Loop-mediated isothermal amplification for influenza A (H5N1) virus.
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ABSTRACT:
SUBMITTER: Jayawardena S
PROVIDER: S-EPMC2792861 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice.
| S-EPMC7111304 | biostudies-literature
Project description:Here we describe the use of the loop-mediated isothermal amplification (LAMP) method to detect human influenza viruses (H1 to H3). Our results were correlated 100% with results deduced from routine clinical diagnostic tests. In addition, we also developed a LAMP assay specific for human beta-actin cDNA as a quality control test.
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Project description:A rapid, convenient and reliable pseudorabies virus (PRV) detection system was developed by using the loop-mediated isothermal amplification (LAMP) method. Six special primers were designed successfully based on the PRV DNA-binding protein (DBP) gene. The assay was optimized to amplify PRV DNA by incubation at 63 degrees C for 1h. The LAMP products had a ladder-like pattern of bands from 188 bp when electrophoresed on an agarose gel and its specificity was confirmed by digestion with Hinc II enzyme. Two naked-eye detection methods were developed for use in the field. The detection limit of the LAMP assay was found to be 10 fg DNA sample which was 100-1,000-fold higher than that of PCR. By using DNA (or cDNA) samples extracted from three different PRV strains and six other viruses known to be related genetically to PRV or to cause similar clinical signals in pig, the system was identified to amplify only the PRV DNA. A comparison between the LAMP and PCR assay using five clinical samples showed good correlation.
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Project description:We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
| S-EPMC102748 | biostudies-literature
Project description:We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription-loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets.
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Project description:Detection of swine influenza virus (SIV) in commercial swine herds is important for understanding the infection status of the herd and for controlling disease. Current molecular diagnostics require that specimens be submitted to a laboratory which provides results to the growers after some time which is generally too late to intercede in disease control. Moreover, current diagnostic assays are time-consuming, typically costly, and require skilled technical expertise. We have instituted a reverse transcription loop-mediated isothermal amplification (RT-LAMP) diagnostic assay based on conserved regions of the SIV matrix (M) gene and H1N1 hemagglutinin (HA) sequences. The RT-LAMP assay was optimized to use both colorimetric and fluorescent endpoints and was validated. The M and HA RT-LAMP assays have a limit-of-detection (LOD) sensitive to 11 and 8-log-fold dilutions of viral RNA, respectively, and are capable of discriminating between H1 and H3 strains of SIV. Additionally, the RT-LAMP assay was optimized for direct amplification of SIV from field samples without the need for viral RNA isolation. The direct RT-LAMP detected >86 % of qRT-PCR validated SIV samples, and >66 % of negative samples when spiked with viral RNA or SIV. The diagnostic RT-LAMP assay is a rapid, sensitive, specific, and cost-effective method for the detection of SIV in herds substantially aiding diagnosis and surveillance.
| S-EPMC7799534 | biostudies-literature
Project description:An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using in vitro RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic.
| S-EPMC3889764 | biostudies-literature
Project description:Considering the fatal human victims and economic loss by the annual epidemic influenza virus, the development of a rapid and convenient genetic analysis methodology is demanding for timely on-site pathogen detection. In this study, we utilized reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for multiplex target gene amplification, and the resultant amplicons were analyzed on the immunochromatographic strip (ICS) for subtyping influenza A virus. Through the optimized primer design, reaction temperature and time, and concentration of enzymes (Bst DNA polymerase and AMV reverse transcriptase) and dNTP, the HA (H1, H3, and H5 gene) and conserved M gene were amplified. The ICS contains two test lines in addition to a control line in order to detect the presence of the HA and M gene, thereby informing us of influenza virus A type as well as its subtype (H1N1, H3N2, and H5N1). The combination of the multiplex RT-LAMP with the ICS could be complete in 40 min and the pathotyping and subtyping of influenza A virus were performed even with 10 copies of viral RNA templates. Moreover, the subtyping of clinical samples, which were obtained from patients infected by influenza A virus was successfully confirmed using the multiplex RT-LAMP and ICS techniques, showing great feasibility of our methodology for real sample analysis with high speed, simplicity and sensitivity.
| S-EPMC7094724 | biostudies-literature
Project description:We present eLAMP, a PERL script, with Tk graphical interface, that electronically simulates Loop-mediated AMPlification (LAMP) allowing users to efficiently test putative LAMP primers on a set of target sequences. eLAMP can match primers to templates using either exact (via builtin PERL regular expressions) or approximate matching (via the tre-agrep library). Performance was tested on 40 whole genome sequences of Staphylococcus. eLAMP correctly predicted that the two tested primer sets would amplify from S. aureus genomes and not amplify from other Staphylococcus species. Open source (GNU Public License) PERL scripts are available for download from the New York Botanical Garden's website.
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Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America. It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment. In this study, a loop-mediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences. Two different techniques, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring, were employed. The specificity and sensitivity of the LAMP assay were determined. The assay's detection limit was 0.5?×?10-9 pmol/µl DNA for NS5 protein coding region and 1.12?×?10-11 pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR. All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV. In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.
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