Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsl mutation of β-lactamase-negative ampicillin-resistant Haemophilus influenzae
Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsl mutation of β-lactamase-negative ampicillin-resistant Haemophilus influenzae
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DOI：
10.1016/j.mimet.2017.08.008
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发表时间：
2017-10-01
影响因子：
2.2
通讯作者：
Nonoyama, Shigeaki
中科院分区：
文献类型：
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作者：
Tamura, Shinsuke;Maeda, Takuya;Nonoyama, Shigeaki
快速简便地检测序列多态性，包括负责与耐药性相关的氨基酸取代的细菌病原体的核苷酸点突变，对于正确使用抗菌药物至关重要。本文建立了一种结合环介导扩增（LAMP）和扩增难治突变系统（ARMS）准确区分靶序列中不同单核苷酸的检测方法，称为ARMS-SNP LAMP。该方法能够对流感嗜血杆菌ftsl基因中的核苷酸（1578 T）进行种属特异性检测，而无需扩增β-内酰胺酶阴性氨苄青霉素耐药（BLNAR）菌株中携带点突变（T1578 G/A）的序列。反应在61 ℃下进行45 min。通过使用浊度计和目视检测测量实时浊度来检测成功的靶基因扩增。该测定具有10.0 pg的基因组DNA每个反应的检测限，并显示出对52种类型的病原体的特异性，而扩增被完全阻断，即使在100.0 ng/μ L的基因组DNA与点突变T1578 G和T1578 A。在实时LAMP程序中通过相同的解链曲线确认预期的ARMS-SNP LAMP产物。该方法也用于分析57株临床分离的H。流感。所有25株具有1578 T初始序列的临床分离株均得到阳性结果。此外，31株T1578 G突变BLNAR菌株和1株T1578 A突变BLNAR菌株获得一致的阴性结果。ARMS-SNP LAMP方法是一种简单快速的临床分离株SNP基因分型方法，可作为床旁检测（POCT）技术。由于其简单和方便，它适用于资源有限的情况和设备齐全的临床环境。
Rapid and easy detection of sequence polymorphisms, including nucleotide point mutations of bacterial pathogens responsible for amino acid substitutions linked to drug resistance, is essential for the proper use of antimicrobial agents. Here, a detection method using loop-mediated amplification (LAMP) combined with amplification refractory mutation system (ARMS) to accurately distinguish a different single nucleotide in the target sequence was established, named ARMS-SNP LAMP. This procedure is capable of species-specific detection of a nucleotide (1578T) in the ftsl gene on Haemophilus influenzae without amplifying the sequence carrying the point mutations (T1578G/A) in beta-lactamase-negative ampicillin resistant (BLNAR) strains. Reactions were performed at 61 degrees C for 45 min. Successful target gene amplifications were detected by measuring real-time turbidity using a turbidimeter and visual detection. The assay had a detection limit of 10.0 pg of genomic DNA per reaction and showed specificity against 52 types of pathogens, whereas amplifications were completely blocked in even 100.0 ng/mu L of genomic DNA with point mutations at T1578G and T1578A. The expected ARMS-SNP LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This novel procedure was also used to analyze 57 clinical isolates of H. influenzae. All 25 clinical isolates with the naive sequence of 1578T gave positive results. In addition, concordant negative results were obtained for 31 of the BLNAR strains with the T1578G mutation and one strain with the T1578A mutation. The ARMS-SNP LAMP method is a simple and rapid method for SNP-genotyping of a clinical isolate as point-of-care testing (POCT) technology. It is suitable for use in both resource-limited situations and well-equipped clinical settings because of its simplicity and convenience.