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    • 10. 发明授权
    • Method of multiplex microorganism detection
    • 多重微生物检测方法
    • US08298758B2
    • 2012-10-30
    • US10584393
    • 2004-12-24
    • Naoko HorikoshiSusumu KawasakiYukio OkadaKazuko TakeshitaTakashi SameshimaShinichi KawamotoKenji Isshiki
    • Naoko HorikoshiSusumu KawasakiYukio OkadaKazuko TakeshitaTakashi SameshimaShinichi KawamotoKenji Isshiki
    • C12Q1/68
    • C12Q1/686C12Q1/689Y02A50/451
    • The present invention is to provide a multiple detection method that can detect contaminating microorganisms existing in foods, including pathogenic Escherichia coli O157, Listeria monocytogenes and Salmonella spp., with high sensitivity comparable or even superior to official methods, comprising the steps of amplifying a plural number of target genes with a single PCR reaction tube and analyzing the same. The following steps are performed consecutively: (A) a step of extracting DNA of the target microorganisms to be detected by treating with at least a lytic enzyme such as Achromopepidase and Lysozyme and/or bacteriocin having lytic activity such as Enterolysine, a surfactant and a protein denaturing agent; and (B) a step of mixing a specific primer to the target microorganisms to be detected to perform multiplex PCR. Further, it is preferable to add a step of culturing with a culture condition where 1 CFU/100 g microorganisms becomes 10.sup.3 CFU/ml or more after 18 to 48 h of culture, for example that the pH after culture becomes 5.1 or more, before the step of extracting DNA of the target microorganisms to be detected.
    • 本发明提供一种可以检测存在于食品中的污染微生物的多重检测方法,包括致病性大肠杆菌O157,单核细胞增生李斯特氏菌和沙门氏菌,具有与官方方法相当或甚至优于其他方法的高灵敏度,包括以下步骤: 使用单个PCR反应管的目标基因数目并进行分析。 连续进行以下步骤:(A)通过用至少一种裂解酶如溶酶酶和溶菌酶和/或具有溶解活性的细菌素(例如肠溶素,表面活性剂和表面活性剂)进行处理来提取要检测的目标微生物的DNA的步骤 蛋白变性剂; 和(B)将特异性引物与要检测的目标微生物混合以进行多重PCR的步骤。 此外,优选在培养18〜48小时后,添加培养条件,其中1CFU / 100g微生物变成10μFCFU / ml以上,例如培养后的pH变为5.1 或更多,在提取要检测的目标微生物的DNA的步骤之前。