Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood.

ABSTRACT: Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) based methods over serological tests and PCR for rapid detection of microbial pathogens. Here, a rapid LAMP assay was developed to detect the hepatitis B virus (HBV) from DNA, and particularly, blood samples from infected patients using a commercially available master mix and portable real-time fluorometer. The final optimized fluorescence-based LAMP assay provided significant amplification time of less than 15 minutes compared with over 1 hour for PCR and an opened tube LAMP system described previously. Results indicated that fluorescence-based LAMP assay was more sensitive than PCR as a rapid, sensitive, efficient, and highly reliable approach for rapid detection of HBV.

SUBMITTER: Quoc NB

PROVIDER: S-EPMC5857714 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood.

Quoc Nguyen Bao NB Phuong Nguyen Doan Nguyen NDN Chau Nguyen Ngoc Bao NNB Linh Do Thi Phuong DTP

Heliyon 20180306 3

Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) based methods over serological tests and PCR for rapid detection of microbial pathogens. Here, a rapid LAMP assay was developed to detect the hepatitis B virus (HBV) from DNA, and particularly, blood samples from infected patients using a commercially available master mix and portable real-time fluorometer. The final optimized fluorescence-based LAMP assay provided significant amp ...[more]

PMID: 29560471

Similar Datasets

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus.

Project description:Hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. An estimated 180 million people are infected worldwide. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of HCV genomic RNA and compared the sensitivity of LAMP with nested-PCR. A total of 30 blood samples from HCV-infected patients were analyzed with six primers targeting conserved sequences of the HCV 5'UTR within 70 min, under isothermal conditions at 62 °C. Then, visualized by gel electrophoresis with ethidium bromide staining and detected by the naked-eye after adding SYBR Green I. All samples positive for HCV by nested PCR were confirmed by LAMP method. When visualized by gel electrophoresis and ethidium bromide staining, the HCV LAMP assay products appeared in a ladder pattern, with many bands of different sizes. The HCV LAMP product could also be detected by the naked-eye after adding SYBR Green I to the reaction tube and observing a color change from orange to green in positive samples. The HCV LAMP had the same sensitivity as a nested-PCR assay, the detection limit for the both systems were found to be 10 copies/mL of HCV RNA. The LAMP assay reported here is superior for rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of HCV in areas with limited resources, such as developing countries.

| S-EPMC3550813 | biostudies-literature

Reverse transcription-loop-mediated isothermal amplification assay for rapid detection of hepatitis E virus.

Project description:The one-step single-tube betaine-free reverse transcription (RT)-loop-mediated isothermal amplification assay was developed for rapid diagnosis of hepatitis E virus. This assay amplified the target gene in less than 45 min (even as short as 20 min) under isothermal conditions at 63 degrees C, and the sensitivity of this assay was 100-fold greater than that of RT-PCR. This assay demonstrated a detection limit of 0.045 fg (nine copies/reaction).

| S-EPMC2708515 | biostudies-literature

Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.

Project description:BACKGROUND: Hepatitis B virus (HBV) is an important blood-borne pathogen that causes hepatic inflammation and can lead to liver cirrhosis and hepatocellular carcinoma. Conventional methods of HBV detection are time consuming and require highly trained personnel and elaborate equipment. This report describes the development of a rapid, simple, specific, and sensitive loop-mediated isothermal amplification assay (LAMP) for detection of HBV genotypes A, B, C, D, E, and F in blood samples. METHODS: HBV standard plasma panels and clinical donor plasma specimens were used for the development and validation of the LAMP assay. Amplification was performed at 60°C for 60 minutes using extracted DNA or heat-treated plasma specimens without DNA extraction. The assay was evaluated for its ability to detect various HBV genotypes and for its sensitivity, specificity, and time-point of detection. RESULTS: The LAMP assay detected HBV genotypes A-F and demonstrated a sensitivity of 10-100 IU per reaction of HBV DNA. The assay also detected 69 of 75 (92%) HBV-positive donor plasma specimens tested and demonstrated a specificity of 100%. CONCLUSIONS: These results demonstrate that our HBV-LAMP assay is rapid, sensitive and specific, and capable of detecting the major HBV genotypes. This assay could be used in clinical point-of-care settings, mainly in endemic and resource-limited environments for HBV diagnostics, donor screening, epidemiological studies, and therapeutic monitoring of patients undergoing antiviral treatment.

| S-EPMC4305128 | biostudies-literature

Rapid detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification assay.

Project description:To improve the diagnosis and treatment of Mycoplasma pneumoniae (Mp) infection and reduce the misuse of antibiotics, we sought to establish a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Mp.Six primers specific for the Mp P1 gene were designed, and the LAMP method was used to rapidly detect Mp. The sensitivity of the LAMP method was determined by serial dilution of the standard Mp strain FH (standard strains of Mycoplasma pneumoniae). Specificity was assessed with 17 common pathogenic microorganisms in the respiratory tract. Patient samples were collected from the Department of Respiratory and Critical Care Medicine at the 307th Hospital of Chinese People's Liberation Army from March 2016 to May 2017, examined prospectively, and compared with diagnosis by quantitative real-time polymerase chain reaction (qRT-PCR).The LAMP assay for Mp detection can be completed within 60?minutes. The minimum detection limit was 39?pg/?L, and no cross-reaction was observed with 17 common respiratory tract pathogens. Of the 125 clinical specimens tested, 43 cases were positive by LAMP assay, and 40 cases were positive by qRT-PCR (P?=?.162). All 43 samples determined as positive by LAMP test were confirmed to be Mp by Mp P1 protein sequencing.The LAMP assay is suitable for rapid detection of Mp. It has high sensitivity and specificity, and the detection results are not inferior to those of qRT-PCR.

| S-EPMC6023700 | biostudies-literature

A closed-tube, single-step, real time, reverse transcription-loop-mediated isothermal amplification assay for infectious bronchitis virus detection in chickens.

Project description:Infectious bronchitis (IB) is a viral infection of the chicken respiratory tract that causes substantial economic burden on the industry. Simple, specific and rapid diagnosis of this disease is critical for the initiation of appropriate control measures. Conventional molecular diagnostic methods require a relatively sophisticated equipment and skilled staff. Here we describe a rapid, simple, semi-quantative, closed-tube, single-step, real-time- reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for IB and compare our assay with quantative, reverse transcription- polymerase chain reaction (RT-qPCR). The limit of detection (LOD) of our RT-LAMP assay is 1 EID50/ ml. Clinical evaluation of samples from diseased chickens with our RT-LAMP showed a very good concordance with RT-qPCR. Our assay enables simple, specific, rapid molecular detection and semi-quantification of the infectious bronchitis virus (IBV) in veterinary diagnostic laboratories. Furthermore, our RT-LAMP detection is carried out in a sealed tube, eliminating the risk of false-positive results in subsequent tests because of any contamination of the work area as in the case of lateral ﬂow strip or gel electrophoresis-based amplicon detection.

| S-EPMC7367007 | biostudies-literature

Detection of hepatitis C virus by an improved loop-mediated isothermal amplification assay.

Project description:An improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (LAMP) assay targeting the 5' untranslated region (UTR) was developed to detect hepatitis C virus (HCV) infection. Based on an accelerating primer (AP), the present assay, named AP-LAMP, has the advantages of rapidity and sensitivity over the routine LAMP method. The possible AP-based amplification pathway during the reaction was revealed by restriction enzyme digestion and eletrophoresis. The detection limit of the AP-LAMP assay was approximately 84 IU/ml, and no cross-detection was observed. The assay was evaluated further with 126 clinical specimens, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for detection of HCV RNA.

| S-EPMC7087303 | biostudies-literature

Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus.

Project description:BACKGROUND: Bovine rotavirus (BRV) infection is common in young calves. This viral infection causes acute diarrhea leading to death. Rapid identification of infected calves is essential to control BRV successfully. Therefore development of simple, highly specific, and sensitive detection method for BRV is needed. RESULTS: A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized for rapid detection of BRV. Specific primer sets were designed to target the sequences of the VP6 gene of the neonatal calf diarrhea virus (NCDV) strain of BRV. The RT-LAMP assay was performed in a water bath for 60 minutes at 63°C, and the amplification products were visualized either directly or under ultraviolet light. This BRV specific RT-LAMP assay could detect 3.32 copies of subtype A BRV. No cross-reactions were detected with other bovine pathogens. The ability of RT-LAMP to detect bovine rotavirus was further evaluated with 88 bovine rectal swab samples. Twenty-nine of these samples were found to be positive for BRV using RT-LAMP. The BRV-specific-RT-LAMP results were also confirmed by real-time RT-PCR assay. CONCLUSIONS: The bovine rotavirus-specific RT-LAMP assay was highly sensitive and holds promise as a prompt and simple diagnostic method for the detection of group A bovine rotavirus infection in young calves.

| S-EPMC3599620 | biostudies-literature

Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses.

Project description:Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited.

| S-EPMC3347125 | biostudies-literature

Validation of SYBR green I based closed-tube loop-mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria.

Project description:BackgroundAs an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi.MethodsA total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes.ResultsLAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange.ConclusionsThese results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

| S-EPMC7995794 | biostudies-literature

Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP).

Project description:A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.

| S-EPMC5854734 | 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