产黄曲霉毒素真菌双重荧光定量PCR检测技术的建立与应用
Establishment and Application of Dual Fluorescence Quantitative PCR for Detection Technologies of Aflatoxin-producing Fungi
陈冠果 1李可 2陆金虎 2金晨晨 2王龑 3帅江冰 2鲍维琪 2程昌勇 1宋厚辉 1张晓峰2
作者信息
- 1. 浙江农林大学动物科技学院·动物医学院;浙江省畜禽绿色生态健康养殖应用技术研究重点实验室;动物健康互联网检测技术浙江省工程研究中心;浙江省动物医学与健康管理国际科技合作基地;中澳动物健康大数据分析联合实验室,杭州 311300
- 2. 浙江省检验检疫科学技术研究院,杭州 310016
- 3. 浙江工业大学食品科学与工程学院,杭州 310014
- 折叠
摘要
为实现产黄曲霉毒素真菌的快速检测,本研究建立了检测产黄曲霉毒素真菌的aflR基因和nor-1基因的双重荧光定量PCR方法.分别针对 aflR 基因和nor-1基因保守序列,各设计2对特异性引物和探针,通过引物对间的有效性实验各筛选出1对最优的aflR引物探针和nor-1引物探针,随后建立并优化双重荧光定量PCR的反应条件,构建标准曲线,测定该方法的重复性、灵敏度和特异性.结果显示,成功筛选出aflR-2和nor1-2 2组引物/探针组合,并优化和确定了引物/探针的最佳反应终浓度.特异性实验结果显示,本研究建立的检测方法仅对产黄曲霉毒素真菌的aflR基因和nor-1基因呈现特异性扩增,对黑曲霉、碳黑曲霉、赭曲霉和镰刀菌等常见产毒真菌的核酸样品均无扩增曲线.建立的方法重复性好,批内和批间变异系数均＜3％,双重反应体系的检测灵敏度均低至1 ×102 copies/μL.利用本研究所建立的方法与ELISA试剂盒分别对80份食品样品进行检测,结果显示,双重荧光定量PCR共检测出9份阳性样品,而ELISA试剂盒只检测出了 7份阳性,表明本研究建立的方法具有更高的灵敏度和准确性.
Abstract
In order to realize the rapid detection of aflatoxin-producing fungi,a dual fluorescence quantitative PCR method was established to detect aflR gene and nor-1 gene of aflatoxin-producing fungi.Two pairs of specific primers and probes were designed for the conservative sequence of aflR gene and nor-1 gene respectively,and one pair of optimal afflR primer probe and nor-1 primer probe were selected by the validity test between primer pairs.Then,the reaction conditions of dual fluorescence quantitative PCR were established and optimized,and the standard curve was established to determine the repeatability,sensitivity and specificity of the method.The results indicated that two groups of primer/probe combinations of afflR-2 and norl-2 were successfully screened,and the optimal fi-nal concentration of the primer/probe was optimized and determined.The specific experimental results indicated that the established method only amplified the aflR gene and nor-1 gene of aflatoxin-producing fungi,but did not am-plify the nucleic acid samples of common fungi,such as Aspergillus niger,Aspergillus carbonarius,Aspergillus ochraceus and Fusarium oxysporum,etc.The established method had good repeatability,the intra-and inter-as-say coefficients of variation were less than 3％,and the detection sensitivity of the dual reaction system was as low as 1 x 102 copies/μL.The method established in this study and ELISA kit were used to detect 80 food samples.The re-sults indicated that a total of 9 positive samples were detected by dual fluorescence quantitative PCR,while only 7 positive samples were detected by ELISA kit,indicating that the method established in this study had a higher sensi-tivity and accuracy.
关键词
产黄曲霉毒素真菌/双重荧光定量PCR/快速检测Key words
aflatoxin-producing fungi/double fluorescent quantitative PCR/rapid detection引用本文复制引用
基金项目
浙江省重点研发计划项目(2021C02058)
出版年
2024
NETL
NSTL
万方数据