Yu Guangchao, Chen Lei, Lin Chii-wann, Li Bing, Cui Hemiao, Chen Siyi, Miao Jian, Bian Huawei, Chen Dingqiang, Deng Yang
Biol Res. 2014 Oct 2;47(1):53. doi: 10.1186/0717-6287-47-53.
The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.
In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%.
The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.
整合子在临床微生物中的发生、流行情况及其在抗菌药物耐药性中所起的作用，近年来已得到充分研究。就整合子的筛选和检测而言，目前的诊断方法存在重大缺陷，需要新的方法来检测整合子。
在本研究中，实施并评估了针对1类、2类和3类整合子的三种环介导等温扩增（LAMP）检测方法。对这些检测方法进行了优化，包括研究反应温度、体积、时间、灵敏度和特异性（引物和靶标）。在总共1082株分离株（先前鉴定为397株整合子阳性和685株整合子阴性菌株）上进一步验证了所建立的LAMP检测方法的应用。根据结果，证实了每种引物的必要性，发现最佳反应温度、体积和时间分别为65°C、45分钟和25μL。就应用而言，携带intI1、intI2和intI3的361株、28株和8株分离株分别产生阳性扩增产物。其他685株整合子阴性细菌的整合子筛选LAMP检测均为阴性，总检测率和特异性均为100%。
本研究建立的intI1-、intI2-和intI3-LAMP检测方法被证明是筛选细菌整合子的有效、快速检测方法。
