环介导等温扩增技术检测藻类制品中 产毒微囊藻的研究
目的 建立环介导等恒温扩增法(loop-mediated isothermal amplification, LAMP)快速、特异地检测藻类制品中的产毒微囊藻。方法 以微囊藻毒素(microcystins, MCs)合成基因mcyE基因为模板设计了1套LAMP引物, 分别用LAMP方法和实时荧光聚合酶链式反应(polymerase chain reaction, PCR)方法进行特异性和灵敏度试验, 并对结果进行比较。对10种实际样品进行LAMP检测, 并与超高效液相色谱串联质谱法测定结果进行对比。结果 在特异性试验中, 6株产毒微囊藻LAMP反应均检出mcyE基因, 12株非产毒微囊藻和其他藻株均未检出mcyE基因, 而实时荧光PCR反应结果则出现非特异性扩增。灵敏度试验中, LAMP方法的最低模板浓度为0.005 ng/μL, 而实时荧光PCR方法的最低模板浓度为0.5 ng/μL。10种实际样品中均未检出mcyE基因和MC-LR。结论 该方法操作简单、特异性高、灵敏度较好, 适用于藻类制品中产毒微囊藻的检测。
Objective To establish a method for the rapid and specifical determination of toxic Microcystis sp. in algae products by loop-mediated isothermal amplification (LAMP). Methods According to the sequence of microcystin synthetase gene mcyE gene, LAMP primers were designed. The results of specificity and sensitivity in LAMP method and real-time PCR method were compared. Ten commodity samples were analyzed by both the LAMP method and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results In specificity test, the mcyE gene was amplified from 6 strains of toxic Microcystis and 12 other algae species did not show non-specific amplification of mcyE gene in LAMP assay. But non-specific amplification was happened in real-time PCR assay. In sensitivity test, the limit of detection by LAMP method was 0.005 ng/μL, while real-time PCR method was 0.5 ng/μL. The result of commodity sample detection was that mcyE gene and MC-LR were not detected in all 10 samples. Conclusion This LAMP method is simple to operation and has high specificity and sensitivity, which is suitable for determination of toxic Microcystis in algae products.
标题：环介导等温扩增技术检测藻类制品中 产毒微囊藻的研究
英文标题：Determination of toxic Microcystis sp. in algae products by loop-mediated isothermal amplification
作者：
梁宇斌 广东产品质量监督检验研究院
伍志航 广东产品质量监督检验研究院
范安妮 广东产品质量监督检验研究院
佘之蕴 广东产品质量监督检验研究院
中文关键词:产毒微囊藻,环介导等温扩增技术,藻类制品,
英文关键词:toxic Microcystis,loop-mediated isothermal amplification method,algae products,
发表日期：2019-03-18
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