水产品中广州管圆线虫微滴数字聚合酶链反应定量检测方法的建立

Establishment of a droplet digital polymerase chain reaction method for the quantitative detection of Angiostrongylus cantonensis in aquatic products

中文关键词:  广州管圆线虫  微滴数字聚合酶链反应  水产品  定量检测

英文关键词:Angiostrongylus cantonensis  droplet digital polymerase chain reaction  aquatic product  quantitative detection

中文摘要:

      目的 建立微滴数字聚合酶链反应(droplet digital polymerase chain reaction, ddPCR)快速、准确定量检测水产品中广州管圆线虫的分析方法。方法 选择广州管圆线虫ITS2基因序列作为靶标序列, 自主设计特异性引物和探针, 建立广州管圆线虫ddPCR定量检测方法, 通过对ddPCR反应条件的优化, 确立了ddPCR最佳反应条件, 从稳定性、特异性、定量限范围、定量检测低限和人工污染样品等方法, 综合评价建立的广州管圆线虫ddPCR定量检测方法的效果。结果 广州管圆线虫DNA质量浓度在0.320~1000.000 pg/μL的范围内, 基因拷贝数与DNA浓度呈良好的线性关系, 相关系数r2=1, 定量检测低限为0.297拷贝/μL, 人工污染样品中3次重复实验分别检出4370、4210、4310拷贝/μL。结论 本研究建立的广州管圆线虫ddPCR定量检测方法特异性强、灵敏度高、稳定性好、定量范围广, 为水产品中广州管圆线虫的定量检测提供新的检测手段。

英文摘要:

      Objective To establish a rapid and accurate method for quantitative detection of Angiostrongylus cantonensis in aquatic products by droplet digital polymerase chain reaction (ddPCR). Methods The ITS2 gene sequence of Angiostrongylus cantonensis was selected as the target sequence, and the specific primers and probes were designed independently, and the quantitative detection method of Angiostrongylus cantonensis droplets was established. By optimizing the reaction conditions of ddPCR, the optimal reaction conditions of ddPCR were established, and the effects of the established quantitative detection method of Angiostrongylus cantonensis were comprehensively evaluated from the aspects of stability, specificity, quantitative limit range, quantitative detection limit and artificially contaminated samples. Results DNA mass concentration of Angiostrongylus cantonensis ranged from 0.320 to 1000.000 pg/μL, and there was a good linear relationship between gene copy number and DNA concentration with correlation coefficient r2=1. The low limit of quantitative detection was 0.297 copies/μL, and 4370, 4210 and 4310 copies/μL were detected in 3 repeated experiments of artificially contaminated samples, respectively. Conclusion The quantitative detection method of Angiostrongylus cantonensis ddPCR established in this study has strong specificity, high sensitivity, good stability and wide quantitative range, which provides a new detection method for Angiostrongylus cantonensis in aquatic products.