中国兽医科学2018，Vol.48Issue(6)：678-685,8.DOI:10.16656/j.issn.1673-4696.2018.0095
副猪嗜血杆菌LAMP检测方法的建立
Development of a loop-mediated isothermal amplification assay for the detection of Haemophilus parasuis
摘要
Abstract
Using the Haemophilus parasuis 16SrRNAgene sequences deposited in GenBank, loop-mediated isothermal amplification (LAMP) primers were designed. Under the optimized reaction system and condition by Loopamp Realtime Turbidimeter LA-320c, the LAMP method was established for the detection of H. parasuis by measuring the results using Loopamp Real-Time Turbidimeter or using the visible fluorescence. The positive clinical specimens could be directly determined in 15 min at 65 ℃. The developed LAMP assay showed good stability and reproducibility and was 1×104 times more sensitive than the PCR assay with a minimum detection limit of 0. 175 fg/ L DNA. Five bacteria(Actinobacillus pleuropneumoniae, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Shigella flexneri) and five viruses (porcine circovirus type 2, canine parvovirus, classical swine fever virus, transmissible gastroenteritis virus of swine, porcine reproductive and respiratory syndrome virus) detected were all no cross-reactive. All nasal swab specimens from 94 diseased pigs were detected by using the LAMP assay developed in this study, which was 96. 8％ identical to the standard NY/T 2417—2013. The successfully developed LAMP assay could be applied to rapid diagnosis of haemophflussuis at ports and grassroots.关键词
副猪嗜血杆菌/16SrRNA基因/LAMPKey words
Haemophilus parasuis/16SrRNAgene/LAMP分类
农业科技引用本文复制引用
刘亚娟,聂福平,王昱,杨俊,吴蕊,唐昌杰,王国民,李应国..副猪嗜血杆菌LAMP检测方法的建立[J].中国兽医科学,2018,48(6):678-685,8.基金项目
质检公益性项目(201310093) （201310093）
国家质检总局科技项目(2015IK332) （2015IK332）
