Loop-mediated Isothermal Amplification Amplification Research Articles

Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Two Clarireedia spp., the Causal Agent of Dollar Spot of Turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.

A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detectionmethod, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the5' endof the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10min faster than several otherprobe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

Rapid DNA extraction and microfluidic LAMP system in portable equipment for GM crops detection

Genetically modified food has come under suspicion because of the potential safety risks. Only a limited number of target genes could be detected by traditional methods. Here, customized-fast DNA extraction methods and a LAMP (Loop-mediated isothermal amplification) system were developed combining with a hand-held chip, a portable analyzer, and an Android App for the qualitative detection of genetically modified crops. DNA of seeds and leaves was obtained within 5 minutes. Primers were designed with high specificity and sensitivity (10 copies) for later LAMP amplification. Crude DNA was added to the chip pre-embedded with customizable primers by quantitative dropper and the whole detection process was less than 40 minutes without any large-scale instrument. The 0.1% quality percentage of GM samples (maize or soybean) could be detected by the detection system. The detection system with its low workload, high accuracy, and stability offers a novel and effective approach for the rapid detection of GM crops and many other food safety areas in the field.

Loop-mediated isothermal amplification linked a nanoparticles-based biosensor for detecting Epstein-Barr virus

Epstein-Barr virus (EBV) is a ubiquitous gamma herpesvirus that maintains a lifelong latent association with B lymphocytes. Here, a rapid and reliable diagnosis platform for detecting EBV infection, employing loop-mediated isothermal amplification (LAMP) combined with a gold nanoparticles–based lateral flow biosensors (AuNPs-LFB) (termed LAMP Amplification Mediated AuNPs-LFB Detection, LAMAD), was developed in the current study. A set of specific LAMP primers targeting the Epstein-Barr nuclear antigen (EBNA) leader protein (EBNA-LP) gene was designed and synthesized. Subsequently, these templates extracted from various pathogens and whole blood samples were used to optimize and evaluate the EBV-LAMAD assay. As a result, the limit of detection (LoD) of the EBV-LAMAD assay was 45 copies/reaction. The EBV-LAMAD assay can detect all representative EBV pathogens used in the study, and of note, no cross-reactions were observed with other non-EBV organisms. Moreover, the whole workflow of the EBV-LAMAD assay can be completed within 70 min, including rapid EBV template preparation, EBV-LAMP amplification, and AuNPs-LFB-mediated detection. Taken together, the EBV-LAMAD assay targeting the EBNA-LP gene is a rapid, simplified, sensitive, reliable, and easy-to-use detection protocol that can be used as a competitive potential diagnostic/screening tool for EBV infection in clinical settings, especially in basic laboratories in resource-limited regions.Key points• A novel, simplified, and easy-to-use AuNPs-LFB biosensor was designed and prepared.• LAMP combined with an AuNPs-LFB targeting the novel EBNA-LP gene was established.• EBV-LAMAD is a rapid, sensitive, and reliable detection protocol for EBV infection.Graphical

On-site detection of Sporothrix globosa using immunoglobulin Y, magnetic separation and loop-mediated isothermal amplification

BackgroundSporothrix globosa (S. globosa) is an agricultural activity-related but neglected pathogenic fungus responsible for sporotrichosis. Timely detection is crucial for managing and preventing its spread. However, due to the lack of efficient recognition elements for enriching S. globosa, the current approaches for detecting S. globosa are not simple and/or sensitive enough. This hinders their wider application of fast screening. ResultsHerein, we successfully prepared immunoglobulin Y (IgY) targeting S. globosa, and developed a rapid and accurate detection method, improving upon current limited and inadequate detection approaches. Our method combined the use of IgY and loop-mediated isothermal amplification (LAMP) to enhance detection sensitivity and specificity simultaneously. The IgY was fabricated on magnetic beads to specifically concentrate S. globosa in samples, while LAMP amplified the captured target after simple boiling DNA extraction. By using our method, as low as 4.66 × 102 Cells mL−1S. globosa was accurately detected in soil and corn straw samples. We further integrated this assay into a portable toolbox for sample-to-result detection in resource-limited areas. By using this toolbox, we have colorimetrically detected soil and corn straw samples contaminated by S. globosa, suggesting the promising on-site detection potential. Significance and noveltyA new IgY recognizing S. globosa was prepared. Through the combination of IgY enrichment and LAMP amplification, the detection sensitivity and specificity were improved simultaneously. This method eliminated thermal cycling, simplified the operation, and reduced the analysis time. Compared to existing methods, our approach is more suitable for on-site detection and can significantly improve public health responses to sporotrichosis outbreaks.

A Hypothetical Approach to Concentrate Microorganisms from Human Urine Samples Using Paper-Based Adsorbents for Point-of-Care Molecular Assays.

This hypothesis demonstrates that the efficiency of loop-mediated isothermal amplification (LAMP) for nucleic acid detection can be positively influenced by the preconcentration of microbial cells onto hydrophobic paper surfaces. The mechanism of this model is based on the high affinity of microbes towards hydrophobic surfaces. Extensive studies have demonstrated that hydrophobic surfaces exhibit enhanced bacterial and fungal adhesion. By exploiting this inherent affinity of hydrophobic paper substrates, the preconcentration approach enables the adherence of a greater number of target cells, resulting in a higher concentration of target templates for amplification directly from urine samples. In contrast to conventional methods, which often involve complex procedures, this approach offers a simpler, cost-effective, and user-friendly alternative. Moreover, the integration of cell adhesion, LAMP amplification, and signal readout within paper origami-based devices can provide a portable, robust, and highly efficient platform for rapid nucleic acid detection. This innovative hypothesis holds significant potential for point-of-care (POC) diagnostics and field surveillance applications. Further research and development in this field will advance the implementation of this technology, contributing to improved healthcare systems and public health outcomes.

Engineering of the LAMP-CRISPR/Cas12b platform for Chlamydia psittaci detection.

Introduction. Chlamydia psittaci (C. psittaci) is a zoonotic infection, that causes psittacosis (parrot fever) in humans, leading to severe clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, in rare cases, death.Gap Statement. Rapid, sensitive and specific detection of C. psittaci facilitates timely diagnosis and treatment of patients.Aim. This study aimed to engineer the LAMP-CRISPR/Cas12b platform for C. psittaci detection.Methodology. The loop-mediated isothermal amplification (LAMP) technique and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 12b (CRISPR-Cas12b) assay were combined to establish two-step and one-tube LAMP-CRISPR/Cas12b reaction systems, respectively, for rapidly detecting C. psittaci.Results. The two-step and one-tube LAMP-CRISPR/Cas12b assay could complete detection within 1 h. No cross-reactivity was observed from non-C. psittaci templates with specific LAMP amplification primers and single-guide RNA (sgRNA) targeting the highly conserved short fragment CPSIT_0429 gene of C. psittaci. The detection limits of the two-step and one-tube LAMP-CRISPR/Cas12b reaction were 102 aM and 103 aM, respectively. The results were consistent with qPCR for nucleic acid detection in 160 clinical samples, including 80 suspected C. psittaci samples, kept in the laboratory.Conclusions. The LAMP-CRISPR/Cas12b assay developed in this study provides a sensitive and specific method for rapidly detecting C. psittaci and offers technical support for its rapid diagnosis.

Boron nitride nanoplate-based improvement of the specificity and sensitivity in loop-mediated isothermal amplification for Vibrio parahaemolyticus detection

BackgroundNucleic acid testing based on DNA amplification is gradually entering people's modern life for clinical diagnosis, food safety monitoring and infectious disease prevention. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are the most powerful techniques that have been the gold standard for quantitative nucleic acid analysis. However, the high nonspecific amplification rate caused by the formation of primer dimers, hairpin structures and mismatched hybridization severely restricts their real-world applications. It is highly desirable to explore a way for improving the specificity and sensitivity of PCR and LAMP assays. ResultsIn this work, we demonstrated that a nanomaterial boron nitride nanoplate (BNNP), due to its unique surface properties, can interact with the main components of the amplification reaction, such as single stranded primers and Bst DNA polymerase, and increase the thermal conductivity of the solution. As a result, the presence of BNNPs dramatically improved the specificity of PCR and LAMP. And BNNPs maintained the specificity even after five rounds of PCR. Moreover, the sensitivity of LAMP was also enhanced by BNNPs, and the detection limit of BNNP-based LAMP was two orders of magnitude lower than that of classical LAMP. Then the BNNP-based LAMP was applied to detect Vibrio parahaemolyticus in contaminated seafood samples with high specificity and a 10-fold increase in sensitivity. SignificanceThis is the first systematic demonstration of BNNPs as a promising additive to enhance the efficiency and fidelity of PCR and LAMP amplification reactions, thereby greatly expanding the application of nucleic acid detection in a wide range of laboratory and clinical settings.

Establishment of two visual interpretation methods of DIV1 LAMP amplification products

Viral diseases have become a significant impediment to the sustainable development of the global shrimp aquaculture industry. Decapod iridescent virus 1 (DIV1) is an emerging shrimp virus that has affected shrimp in China recent years. Rapid detection of DIV1 could improve enhance the effectiveness of prevention, control and treatment in the absence of good prevention and control measures. This study established loop-mediated isothermal amplification (LAMP) along with two visual interpretation methods, LAMP-dye and LAMP-LFD, to detect DIV1. The newly developed method would not cause cross-reactions with other shrimp pathogens such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VpAHPND). The detection limit of DIV1 LAMP was as low as 103 copies of DIV1 per reaction, with a reaction time of less than 40 min. The diagnostic sensitivity and diagnostic specificity of this method were determined to be 88% and 100%, respectively, when compared with the conventional PCR. Both of the LAMP-dye and LAMP-LFD methods are cost-effective and do not require expensive amplification equipment. They can be combined with LAMP and other temperature amplification methods for rapid on-site detection, effectively prevent aerosol contamination, and which are convenient and suitable for field testing or preliminary infection rish prediction experiments to predict the risk of infection.

Integration of in-cassette lysis, purification, and lateral flow strips-based sensor for rapid and on-site detection of yak milk adulteration

Amplification-based nucleic acid testing has become a widely used method for food authenticity analysis. However, due to the need for specialized instrumentation, technical expertize, and trained operators, routine nucleic acid analysis can only be performed in a laboratory setting. For perishable products like yak milk, rapid and accurate detection of dairy components on-site is crucial for the automated management of the production process and improving the milk's quality at dairies. As a feasibility study, we developed an assay strategy for the simultaneous qualitative detection of yak and cow derived components in yak milk. We presented a handheld integrated cassette that combined rapid extraction and purification of dairy nucleic acids, loop-mediated isothermal amplification (LAMP), and visual detection of lateral flow strips (LFS). To enable visual detection of LAMP amplification products on LFS, we utilized a unique primer modification method that labeled the amplification terminator and single oligonucleotide sequence at the 5′ end of the loop primer. This modification avoids the use of antibodies and conventional fluorescein/biotin. The cassette allowed for DNA transfer, fluid drive, and liquid mixing by magnetic force and gravity alone, without the need for complex pumps and valves. The disposable cassette is easy to use, costs less than $1 to produce, has low instrument dependency, and is well suited to on-site detection. The platform can detect 0.1% of cow milk adulteration in yak milk within 50 min. This fully integrated "sample in-result out" detection procedure can also be extended to other detection areas.

Rapid Screening Detection of Genetically Modified Papaya by Loop-Mediated Isothermal Amplification.

A loop-mediated isothermal amplification (LAMP)-mediated screening detection method for genetically modified (GM) papaya was developed targeting the 35S promoter (P35S) of the cauliflower mosaic virus. LAMP products were detected using a Genie II real-time fluorometer. The limit of detection (LOD) was evaluated and found to be ≤0.05% for papaya seeds. We also designed a primer set for the detection of the papaya endogenous reference sequence, chymopapain, and the species-specificity was confirmed. To improve cost-effectiveness, single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) system, which is a lateral flow DNA chromatography technology, was applied. LAMP amplification was clearly detected by the system at the LOD level, and a duplex detection of P35S and chymopapain was successfully applied. This simple and quick method for the screening of GM papaya will be useful for the prevention of environmental contamination of unauthorized GM crops.

Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM).

Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population's health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

Loop-mediated isothermal amplification combined with lateral flow biosensor for rapid and sensitive detection of monkeypox virus.

The ongoing outbreak of the monkeypox, caused by monkeypox virus (MPXV), has been a public health emergency of international concern, indicating an urgent need for rapid and sensitive MPXV detection. Here, we designed a diagnostic test based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor(LFB)for diagnosis of MPXV infection, termed MPX-LAMP-LFB. A set of six LAMP primers was designed based the ATI gene of MPXV, and LAMP amplification of MPXV templates was performed at 63°C for only 40 min. The results were rapidly and visually decided using the LFB test within 2 min. The MPX-LAMP-LFB assay can specifically detect MPXV strains without cross-reaction with non-MPXV pathogens. The sensitivity of the MPX-LAMP-LFB assay is as low as 5 copies/μl of plasmid template and 12.5 copies/μl of pseudovirus in human blood samples. The whole process of the MPX-LAMP-LFB assay could be completed ~1 h, including rapid template preparation (15 min), LAMP reaction (40 min)and result reporting (<2 min). Collectively, MPX-LAMP-LFB assay developed here is a useful tool for rapid and reliable diagnosis of MPXV infection.

Ultrasensitive and Specific Identification of Monkeypox Virus Congo Basin and West African Strains Using a CRISPR/Cas12b-Based Platform.

Human monkeypox (MPX) is a severe and reemerging infectious disease caused by monkeypox virus (MPXV) and forms two distinct lineages, including Congo Basin and West African clades. Due to the absence of specific vaccines and antiviral drugs, developing a point-of-care (POC) testing system to identify MPXV is critical for preventing and controlling MPX transmission. Here, a CRISPR/Cas12b diagnostic platform was integrated with loop-mediated isothermal amplification (LAMP) to devise a novel CRISPR-MPXV approach for ultrasensitive, highly specific, rapid, and simple detection of MPXV Congo Basin and West African strains, and the detection results were interpreted with real-time fluorescence and a gold nanoparticle-based lateral flow biosensor (AuNP-LFB). The optimal detection process, including genomic DNA extraction (15 min), LAMP preamplification (35 min at 66°C), CRISPR/Cas12b-based detection (5 min at 45°C), and AuNP-LFB readout (~2 min), can be completed within 60 min without expensive instruments. Our assay has a limit of detection of 10 copies per test and produces no cross-reaction with any other types of pathogens. Hence, our CRISPR-MPXV assay exhibited considerable potential for POC testing for identifying and distinguishing MPXV Congo Basin and West African strains, especially in regions with resource shortages. IMPORTANCE Monkeypox (MPX), a reemerging zoonotic disease caused by monkeypox virus (MPXV), causes a smallpox-like disease in humans. Early diagnosis is critical to prevent MPX epidemics. Here, CRISPR/Cas12b was integrated with LAMP amplification to devise a novel CRISPR-MPXV approach to achieve highly specific, ultrasensitive, rapid, and visual identification of MPXV Congo Basin and West African strains.

Loop mediated isothermal amplification (LAMP) of DNA: An advanced gene amplification technique

The amplification of DNA provides method for gene based specific detection. Besides Polymerase Chain Reaction (PCR), amplification can be achieved by several isothermal techniques. The PCR suffers from various drawbacks like requirement of laboratory and expertise to perform. Loop Mediated Isothermal Amplification (LAMP) of DNA is one of the promising isothermal techniques rapidly gaining popularity among researchers. The technique requires Bst DNA polymerase and a set of four primers recognizing 6-8 regions of target DNA. LAMP technique of DNA gained surprising popularity for amplifying a desired gene at constant temperature between 60-65 °C & maintain a constant temperature can be easily fulfilled by a water bath or a heat block. The LAMP amplification of the desired DNA fragment can be achieved as high as 109 copies in merely 30 min with higher specificity as compared with the existing PCR technique. The technique of LAMP averts the use of costly thermal cycler instrument, facilitating establishment of a field-friendly technique and is promising technique for gene detection in a resource-limited laboratory.

Rapid identification of Staphylococcus aureus based on a fluorescence imaging/detection platform that combines loop mediated isothermal amplification assay and the smartphone-based system

Food associated diseases pose significant public health threat in the United States. Health risks associated with food-borne pathogens drive the need for constant monitoring of food products. An efficient method that can diagnose food-borne pathogens rapidly will be invaluable and in high demand. In this study, we showed the feasibility of a novel rapid detection platform based on fluorescence imaging/detection that combines a user-friendly, portable loop mediated isothermal amplification (LAMP) reaction device and a smartphone-based detection system. The proposed platform was used to detect Staphylococcus aureus which is one of the most important food-borne pathogen especially dairy products. The complete protocol is quicker; the reaction is performed under isothermal conditions and completed in 1 h or less. Experimental results show that LAMP assays were ten-fold more sensitive than PCR-based detection. The proposed smartphone detection system was able to detect and quantify LAMP assay samples containing three different concentrations of S. aureus from 109 CFU/mL down to 103 CFU/mL. The present proof-of-concept study demonstrated that this platform offers a portable, easy to use method for measuring target pathogens with LAMP amplification.

Development and clinical application of a endonuclease restriction real-time loop-mediated isothermal amplification (ERT-LAMP) assay for rapid detection of Haemophilus influenzae.

Haemophilus influenzae is a main human pathogen that results in a series of diseases in children and adults, such as pneumonia, bacteremia, and meningitis. Although there are many detection methods, they cannot meet the requirements of an early diagnosis. For the prevention and control of H. influenzae infection, quick, sensitive, and particular diagnostics are crucial. Loop-mediated isothermal amplification (LAMP) coupled with restricted endonuclease digestion and real-time fluorescence (H. influenzae-ERT-LAMP) detection was employed to diagnose H. influenzae. H. influenzae-ERT-LAMP combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence identification into a single-pot reaction, allowing for the rapid identification of H. influenzae in 40 min. The outer membrane protein (OMP) P6 gene of H. influenzae was employed to build a sequence of H. influenzae-ERT-LAMP primers. The limit of detection (LoD) of H. influenzae-ERT-LAMP test was 40 fg of genomic DNA per reaction, and the non-H. influenzae templates did not provide positive outcomes. To investigate the applicability of H. influenzae-ERT-LAMP method in clinical sample detection, 30 sputum specimens were obtained from individuals suspected of being infected with H. influenzae. H. influenzae-ERT-LAMP outcomes were in total agreement with LAMP-LFB and PCR. The H. influenzae-ERT-LAMP assay provides rapid, accurate, and sensitive detection making it a promising screening strategy in clinical and basic lab settings.