Isothermal Amplification Technique Research Articles

DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL−1, 1.5 copies⋅μL−1, and 0.17 copies⋅μL−1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens.

Loop-mediated isothermal amplification, LAMP, is nowadays the most popular isothermal nucleic acid amplification technique, and as such, several commercial, ready-to-use master mixes have flourished. Unfortunately, independent studies to determine their performance are limited. The current study performed an independent evaluation of the existing ready-to-use commercial LAMP master mixes WarmStart® LAMP Kit, LavaLAMP™ DNA Master Mix, Saphir Bst Turbo GreenMaster, OptiGene Fast Master Mix ISO-004, and SynLAMP Mix. To reduce bias, three different genes, namely ttr (Salmonella spp.), rfbE (E. coli O157), and hly (Listeria monocytogenes), were targeted. The comparison was based on amplification speed, performance with decreasing DNA concentrations, and the effect of five typical LAMP reaction additives (betaine, DMSO, pullulan, TMAC, and GuHCl). Significant differences were observed among the different master mixes. OptiGene provided the fastest amplification and showed less detrimental effects associated with the supplements evaluated. Out of the chemicals tested, pullulan provided the best results in terms of amplification speed. It is noteworthy that the different additives impacted the master mixes differently. Overall, the current study provides insights into the performance of commercial LAMP master mixes, which can be of value for the scientific community to better select appropriate reagents when developing new methods.

Endonuclease Q as a robust enhancer for nucleic acid amplification

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites

BackgroundNatural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. MethodsBy using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. ResultsThere was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. ConclusionThis is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

Recombinase polymerase amplification: method’s characteristics and applications in diagnostics of infectious diseases

Isothermal amplification techniques have been actively developed in recent years and are gradually introduced into the range of methods for infectious disease diagnostics. One of the fastest isothermal methods is recombinase polymerase amplification (RPA). This review contains information about the principle of RPA, the role of individual reaction components and primer design considerations. It provides information on characteristics of various methods of RPA results detection, effects of inhibitors, temperature and agitation on the efficiency of reaction. Approaches to quantitative and multiplex RPA are described, as well as some variants of portable devices designed to identify infectious agents. The conclusion summarizes advantages and disadvantages of RPA in comparison with other amplification methods.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

LAMP based electrochemical sensor for extraction-free detection of Listeria monocytogenes in food samples

Listeria monocytogenes (LMO) represents one of the most serious hazards for food health due to the high mortality rate and its ability to survive at limiting conditions. In this study, an electrochemical sensor for the detection of LMO in food samples using loop mediated isothermal amplification (LAMP) technique is presented. The extraction step for the release of the nucleic acids of the sample was simplified using three different alternatives (QuickExtract buffer lysis, heating method and an extraction-free option). The three protocols were tested with the LAMP reaction in seven different food matrices (Dairy milk, fresh cheese, ham, hummus, lettuce, milk powder and pâté) and were able to detect 1 cfu/25 g as stipulated by the food safety regulations. It was proved that all three alternatives were compatible with the electrochemical detection using methylene blue redox-active molecule in dairy milk samples. The final validation of the sensor was carried out with the extraction-free option in nine different food matrices, which were able to detect 1 cfu/25 g of food sample in all cases. This validation demonstrates the feasibility of the sensor for its application in the food industry as a point-of-test solution.

Comparison of Elva Diagnostic SARS-CoV-2 Saliva Nucleic Acid Test Kit with RT-qPCR and Rapid Antigen Tests in COVID-19 Patients

The practical application of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique has been confirmed in diagnosing different viral infections. Nevertheless, its accuracy in identifying SARS-CoV-2, particularly in practical clinical situations, has not been thoroughly investigated. This study aims to investigate the sensitivity and specificity of the Elva Diagnostic SARS-CoV-2 Saliva Nucleic Acid Test Kit, utilizing the RT-LAMP and Rapid Antigen Test (RAT) methods for in vitro diagnostic testing, compared to the real-time quantitative polymerase chain reaction (RT-qPCR) method throughout the progression of COVID-19. Method: This study employed an analytical observational diagnostic test at Dr. Soetomo Regional Public Hospital, Surabaya, from March 2022 to May 2022. This research involved 54 samples of saliva and nasopharyngeal swabs obtained from 36 patients confirmed positive for COVID-19 and 18 samples from subjects not confirmed to have COVID-19, tested using the RT-qPCR method. The diagnostic performance of both the RT-LAMP and RAT methods was assessed by calculating their sensitivity and specificity in comparison to RT-qPCR, beginning from the time the patient was confirmed positive for COVID-19. The suitability of each method was analyzed using Cohen’s kappa. The nucleocapsid (N) protein gene from SARS-CoV-2 RNA was detected with RT-LAMP and RAT test kits which showed incompatibilities with the RT-qPCR method (p value 0.308). The positive and negative results with the RT-LAMP and RAT method examinations were similar in number compared to the RT-qPCR method, where the positive results in the RT-LAMP and RAT methods were 2 subjects and the negative results were 52 subjects. Based on the results, only 2 confirmed cases had positive results with RT-LAMP and RAT, which means the sensitivity of both tests is only 5.5% and both are poor screening tests for patients suspected of having COVID-19. In addition, the specificity of RT-qPCR as the gold standard examination method for diagnosing COVID-19 cannot be replaced by the RT-LAMP and RAT methods.

DNA-functionalized MOF fluorescent probes for the enzyme-free and pretreatment-free detection of MicroRNA in serum

MicroRNA (miRNA) is a promising biomarker that plays an important role in various biomedical applications, especially in cancer diagnosis. However, the current miRNA detection technology has inherent limitations such as complex operation, expensive testing cost and excessive detection time. In this study, a dual signal amplification biosensor based on DNA-functionalized metal–organic frameworks (MOFs) fluorescent probes, MFPBiosensor, was established for the enzyme-free and pretreatment-free detection of the colon cancer (CC) marker miR-23a. DNA-functionalized MOFs NH2-MIL-53(Al) (DNA@MOFs) were synthesized as fluorescent probes with specific recognition functions. A single DNA@MOF carries a large number of fluorescent ligands 2-aminoterephthalic acid (NH2–H2BDC), which can generate strong fluorescence signals after alkaline hydrolysis. Combined with catalyzed hairpin assembly (CHA), an efficient isothermal amplification technique, the dual signal enhancement strategy reduced matrix interference and sensitized the signal response. The established MFPBiosensor successfully detected extremely low levels of miRNA in complex biological samples with acceptable sensitivity and specificity. With a single detection cost of $0.583 and a test time of 50 min, the excellent inexpensive and rapid advantage of the MFPBiosensor is highlighted. More importantly, the subtle design enables the MFPBiosensor to achieve convenient batch detection, where miRNA in serum can be directly detected without any pretreatment process or enzyme. In conclusion, MFPBiosensor is a promising biosensor with substantial potential for commercial miRNA detection and clinical diagnostic applications of CC.

Hyperbranched RCA-mediated construction of a dendritic DNA nanostructure for ratiometric fluorescence biosensing

As a robust isothermal strand amplification technique, rolling circle amplification (RCA) is popularly adopted for constructing various homogeneous fluorescence biosensing strategies. However, the limited functional unit decoration on linear RCA products and the slow homogeneous reaction kinetics usually restrict their performance. Herein, by designing a target biorecognition-triggered hyperbranched RCA reaction to construct an Mg2+-dependent DNAzyme (MNAzyme)-decorated dendritic DNA nanostructure, a novel ratiometric fluorescence biosensing method was proposed. The catalyzed cleavage of MNAzymes toward a short stand labeled with the FAM fluorophore and its quencher realized a “signal-on” fluorescence output. Meanwhile, a “signal-off” fluorescence output of 2-aminopurine (2-AP) labeled at a DNA hairpin was realized through the DNA hybridization with the nanostructure. Besides efficient acceleration of homogeneous reaction kinetics, these fluorescence responses were dramatically amplified by the high decoration of MNAzymes on the dendritic nanostructure and its strong signal suppression toward 2-AP. Together with the exonuclease III-amplified target biorecognition reaction, a very low detection limit of 0.53 fg mL–1 and a six-order magnitude linear range were endowed for kanamycin antibiotic assay. This excellent analytical performance and its high selectivity, and good operability determine promising prospect of the method for practical applications.

Validation and optimization of the loop-mediated isothermal amplification (LAMP) technique for rapid detection of wheat stripe mosaic virus, a wheat-infecting pathogen

BackgroundWheat stripe mosaic virus (WhSMV) is a significant wheat pathogen that causes substantial yield losses in Brazil and other countries. Although several detection methods are available, reliable and efficient tools for on-site WhSMV detection are currently lacking. In this study, a Loop-Mediated Isothermal Amplification (LAMP) method was developed for rapid and reliable field detection of WhSMV. We designed WhSMV-specific primers for the LAMP assay and optimized reaction conditions for increased sensitivity and specificity using infected plant samples. ResultsWe have developed a diagnostic method utilizing the Loop-Mediated Isothermal Amplification (LAMP) technique capable of rapidly and reliably detecting WhSMV. The LAMP assay has been optimized to enhance sensitivity, specificity, and cost-effectiveness. ConclusionThe LAMP assay described here represents a valuable tool for early WhSMV detection, serving to mitigate the adverse economic and social impacts of this viral pathogen. By enabling swift and accurate identification, this assay can significantly improve the sustainability of cereal production systems, safeguarding crop yields against the detrimental effects of WhSMV.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma

BackgroundPoint-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow. However, sample dilution is often needed in extraction-free amplification to reduce assay inhibition from sample matrices. Since NAATs are typically run at small volumes around 20 μL, the input sample quantity is therefore limited, resulting in an inevitable sensitivity loss. ResultsHere we explore the potential to perform isothermal amplification in larger reaction volumes to accommodate larger sample quantities, thereby improving sensitivity in extraction-free amplification. We demonstrated the approach by developing large-volume reverse transcription loop-mediated isothermal amplification (RT-LAMP) for HIV RNA detection from fingerstick plasma. We found that LAMP at reaction volumes up to 1 mL maintained the same performance. We then identified plasma dilution conditions needed to maintain the limit of detection in RT-LAMP. Subsequently, using inactivated HIV virus, we showed the successful detection of 24 HIV RNA copies in a 500 μL RT-LAMP reaction in the presence of 20 μL plasma (fingerstick volumes), translating to a viral load of 1200 copies per mL. To reduce the increased reagent cost with expanded reaction volumes, we further identified lower-cost reagents with maintained assay performance. Moreover, we showed that large-volume LAMP, compared to 20 μL reactions, could tolerate higher concentrations of various inhibitors in the sample, such as albumin and GuSCN. Significance and noveltyNAATs are conventionally conducted at small reaction volumes. Here we demonstrated that LAMP can be run at large reaction volumes (over 100 μL) with maintained assay performance, allowing sample inhibition to be mitigated while accommodating larger sample quantities. The same strategy of expanding reaction volumes could be applied to other isothermal amplification methods and various POC applications, to streamline test workflows and/or improve assay sensitivity.

Isothermal amplification of long DNA fragments at low temperature by improved strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal amplification technique wherein amplification of a nucleic acid is initiated by nicking enzyme activity at sites flanking the target. Diagnostic SDA is very fast but requires precise optimization and is limited to very short amplicons. Here we report an enhanced approach by addition of single-stranded DNA binding protein, crowding agents and dUTP to enable amplification of kilobase-length products at low temperatures. Additionally, we pair this improved SDA with a novel carryover contamination prevention, eliminating amplifiable DNA at the end of the reaction to reduce contamination risk. Taken together these developments increase the utility and versatility of SDA, broadening the reach of this powerful but uncommonly used method.

Simultaneous Dual-Gene Test of Methicillin-Resistant Staphylococcus Aureus using an Argonaute-Centered Portable and Visual Biosensor.

The development of novel method for drug-resistant bacteria detection is imperative. A simultaneous dual-gene Test of methicillin-resistant Staphylococcus aureus (MRSA) is developed using an Argonaute-centered portable biosensor (STAR). This is the first report concerning Argonaute-based pathogenic bacteria detection. Simply, the species-specific mecA and nuc gene are isothermally amplified using loop-mediated isothermal amplification (LAMP) technique, followed by Argonaute-based detection enabled by its programmable, guided, sequence-specific recognition and cleavage. With the strategy, the targeted nucleic acid signals gene are dexterously converted into fluorescent signals. STAR is capable of detecting the nuc gene and mecA gene simultaneously in a single reaction. The limit of detection is 10 CFU/mL with a dynamic range from 10 to 107 CFU/mL. The sample-to-result time is <65min. This method is successfully adapted to detect clinical samples, contaminated foods, and MRSA-infected animals. This work broadens the reach of Argonaute-based biosensing and presents a novel bacterial point-of-need (PON) detection platform.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status

BackgroundAnalysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. ResultsMBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. Significance and noveltyReal-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

Comparative molecular study based on Loop-mediated isothermal amplification (LAMP) and PCR technique for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA)

Comparative molecular study based on Loop-mediated isothermal amplification (LAMP) and PCR technique for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA)

Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies.

The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic.

Highly Multiplexed Reverse-Transcription Loop-Mediated Isothermal Amplification and Nanopore Sequencing (LAMPore) for Wastewater-Based Surveillance.

Wastewater-based surveillance (WBS) has gained attention as a strategy to monitor and provide an early warning for disease outbreaks. Here, we applied an isothermal gene amplification technique, reverse-transcription loop-mediated isothermal amplification (RT-LAMP), coupled with nanopore sequencing (LAMPore) as a means to detect SARS-CoV-2. Specifically, we combined barcoding using both an RT-LAMP primer and the nanopore rapid barcoding kit to achieve highly multiplexed detection of SARS-CoV-2 in wastewater. RT-LAMP targeting the SARS-CoV-2 N region was conducted on 96 reactions including wastewater RNA extracts and positive and no-target controls. The resulting amplicons were pooled and subjected to nanopore sequencing, followed by demultiplexing based on barcodes that differentiate the source of each SARS-CoV-2 N amplicon derived from the 96 RT-LAMP products. The criteria developed and applied to establish whether SARS-CoV-2 was detected by the LAMPore assay indicated high consistency with polymerase chain reaction-based detection of the SARS-CoV-2 N gene, with a sensitivity of 89% and a specificity of 83%. We further profiled sequence variations on the SARS-CoV-2 N amplicons, revealing a number of mutations on a sample collected after viral variants had emerged. The results demonstrate the potential of the LAMPore assay to facilitate WBS for SARS-CoV-2 and the emergence of viral variants in wastewater.

Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.