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J Microbiol Methods. 2017 Oct;141:108-114. doi: 10.1016/j.mimet.2017.08.008. Epub 2017 Aug 12.

Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsI mutation of β-lactamase-negative ampicillin-resistant Haemophilus influenzae.

Author information

1
Department of Pediatrics, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.
2
Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan; Department of Microbiology, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan; Center for Clinical Infectious Diseases and Research, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan. Electronic address: t_maeda@saitama-med.ac.jp.
3
Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.
4
Department of Laboratory Medicine, National Defense Medical College Hospital, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.

Abstract

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 ftsI gene on Haemophilus influenzae without amplifying the sequence carrying the point mutations (T1578G/A) in β-lactamase-negative ampicillin resistant (BLNAR) strains. Reactions were performed at 61°C for 45min. Successful target gene amplifications were detected by measuring real-time turbidity using a turbidimeter and visual detection. The assay had a detection limit of 10.0pg of genomic DNA per reaction and showed specificity against 52 types of pathogens, whereas amplifications were completely blocked in even 100.0ng/μL 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 naïve 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.

KEYWORDS:

ARMS; Haemophilus influenzae; Loop-mediated amplification; PBP3; Single nucleotide polymorphism; ftsI

PMID:
28807759
DOI:
10.1016/j.mimet.2017.08.008
[Indexed for MEDLINE]

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