared by the above method as the fluorescent quantum dot labeled antibody,coating the detection line(T line)with another monoclonal antibody paired with this labeled monoclonal antibody,and coating the quality control line(C line)with goat anti-mouse IgG.The results showed that there was no cross-reaction between the prepared test strips and inactivated vaccines for immunization of eight common swine diseases,such as classical swine fever.The recombinant protein VP72 of 8μg/mL can be detected.Compared with commercial ASFV fluorescent quantitative PCR detection kit,the positive coincidence rate of clinical samples was 82.1%(23/28)and the negative coincidence rate was 93.8%(75/80).The test strip has good specificity,high sensitivity and stability,which provides a technical support for the rapid preliminary diagnosis of ASFV on site.