首先根据空肠弯曲菌mapA基因序列设计LAMP引物,建立检测空肠弯曲菌的LAMP检测方法。继而对LAMP方法中不同反应组分的浓度分别进行筛选,结果显示,当内外引物浓度分别为1.6,0.2μmol/L、Mg2+。浓度为8mmol/L、dNTP浓度为1.4 mmol/L、Betaine浓度为0.8 mol/L时获得最佳的反应结果。最后对LAMP反应的特异性和敏感性进行检测,特异性检测结果显示,对其他13种革兰氏阴性和革兰氏阳性细菌扩增结果均为阴性;敏感性检测结果显示,对空肠弯曲菌基因组DNA的检测限为每个反应管100 fg,对空肠弯曲菌培养菌液检测限为每个反应管7.5cfu。试验建立的LAMP方法为快速简便地自动物源性产品中检测空肠弯曲菌奠定了基础。
In this study， the specific mapA gene was used to design a set of primers to develop a loop-mediated isothermal amplification ( LAMP) assay for detection of C． jejuni． To optimize the LAMP assay， the reaction components in the assay were screened to determine the optimal concentration． The final LAMP assay comprised 1．6μmol /L each of inner primers FIP and BIP，0．2 μmol /L each of outer primers F3 and B3,8 mmol /L Mg2 + ，1．4mmol /L dNTP，0 ．8 mol /L Betaine． The specificity test showed that the assay correctly identified all C． jejuni strains but not 13 other bacterial species． The sensitivity of the assay was 100 fg per test tube for C． jejuni genomic DNA and 7． 5 cfu per test tube for C． jejuni bacterial culture． This LAMP assay is a rapid and simple tool for detection of C． jejuni and will provide an essential role in facilitating early diagnosis of this organism from animal products．
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