Isothermal Amplification Assay For Detection Research Articles

Aptamer-associated colorimetric reverse transcription loop-mediated isothermal amplification assay for detection of dengue virus.

Dengue is a neglected tropical disease of significant epidemiological importance in tropical and subtropical countries. Current diagnostics for this infection present challenges, such as cross-reactivity in serological tests. Finding ways to enhance the diagnosis of this disease is crucial, given the absence of specific treatments. An accurate, simple, and effective diagnosis contributes to the improved management of infected individuals. In this context, our work combines molecular biology techniques, such as isothermal loop amplification, with aptamers to detect the dengue virus in biological samples. Our method produces colorimetric results based on a color change, with outcomes available in less than 2 hours. Moreover, it requires simpler equipment compared to molecular PCR tests.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites

BackgroundNatural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. MethodsBy using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. ResultsThere was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. ConclusionThis is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

Sensitive and Specific Loop-Mediated Isothermal Amplification Assays for Detection of Salmonella, CTX-M-1 Group Genes, mph(A), and ermB in Stool and Blood Samples Based on Orange to Green Visible Dye.

Salmonella is a globally prevalent foodborne bacterium, and ceftriaxone and azithromycin have been regarded as drugs of choice for treating Salmonella infections, particularly in children. With the growing incidence of ceftriaxone and azithromycin resistance in Salmonella, there is an urgent requirement for a rapid and dependable gene testing approach to enhance the efficacy of treating Salmonella infections. Utilizing the orange to green visible dye approach, this study developed loop-mediated isothermal amplification (LAMP) assays for the sensitive and specific detection of Salmonella, ceftriaxone and azithromycin resistance genes (including CTX-M-1 group, mph(A), and ermB genes) in stool and blood samples. The specificity and sensitivity of primers during the LAMP assays for detection of Salmonella, CTX-M-1 group, mph(A), and ermB genes were determined in this study. The detection threshold for Salmonella was found to be 1.5 × 103 colony-forming units (CFU)/mL, while it was 1.5 × 102 CFU/mL for CTX-M-1 group genes (including blaCTX-M-3, blaCTX-M-15, and blaCTX-M-55), 1.5 × 102 CFU/mL for mph(A), and 1.5 × 102 CFU/mL for ermB, showing 10-103-fold, 103-fold, and 105-fold increased sensitivity compared with the polymerase chain reaction assay, respectively. Results indicated that the LAMP primers designed for Salmonella, CTX-M-1 group, mph(A), and ermB genes possess high specificity (100%) and sensitivity (over 94%). This novel approach advocates its application in detecting Salmonella, CTX-M-1 group, mph(A), and ermB genes.

Evaluation of Loop-Mediated Isothermal Amplification Assay for Detection of Mycoplasma hominis in Vaginal Samples in Comparison with Real Time PCR

Mycoplasma hominis as one of the etiologic agents of non-gonococcal urethritis in women should be identified for early treatment that led to the patient does not suffer from serious complications such as pelvic inflammatory disease and infertility. With regard to the importance of M. hominis in genitourinary diseases, is required for the choice of the appropriate, reliable, simple, and repeatable laboratory assay for diagnosis. The focus of this study was to investigate 16srRNA-based LAMP assay for the identification of M. hominis from vaginal swabs in comparison with quantitative real-time PCR (qPCR). The eighty vaginal swabs were taken and analyzed by cultures, qPCR, and LAMP assay. Totally, 13 (16.3%), 14 (17.5%), and 25 (31.3%) samples were positive for M. hominis by culture, qPCR, and LAMP assay, respectively. The results indicate a sensitivity of 100%, a specificity of 83.3%, a negative predictive value (NPV) of 100%, and a Positive predictive value (PPV) of 56% of the developed LAMP assays. According to the result, the LAMP assay is significantly more specific, and sensitive than both qPCR and culture methods for diagnosis of M. hominis.

SAT-499 - Development of loop-mediated isothermal amplification assay for detection of hepatitis delta virus

SAT-499 - Development of loop-mediated isothermal amplification assay for detection of hepatitis delta virus

Dry loop-mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens.

To establish a point-of-care test for coronavirus disease 2019 (COVID-19), we developed a dry loop-mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture, except for the primers, is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viruses associated with respiratory infections, including SARS-CoV-2, were tested. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. No LAMP product was detected in reactions performed with RNA from any pathogens other than SARS-CoV-2. After completing an initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Of the 24 samples, 19 (79.2%) were determined by real-time RT-PCR analysis as being positive for SARS-CoV-2 RNA. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive values of the Loopamp 2019-CoV-2 detection reagent kit were 78.9%, 100%, 100%, and 55.6%, respectively. The dry LAMP method for detecting SARS-CoV-2 RNA is fast and easy to use, and its reagents can be stored at 4°C, solving the cold chain problem; thus, it represents a promising tool for COVID-19 diagnosis in developing countries.

Development of a loop-mediated isothermal amplification assay for detection of Austropeplea tomentosa from environmental water samples

Lymnaeid snails are key intermediate hosts for the development and survival of Fasciola spp., the causative agent of Fascioliasis which are economically important parasites infecting humans and livestock globally. The current control method for treating Fascioliasis is heavily reliant on anthelmintic drugs, particularly Triclabendazole (TCBZ) which has resulted in drug-resistant parasites and poses significant risk as there are no long-term efficacious alternatives available. Sustainable control measures at the farm level could include both parasite and snail control will play an important role in Fasciola spp. control and reduce the reliance on anthelmintic drugs. Implementation of such sustainable control measures requires effective identification of snails on the property however Lymnaeid snails are small and difficult to physically locate. Snail identification using an environmental DNA approach is a recent approach in which physically locating snails are not required. Austropeplea tomentosa, is the primary intermediate snail host for F. hepatica transmission in South-East Australia and we present an in-field loop-mediated isothermal amplification and water filtering method for the detection of A. tomentosa eDNA from water samples to improve current surveillance methods. This methodology is highly sensitive with a detection limit of 5 × 10− 6 ng/μL, detected in < 20 minutes, with cumulative sample preparation and amplification time under 1 hour. This proposed workflow could assist in monitoring areas to determine the risk of Fascioliasis infection and implement strategies to manage snail populations to ultimately reduce the risk of infection for humans and livestock.

Colorimetric loop-mediated isothermal amplification assay for detection and ecological monitoring of Sarocladium oryzae, an important seed-borne pathogen of rice.

Accurate and timely disease detection plays a critical role in achieving sustainable crop protection. Globally, rice has been a staple crop for centuries plagued by the diseases that greatly hamper its productivity. Sheath rot, an emerging disease of rice caused by the seed-borne pathogen Sarocladium oryzae, has reportedly caused heavy losses to agricultural produce in recent years. Our study has led to the development and validation of a LAMP assay for early detection of S. oryzae, the causal agent of sheath rot from the live-infected tissues, seeds, weeds, and environmental samples. The assay could detect as low as 1.6 fg/μl of the pathogen in 15 min. The assay was implemented to bio-surveil the presence of this pathogen by testing it on three weed species (Echinochloa colona, Echinochloa crus-galli, and Cyperus teneriffae) growing around the rice fields. The results showed the presence of the pathogen in two of the weed species viz. E. colona and E. crus-galli. The assay was used to test 13 different rice varieties for the presence of S. oryzae in seeds. In total, three of the varieties did not show the presence of S. oryzae in their seeds while the rest were found to harbor the pathogen. The developed assay can effectively be used to detect and screen the presence of S. oryzae in live samples including seeds and field soil.

Development of a Ladder-Shape Melting Temperature Isothermal Amplification Assay for Detection of Duck Adulteration in Beef

Ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed technology, and the objective of this study was to establish its effectiveness for detection of duck adulteration in beef. LMTIA primers were designed with the prolactin receptor gene of Anas platyrhynchos as the target. The LMTIA reaction system was optimized, and its performance was compared with that of the loop-mediated isothermal amplification (LAMP) assay in terms of specificity, sensitivity, and limit of detection (LOD). Our results showed that the LMTIA assay was able to specifically detect 10 ng of genomic DNAs (gDNAs) of A. platyrhynchos, without detecting 10 ng of gDNAs of Bos taurus, Sus scrofa, Gallus gallus, Capra hircus, Felis catus, and Canis lupus familiaris. The sensitivity of the LMTIA assay was 1 ng of gDNAs of A. platyrhynchos; it was able to detect duck adulteration in beef with a 0.1% LOD. Although the LAMP assay could not clearly distinguish A. platyrhynchos from G. gallus, it had a sensitivity of 10 ng of gDNAs of A. platyrhynchos and a LOD of 1% duck adulteration in beef. This study may help facilitate the surveillance of commercial adulteration of beef with duck meat.

Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

Assessment of a Smartphone-Based Loop-Mediated Isothermal Amplification Assay for Detection of SARS-CoV-2 and Influenza Viruses

A critical need exists in low-income and middle-income countries for low-cost, low-tech, yet highly reliable and scalable testing for SARS-CoV-2 virus that is robust against circulating variants. To assess whether a smartphone-based assay is suitable for SARS-CoV-2 and influenza virus testing without requiring specialized equipment, accessory devices, or custom reagents. This cohort study enrolled 2 subgroups of participants (symptomatic and asymptomatic) at Santa Barbara Cottage Hospital. The symptomatic group consisted of 20 recruited patients who tested positive for SARS-CoV-2 with symptoms; 30 asymptomatic patients were recruited from the same community, through negative admission screening tests for SARS-CoV-2. The smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) was first optimized for analysis of human saliva samples spiked with either SARS-CoV-2 or influenza A or B virus; these results then were compared with those obtained by side-by-side analysis of spiked samples using the Centers for Disease Control and Prevention (CDC) criterion-standard reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. Next, both assays were used to test for SARS-CoV-2 and influenza viruses present in blinded clinical saliva samples obtained from 50 hospitalized patients. Statistical analysis was performed from May to June 2021. Testing for SARS-CoV-2 and influenza A and B viruses. SARS-CoV-2 and influenza infection status and quantitative viral load were determined. Among the 50 eligible participants with no prior SARS-CoV-2 infection included in the study, 29 were men. The mean age was 57 years (range, 21 to 93 years). SmaRT-LAMP exhibited 100% concordance (50 of 50 patient samples) with the CDC criterion-standard diagnostic for SARS-CoV-2 sensitivity (20 of 20 positive and 30 of 30 negative) and for quantitative detection of viral load. This platform also met the CDC criterion standard for detection of clinically similar influenza A and B viruses in spiked saliva samples (n = 20), and in saliva samples from hospitalized patients (50 of 50 negative). The smartphone-based LAMP assay was rapid (25 minutes), sensitive (1000 copies/mL), low-cost (<$7/test), and scalable (96 samples/phone). In this cohort study of saliva samples from patients, the smartphone-based LAMP assay detected SARS-CoV-2 infection and exhibited concordance with RT-qPCR tests. These findings suggest that this tool could be adapted in response to novel CoV-2 variants and other pathogens with pandemic potential including influenza and may be useful in settings with limited resources.

Development of a reverse transcription-loop mediated isothermal amplification assay for detection of wisteria vein mosaic virus

Development of a reverse transcription-loop mediated isothermal amplification assay for detection of wisteria vein mosaic virus

Immunocapture-Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Plant RNA Viruses.

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.

Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

BackgroundCurrent assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step.MethodsIn the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets.ResultsHere, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9–96.1%) and 67.4% sensitive (95% CI: 51.5–80.9%) for E gene and RdRp gene, respectively.ConclusionWithout the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

Research Note: Development of rapid isothermal amplification assay for detection of duck circovirus

Duck circovirus disease (DuCVD) caused by duck circovirus (DuCV) continues to spread in recent years, which brings serious harm to the poultry industry, so early diagnosis of DuCVD is of great significance for the prevention and control of this disease. Specific primers and probes for DuCV were designed in this study. Reverse primers and probes were modified at the 5′ ends with biotin and fluorescein, respectively, and they were combined with dipsticks labeled with biotin antibodies and fluorescein antibodies to establish a recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay for detection of duck circovirus. By using this method, the reaction products reached detectable levels in about 20 min as a result of rapid amplification at a constant temperature of 37℃. The detection results could be observed by dripping the reaction products onto the dipstick within 2 to 3 min. The RAA-LFD method has good specificity and high sensitivity (102 copies/μL). Compared with conventional polymerase chain reaction (PCR), RAA-LFD has no power limit on the testing instrument, and is easy to use, saving more time and manpower, so it is more suitable for clinical detection.

Evaluation of loop-mediated isothermal amplification assay for detection of Clostridioides difficile infection: A prospective diagnostic performance study

A novel Loop-mediated isothermal amplification (LAMP) assay, HiberGene's CD was evaluated with 82 unformed stools from patients suspected of C. difficile infection (CDI). Compared to glutamate dehydrogenase (GDH) toxins A/B test (C.diff Quik Chek®), HiberGene's LAMP showed 100% of sensitivity and 95,8% of specificity; and compared to FilmArray™ GI panel ® (BioFire), a sensitivity of 81,2% and a specificity of 100%, with 96.38% of agreement.

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus-baumannii Complex and Associated Carbapenem Resistance.

Background: Acinetobacter calcoaceticus–baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by bla OXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by bla OXA-23. Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S–23S rRNA and bla OXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR). Results: The optimal temperature for the LAMP assay was 65°C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg/μl and 1 ng/μl of DNA concentration and 104 cfu/ml and 108 cfu/ml of colony count, respectively. The LAMP assay was 10- and 104-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species. Significance of the study: The LAMP assay can be applied for early detection of significant species of the ACB complex from clinical samples and their carbapenem-resistant variants. Depending on the emerging pathogen and locally prevalent resistance genes, the LAMP assay can be modified for detection of colonization or infection by various resistant bugs.

Establishment of loop-mediated isothermal amplification assay for detection of parasitic ciliate Ichthyophthirius multifiliis in cyprinid fish

The objective of this research was to establish loop-mediated isothermal amplifications (LAMP) that could be used to detect parasitic ciliate Ichthyophthirius multifiliis (I. multifiliis) in freshwater cyprinid fish. Primers were developed from the distinguishing fragments of 18S ribosomal RNA of I. multifiliis and the LAMP test was then used to evaluate and optimize various concentrations of chemicals, time and temperature. The results indicated that LAMP required 1.6 μM of FIP primers and BIP primers, 0.2 μM of F3 and B3, 2 mM of Mg2+, 1 M of Betaine, and 0.6 mM of dNTP. This assay was able to detect parasite DNA within a 40 min period of incubation and at a constant optimal temperature of 64oC. The positive sample appeared as a clear ladder like pattern on gel electrophoresis, while a yellowish green color appeared with SYBR Green I under ultraviolet light with the use of a heating block. The LAMP test was determined to be more sensitive than conventional PCR in the detection of I. multifiliis. In conclusion, we have presented a sensitive and specific rapid detection system for I. multifiliis based on isothermal DNA amplification. Importantly, this system could then be employed as an alternative and effective diagnostic method in place of other molecular techniques.

Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.

Establishment and Application of a Loop-mediated Isothermal Amplification Assay for Detection of Ganoderma pseudoferreum on Hevea

Establishment and Application of a Loop-mediated Isothermal Amplification Assay for Detection of Ganoderma pseudoferreum on Hevea