Zhao Yueming, Jiang Xia, Qu Yanyan, Pan Ruili, Pang Xinyi, Jiang Yujun, Man Chaoxin
Key Laboratory of Dairy Science, Ministry of Education, Department of Food Science, Northeast Agricultural University, Harbin, 150030, China.
Key Laboratory of Dairy Science, Ministry of Education, Department of Food Science, Northeast Agricultural University, Harbin, 150030, China; National Research Center of Dairy Engineering and Technology, Northeast Agricultural University, Harbin, 150028, China.
J Dairy Sci. 2017 May;100(5):3480-3496. doi: 10.3168/jds.2016-12535. Epub 2017 Mar 9.
In this study, we developed a rapid, specific, and sensitive loop-mediated isothermal amplification technique combined with a lateral flow dipstick (LAMP-LFD) method to detect Salmonella targeting the siiA gene in powdered infant formula (PIF). The specificity of the detection method (LAMP-LFD) approached 100% using 21 Salmonella and 31 non-Salmonella bacterial strains. This detection method exhibited high sensitivity limits for pure cultures at 3.7 cfu/mL and in PIF at 2.2 cfu/g without enrichment. To evaluate the applicability of the LAMP-LFD method, we detected 60 positive PIF samples and 20 negative PIF samples. The results showed that the method of LAMP-LFD had a high diagnostic specificity of 100% for detection of Salmonella in PIF. To reduce incidence of LAMP contamination, we applied propidium monoazide (PMA) to eliminate carryover contamination of LAMP. At the same time, we found that PMA does not affect observation of LFD for measurement of LAMP signal. The results verified that the method of LAMP-LFD targeting the siiA gene is rapid, accurate, and sensitive for Salmonella detection in PIF, and that PMA shows great potential to be widely used to eliminate the amplicon contamination risk generated by the highly sensitive LAMP reaction in the detection process.
在本研究中,我们开发了一种快速、特异且灵敏的环介导等温扩增技术结合侧向流动试纸条(LAMP-LFD)方法,用于检测婴儿配方奶粉(PIF)中靶向siiA基因的沙门氏菌。使用21株沙门氏菌和31株非沙门氏菌菌株,该检测方法(LAMP-LFD)的特异性接近100%。此检测方法对纯培养物的灵敏度极限为3.7 cfu/mL,对未富集的PIF中的灵敏度极限为2.2 cfu/g。为评估LAMP-LFD方法的适用性,我们检测了60份阳性PIF样本和20份阴性PIF样本。结果表明,LAMP-LFD方法对PIF中沙门氏菌检测的诊断特异性高达100%。为降低LAMP污染发生率,我们应用单叠氮化丙锭(PMA)消除LAMP的残留污染。同时,我们发现PMA不影响用于测量LAMP信号的LFD观察。结果证实,靶向siiA基因的LAMP-LFD方法在检测PIF中的沙门氏菌时快速、准确且灵敏,并且PMA在消除检测过程中高灵敏度LAMP反应产生的扩增子污染风险方面具有广泛应用的巨大潜力。