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

Tamura, Shinsuke; Maeda, Takeshi; Misawa, Kazuhisa; Osa, Morichika; Hamamoto, Takaaki; Yûki, Atsushi; Imai, K.; Mikita, Kei; Morichika, Kyoko; Kawana, Akihiko; Matsumoto, Hiroshi; Nonoyama, Shigeaki

doi:10.1016/j.mimet.2017.08.008

Cited by 19 publications

(16 citation statements)

References 38 publications

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“…Using the high specificity of the LAMP method, techniques for genotyping gene polymorphisms without sequence analysis have been developed, including LAMP with FIP and/or BIP primers, which are designed to contain a single-nucleotide polymorphism at each 5′ end (SNPs-LAMP) ( http://loopamp.eiken.co.jp/ ) or insert artificial mutations using FIP and/or BIP primers (ARMS-LAMP) [ 46 , 47 ]. However, the design of LAMP primers for the above-mentioned protocol is not only quite difficult and limited, but also less sensitive than conventional LAMP assays [ 48 ].…”

Section: Discussionmentioning

confidence: 99%

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer

Imai

Tarumoto

Runtuwene

et al. 2018

Malar J

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BackgroundThe recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer.ResultsA LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia.ConclusionsAn innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2362-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer

Imai

Tarumoto

Runtuwene

et al. 2018

Malar J

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“…The POI values are well linearly dependent on the logarithm (lg) of the concentrations of target in the range from 100aM to 1 pM which indicates the linear range spans over 4 orders of magnitude. It is worth to notice the reaction time is longer than typical LAMP that is due to the lower concentration of Bst DNA polymerase and primers than previous reported LAMP reactions . With such high sensitive method in hand, only several nanograms of genomic DNA would be required for mutation detection, which make our method adequate as a practical approach for one‐stepquantitative SNP diagnosis.…”

Section: Methodsmentioning

confidence: 94%

“…Loop‐mediated isothermal amplification (LAMP) is an outstanding exponential amplification technique under isothermal conditions, which has high specificity due to use four primers recognizing six distinct regions in the DNA target 15,16 and also is simple and cost‐efficient since only one‐typed DNA polymerase is required ,,. AS‐LAMP has been widely used to type single nucleotide polymorphisms as a one‐step method, which need to design the primer FIP or BIP containing an SNP nucleotide at each 3’ end .…”

Section: Methodsmentioning

confidence: 99%

One‐Step Quantitative Single Nucleotide Polymorphism (SNP) Diagnosis By Modified Loop‐Mediated Isothermal Amplification (mLAMP)

Ren

Duan

et al. 2019

ChemistrySelect

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Single nucleotide polymorphism (SNP) detection usually needs two steps: amplification (mostly by polymerase chain reaction) and genotyping of SNP by using the amplification products. To shorten the time and simplify the detection, it is ideal to develop a one-step method, in which the amplification itself can be the SNP detection signal. Meanwhile the difficulty in developing one-step technology is the suppression of the background amplification. In this work, we designed an artificial mismatch into the third position from the 3' terminus of the probes to prevent false positive extension reaction of the probes, which greatly improved the selectivity of mutation detection. We integrated this high selective version of primer extension reaction with a rapid and isothermal nucleic acid amplification loop-mediated isothermal amplification (LAMP) method to distinguish between wild-type and mutant DNA. SYBR ® Green I is selected as the fluorescent dye that is tested for real-time measurement of the LAMP reaction by its fluorescence intensity.100aM single strand DNA (ssDNA) or double strand DNA (dsDNA) targets can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LAMP and gives a wide dynamic range for detection of DNA targets (from 100aM to 1 pM).Single nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, which are single nucleotide change with an estimated frequency of one to two polymorphic nucleotides per kilobase. [1] They can be used as not only high-resolution genetic markers for mapping gene, defining population structure, and performing gene association studies but also a fundamental tool for drug discovery and identification of numerous genetic diseases. [2] SNP detection generally needs two steps: amplification of target DNA and genotyping of SNP by using the amplification products. [3] Amplification, usually by the polymerase chain reaction (PCR), is a powerful tool to achieve enough sensitivity for detection of genomic DNA. In principle, the allele-specific PCR, by designing a primer with the nucleotide at its 3'-end complementary to the base at the SNP site in the detected targets, can directly detect SNP. However, the DNA polymerase cannot provide enough specificity to accurately discriminate single-base mis-match. The mismatched primer extension often occurs and the extension products can subsequently be exponentially amplified with PCR prone to generate of false positive results. [4] Therefore, the genomic DNA region that spans the SNP sites of interest needed to be amplified by PCR, and then the amplification products were genotyped at the SNP sites by using allele-specific hybridization, oligonucleic ligation, [5] invasive cleavage, [6] primer extension, and so on, [7] The padlock probes can be specifically ligated with DNA ligase when they are perfectly complementary to the DN...

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“…36 Monitoring of this method is generally performed using intercalating dyes, 33,36 which can inhibit LAMP reactions, 43e45 or real-time turbidimetry. 35 Both of these monitoring approaches, however, do not facilitate multiplex detection and require extensive optimization to avoid non-specific amplification. Yamanaka et al 40 recently reported SNP LAMP techniques using allelespecific oligonucleotide hybridization and annealing selectivity of allele-specific inner primers with 3 0 -end or 5 0 -end mismatches (3 0 AS-LAMP and 5 0 AS-LAMP, respectively).…”

Section: Discussionmentioning

confidence: 99%

Loop-Primer Endonuclease Cleavage–Loop-Mediated Isothermal Amplification Technology for Multiplex Pathogen Detection and Single-Nucleotide Polymorphism Identification

Higgins

Smith

2020

The Journal of Molecular Diagnostics

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Loop-mediated isothermal amplification (LAMP) provides effective diagnostic technology for infectious disease pathogen identification and is compatible with inexpensive instrumentation for use in diseaseprevalent developing regions. However, simultaneous multiple-target detection and single-nucleotide polymorphism (SNP) identification, essential properties of nucleic acid diagnostics, are difficult to achieve using LAMP. This study introduces loop-primer endonuclease cleavage (LEC)eLAMP, a singleplex or multiplex LAMP technology with single-base specificity for variable SNP identification. We developed a singleplex LEC-LAMP Neisseria meningitidis assay that demonstrated complete analytical specificity and a limit of detection of 3.1 genome copies per reaction. Small-scale clinical testing of this assay demonstrated 100% diagnostic specificity and sensitivity when assessed with anonymized DNA extracts from confirmed cases of bacterial meningitis infection. The single-base specificity of this assay indicated effective SNP identification properties when challenged with DNA templates containing SNPs located within a specific six-base region. This assay was modified to generate an allele-specific LEC-LAMP N. meningitidis assay that successfully demonstrated single-tube differentiation of wildtype and mutant allele templates. The singleplex assay was further modified to generate a multiplex LEC-LAMP assay that successfully demonstrated simultaneous multiple-target detection of three bacterial targets, N. meningitidis, Streptococcus pneumonia, and Hemophilus influenzae. LEC-LAMP is the first report of single-tube, real-time, singleplex or multiplex LAMP technology with single-base specificity for variable SNP identification.

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

Cited by 19 publications

References 38 publications

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer

One‐Step Quantitative Single Nucleotide Polymorphism (SNP) Diagnosis By Modified Loop‐Mediated Isothermal Amplification (mLAMP)

Loop-Primer Endonuclease Cleavage–Loop-Mediated Isothermal Amplification Technology for Multiplex Pathogen Detection and Single-Nucleotide Polymorphism Identification

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