Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.

ABSTRACT: Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.

SUBMITTER: Hayashida K

PROVIDER: S-EPMC6564047 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

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

Project description:The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 x 10(8) to 2 x 10(2) copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 x 10(8) to 2 x 10(1) copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63 degrees C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.

| S-EPMC1829040 | biostudies-literature

Rapid detection of Akabane virus by a novel reverse transcription loop-mediated isothermal amplification assay (RT-LAMP).

Project description:BACKGROUND: Akabane disease, caused by Akabane virus, is an insect-transmitted disease of ruminants that is primarily characterized by fetal damage. METHODS AND RESULTS: In this study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Akabane virus was successfully developed. The primers were designed to target the highly conserved fragment of nucleoprotein from the Akabane virus. The results indicate that the assay is highly specific and sensitive with a detection limit of 5.0 TCID50/mL within a 60-min incubation time. A total of 126 abortive samples collected from Xinjiang province were detected by the established RT-LAMP. The results of RT-LAMP assay showed 96.8% agreement with the semi-nested RT-PCR. CONCLUSION: This study is to first to develop a rapid, sensitive, and accurate method for the detection of Akabane virus, which may be used to screen clinical samples in developing countries or regions.

| S-EPMC3848447 | biostudies-literature

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus.

Project description:A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63? for 60?min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.

| S-EPMC7113777 | biostudies-literature

Colorimetric and fluorometric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for diagnosis of SARS-CoV-2.

Project description:The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since it infected humans almost 3 years ago. Improvements of current assays and the development of new rapid tests or to diagnose SARS-CoV-2 are urgent. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and propitious assay, allowing to detect both colorimetric and/or fluorometric nucleic acid amplifications. This study describes the analytical and clinical evaluation of RT-LAMP assay for detection of SARS-CoV-2, by designing LAMP primers targeting N (nucleocapsid phosphoprotein), RdRp (polyprotein), S (surface glycoprotein), and E (envelope protein) genes. The assay's performance was compared with the gold standard RT-PCR, yielding 94.6% sensitivity and 92.9% specificity. Among the tested primer sets, the ones for S and N genes had the highest analytical sensitivity, showing results in about 20 min. The colorimetric and fluorometric comparisons revealed that the latter is faster than the former. The limit of detection (LoD) of RT-LAMP reaction in both assays is 50 copies/µl of the reaction mixture. However, the simple eye-observation advantage of the colorimetric assay (with a color change from yellow to red) serves a promising on-site point-of-care testing method anywhere, including, for instance, laboratory and in-house applications.

| S-EPMC9464610 | biostudies-literature

LAMP (Loop-mediated isothermal amplification) assay for rapid identification of Varroa mites.

Project description:Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V. destructor and V. jacobsoni, providing the ability to tell them apart from other non-target beehive associated mite and insect species. This LAMP assay is specific in detecting these Varroa species and has been tested on specimens originating from multiple countries. It produces amplification of V. destructor and V. jacobsoni in 16 ± 3.4 min with an anneal derivative of 78 ± 0.5 °C whilst another Varroa species,V. underwoodi, showed late amplification. A gBlock gene fragment, used here as a positive control has a different anneal derivative of 80 °C. Three non-destructive DNA extraction methods (HotShot, QuickExtract and Xtract) were tested and found to be suitable for use in the field. The LAMP assay was sensitive to very low levels of Varroa DNA, down to 0.24 picogram (~ 1 × 10 copies/µL of Varroa gBlock). This is a new molecular tool for rapid and accurate detection and identification of Varroa mites for pest management, in areas where these mites do not occur.

| S-EPMC10366197 | biostudies-literature

Electric LAMP: Virtual Loop-Mediated Isothermal AMPlification.

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.

| S-EPMC4417551 | biostudies-literature

A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer.

Project description:A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer.We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species.Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR.Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

| S-EPMC5598014 | biostudies-literature

One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

Project description:Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

| S-EPMC5220095 | biostudies-literature

Application of the colorimetric loop-mediated isothermal amplification (LAMP) technique for genotyping Cre-driver mice.

Project description:The Cre-loxP system is widely used to investigate the cell-type specific roles of genes of interest. Cre-driver mice are required for cell-type specific knockout during the Cre-loxP reaction. To maintain Cre-driver mouse strains, Polymerase chain reaction (PCR)-oriented genotyping targeting the Cre gene cassette is usually conducted. In this study, we instead applied a colorimetric loop-mediated isothermal amplification (LAMP) method for Cre-genotyping. Among four sets of primers designed by the in silico program, one set effectively amplified the Cre cassette of three Cre-driver strains, but not of C57BL/6 mice. This LAMP-oriented method reduces assay time by less than half compared to the PCR-based method, and can be carried out using a conventional isothermal incubator. Applying this LAMP method may accelerate genotyping of Cre-driver mice.

| S-EPMC9096032 | biostudies-literature

Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.

Project description:Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.

| S-EPMC2844582 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data