Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Loop-mediated isothermal amplification (LAMP): Expansion of its practical application as a tool to achieve universal health coverage.

Since its invention in 2000, loop-mediated isothermal amplification (LAMP) has attracted great interest from researchers and has been used as a simple and rapid diagnostic tool for detection of infectious and non-infectious diseases. Here we review the recent circumstances and outcomes of these applications of LAMP to show the potential of LAMP as a tool for achieving universal health coverage (UHC). A future application of LAMP, such as in an automated multiplex format, is also discussed.

Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections. METHODS: Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region. RESULTS: The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR. CONCLUSIONS: These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.

Loop-mediated isothermal amplification (LAMP): recent progress in research and development.

Loop-mediated isothermal amplification (LAMP) is an established technology that continues to attract the attention of researchers in many fields. Research and development efforts on LAMP technology in recent years have focused on two major areas; first, the study of its clinical application as an approved in vitro diagnostics tool in Japan and certain other countries; and second, research aimed at further simplifying the LAMP test process. This review provides an overview of the status of LAMP on these two topics by summarizing research work conducted, in the main, after our previous review article.

Mitochondrial DNA targets increase sensitivity of malaria detection using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per microl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per microl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays.

Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Sensitive, Specific, and Cost-Effective Point-of-Care Test for Coronaviruses in the Context of COVID-19 Pandemic.

The rampant spread of COVID-19 and the worldwide prevalence of infected cases demand a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. The most common molecular tests approved by regulatory bodies across the world for COVID-19 diagnosis are based on Polymerase Chain Reaction (PCR). While PCR-based tests are highly sensitive, specific, and remarkably reliable, they have many limitations ranging from the requirement of sophisticated laboratories, need of skilled personnel, use of complex protocol, long wait times for results, and an overall high cost per test. These limitations have inspired researchers to search for alternative diagnostic methods that are fast, economical, and executable in low-resource laboratory settings. The discovery of Loop-mediated isothermal Amplification (LAMP) has provided a reliable substitute platform for the accurate detection of low copy number nucleic acids in the diagnosis of several viral diseases, including epidemics like Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). At present, a cocktail of LAMP assay reagents along with reverse transcriptase enzyme (Reverse Transcription LAMP, RT-LAMP) can be a robust solution for the rapid and cost-effective diagnosis for COVID-19, particularly in developing, and low-income countries. In summary, the development of RT-LAMP based diagnostic tools in a paper/strip format or the integration of this method into a microfluidic platform such as a Lab-on-a-chip may revolutionize the concept of PoCT for COVID-19 diagnosis. This review discusses the principle, technology and past research underpinning the success for using this method for diagnosing MERS and SARS, in addition to ongoing research, and the prominent prospect of RT-LAMP in the context of COVID-19 diagnosis.

Fully Automated Molecular Diagnostic System "Simprova" for Simultaneous Testing of Multiple Items.

Nucleic acid amplification-based diagnostics is known as one of the molecular diagnostic systems that allows higher sensitive detection of pathogens than test methods such as immunoassay. However, it has not been widely used because it is complicated to use and takes a long time to generate results. On the other hand, development of fully automated molecular diagnostic systems has been growing around the world as demand for such systems from physicians and laboratory technicians has increased. To meet this demand, we have developed the "Simprova" fully automated molecular diagnostic system, which takes advantage of LAMP (Loop-mediated Isothermal Amplification), a method Eiken Chemical Co., Ltd. invented. Simprova comprises a master unit that controls the entire system and a test unit that extracts and purifies nucleic acid from samples (pretreatment), and uses the LAMP method to detect and amplify nucleic acid. Users can obtain test results automatically by simply installing a pretreatment cartridge, a multi-well testing chip and the sample in the test unit. The multi-well testing chip has 25 reaction wells connected by channels and enables simultaneous testing of multiple targets with one sample. Turnaround time for one test is approximately 30 minutes. Since a conventional extraction and purification method using magnetic-bead separation is used for the pretreatment, nucleic acid can be extracted from serum, plasma, whole blood, urine, and sputum, for example. In addition, the system can perform random-access testing by connecting four test units to the master unit to realize near-the-patient testing. Simprova is therefore a robust and useful system for a wide variety of applications.

Publisher Correction: Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

Influenza virus, respiratory syncytial virus, and human metapneumovirus commonly cause acute upper and lower respiratory tract infections, especially in children and the elderly. Although rapid antigen detection tests for detecting these infections have been introduced recently, these are less sensitive than nucleic acid amplification tests. More recently, highly sensitive point-of-care testings (POCTs) have been developed based on nucleic acid amplification tests, which are easy to use in clinical settings. In this study, loop-mediated isothermal amplification (LAMP)-based POCT "Simprova" to detect influenza A and B viruses, respiratory syncytial virus, and human metapneumovirus was developed. Simprova system is fully automated and does not require skilled personnel. In addition, positive results can be achieved faster than with PCR. In this study, the accuracy of the POCT was retrospectively analyzed using 241 frozen stocked specimens. Additionally, the usability of the Simprova at clinical sites was assessed in a prospective clinical study using 380 clinical specimens and compared to those of real-time PCR and rapid antigen detection test. The novel LAMP-based POCT demonstrated high sensitivity and specificity in characterizing clinical specimens from patients with influenza-like illnesses. The Simprova is a powerful tool for early diagnosis of respiratory viral infections in point-of-care settings.

Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus.

Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.

Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay.

A one-step, single tube, real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting sequences of the untranslated region in the genome of hepatitis A virus (HAV). The RT-LAMP assay reported in this study was very simple and rapid; the HAV-specific amplification was obtained in 50 min under isothermal conditions at 62.5 degrees C by employing a set of seven primers. The RNAs of three cell-adapted HAV strains belonging to different subgenotypes (IA, IB and IIIB) were equally well amplified. The detection limits of the RT-LAMP assay for these HAV strains were 0.4-0.8 focus forming units (FFU)/reaction. The results of the calibration using the WHO international standard indicated that the RT-LAMP assay had similar sensitivity to the conventional RT-PCR method. A comparison of the results from the RT-LAMP and the LightCycler PCR assay using clinical samples in feces revealed that the findings were similar between the two methods. Although several genotypes remain to be tested, it is concluded that the new real-time RT-LAMP assay is very suitable for detection and quantitation of most prevalent genotypes of HAV in diagnostic laboratories.

Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.

[Loop-mediated isothermal amplification].

We have developed the loop-mediated isothermal amplification (LAMP) method, an innovative gene amplification technique, which has the potential to become important in genetic testing. This method allows highly efficient amplification by a single enzyme at a constant temperature. It also has features such as high specificity, rapidity, and simplicity of detection. Therefore, application to various life sciences, such as clinical medicine, is expected.

Sequence specific visual detection of LAMP reactions by addition of cationic polymers.

BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device.

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.

Detection of monoclonality of the immunoglobulin heavy chain gene in thyroid malignant lymphoma by vectorette polymerase chain reaction.

Distinguishing between thyroid malignant lymphoma and lymphocytic thyroiditis (Hashimoto's thyroiditis) is quite difficult and problematic. B cell lymphomas display clonal Ig heavy-chain (IgH) gene rearrangement, and Southern blot hybridization (SBH) is often used for detection of the monoclonality of the IgH gene. However, SBH is often problematic because it requires a large volume of samples. We examined the efficiency in detecting the monoclonality of IgH gene in thyroid malignant lymphomas by vectorette PCR, which we started with only 200 ng of genomic DNA. Monoclonality was detected in 36 of 47 (76.6%) malignant lymphomas, whereas it was not detected in 10 samples of tissue of Hashimoto's thyroiditis. The sensitivity was almost the same as that with SBH in which monoclonality was detected in 33 of 45 (73.3%) malignant lymphomas. These results suggest that vectorette PCR may be a substitute for SBH, and because it requires only a small volume of samples, it may be used in the analysis of aspiration biopsies.

Recombinant nucleocapsid protein-based IgG enzyme-linked immunosorbent assay for the serological diagnosis of SARS.

The recombinant nucleocapsid protein (rNP) of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) was expressed in a baculovirus system. The purified SARS-CoV rNP was used as an antigen for detection of SARS-CoV antibodies in IgG enzyme-linked immunosorbent assay (ELISA). The ELISA was evaluated in comparison with neutralizing antibody assay and the authentic SARS-CoV antigen-based IgG ELISA. Two-hundred and seventy-six serum samples were collected from health care workers in a hospital in which a nosocomial SARS outbreak took place and used for evaluation. The SARS-CoV rNP-based IgG ELISA has 92% of sensitivity and specificity compared with the neutralizing antibody assay and 94% sensitivity and specificity compared with the authentic SARS-CoV antigen-based IgG ELISA. The results suggest that the newly developed SARS-CoV rNP-based IgG ELISA is a valuable tool for the diagnosis and seroepidemiological study of SARS. The SARS-CoV rNP-based IgG ELISA has an advantage over the conventional IgG ELISA in that the antigen can be prepared by laboratory workers without the risk of infection.

Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.

Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3'-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.

Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.

BACKGROUND: Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients' cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF). METHODOLOGY/PRINCIPAL FINDINGS: We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively. CONCLUSIONS/SIGNIFICANCE: Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

Real-time turbidimetry of LAMP reaction for quantifying template DNA.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the synthesis of large amounts of DNA in a short period of time with high specificity. As the LAMP reaction progresses, the reaction by-product pyrophosphate ions bind to magnesium ions and form a white precipitate of magnesium pyrophosphate. We designed an apparatus capable of measuring the turbidity of multiple samples simultaneously while maintaining constant temperature to conduct real-time measurements of the changes in the turbidity of LAMP reactions. The time (Tt) required for the turbidity of the LAMP reaction solution to exceed a given value was dependent on the quantity of the initial template DNA. That is, a graph with the plot of Tt versus the log of the amount of initial template DNA was linear from 2 x 10(3) copies (0.01 pg/tube) to 2 x 10(9) copies (100 ng/tube) of template DNA. These results indicate that real-time turbidity measurements of the LAMP reaction permit the quantitative analysis of minute amounts of nucleic acids present in a sample, with a high precision over a wide range, using a simple apparatus reported in this study.