RT-LAMP Assay Research Articles

Rapid Detection of Lily mottle virus and Arabis mosaic virus Infecting Lily (Lilium spp.) Using Reverse Transcription Loop-Mediated Isothermal Amplification

The Lily mottle virus (LMoV) impedes the growth and quality of lily crops in Lanzhou, China. Recently Arabis mosaic virus (ArMV) has been detected in LMoV-infected plants in this region, causing plant stunting as well as severe foliar symptoms, and likely posing a threat to lily production. Consequently, there is a need to develop simple, sensitive, and reliable detection methods for these two viruses to prevent them from spreading. Reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assays have been developed to detect LMoV and ArMV using two primer pairs that match six conserved sequences of LMoV and ArMV coat proteins, respectively. RT-LAMP assay results were visually assessed in reaction tubes using green fluorescence and gel electrophoresis. Our assays successfully detected both LMoV and ArMV in lily plants without the occurrence of viral cross-reactivity from other lily viruses. Optimal conditions for LAMP reactions were 65°C and 60°C for 60 min for LMoV and ArMV, respectively. Detection sensitivity for both RT-LAMP assays was a hundredfold greater than that of our comparative RT-polymerase chain reaction assays. We have also found this relatively rapid, target specific and sensitive method can also be used for samples collected in the field and may be especially useful in regions with limited or no laboratory facilities.

Reverse transcription loop-mediated isothermal amplification based rapid detection of Sugarcane mosaic virus and Sugarcane streak mosaic virus associated with mosaic disease of sugarcane

Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) belonging to the family Potyviridae are the causal agents of mosaic disease complex of sugarcane reported from different Asian countries which causes significant economic losses to sugarcane production. In the present study, a sensitive RT-LAMP based assay was developed for rapid diagnosis of SCMV and SCSMV causing mosaic disease of sugarcane. Primers were designed to amplify the six different regions of the coat protein genes of SCMV and SCSMV and both the viruses were successfully detected by this technique. Comparative analysis of RT-LAMP with the conventional reverse transcription polymerase chain reaction (RT-PCR) and quantitative real time PCR (RT-qPCR) showed that RT-LAMP was more sensitive than the other methods due to its rapid visual detection of the viruses. The sensitivity of RT-LAMP assay developed in the present study was tenfold more than that of conventional RT-PCR and RT-qPCR techniques. This is the first report of RT-LAMP based diagnosis of SCMV and SCSMV from India.

A rapid RT-LAMP assay for the detection of all four lineages of Peste des Petits Ruminants Virus

Peste des petits ruminants (PPR) is a viral disease of small ruminants that is caused by the PPR virus (PPRV) and is a significant burden on subsistence farmers across the developing world. Loop-mediated isothermal amplification (LAMP) provides cost-effective, rapid, specific and sensitive detection of nucleic acid and has been demonstrated to have field application for a range of viruses. We describe the development of a novel PPRV RT-LAMP assay utilising carefully-selected primers (targeting the N-gene) allowing for the detection of all known PPRV lineages in < 20 min. The assay was evaluated in comparison with a “gold standard” real-time RT-PCR assay using more than 200 samples, comprising samples from recent PPRV outbreaks, experimentally-infected goats, well-characterised cell culture isolates and samples collected from uninfected animals. The RT-LAMP assay demonstrated 100% diagnostic specificity and greater than 97% diagnostic sensitivity in comparison with the real-time RT-PCR assay. The limit of detection was between 0.3 and 0.8 log10 TCID50 ml−1 equating to a CT value of 31.52 to 33.48. In experimentally-infected animals, the RT-LAMP could detect PPRV as early as 4 days post infection (dpi) - before clinical signs were observed at 7 dpi. The RT-LAMP assay can support the global PPR eradication campaign.

A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.

Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting resulting in significant economic losses in citrus production. A immunocapture reverse-transcriptase loop-mediated amplification (IC-RT-LAMP) assay was developed in this study to detect the severe VT strains that are typically associated with severe CTV symptoms. The sensitivity of RT-LAMP assay was determined by ten-fold serial dilutions of CA-VT-AT39 RNA, in comparison to one-step RT-droplet digital (dd) PCR. RT-LAMP detected up to 0.002 ng RNA with an amplification time of 10:35 (min:sec.), equivalent to 11.3 copies as determined by one step RT-ddPCR. The RT-LAMP assay specifically detected CA-VT-AT39 RNA and did not cross react with other CTV genotypes tested (T36, T30, RB, S1 and T68). To facilitate rapid on-site detection, the RT-LAMP assay was improved by first capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP), thereby eliminating nucleic acid extraction steps. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all dilutions tested. The minimum amplification time was 6:45 (min:sec) with 1:500 and 1:1000 of CTV-IgG dilutions. The limit of detection of IC-RT-LAMP assay with crude leaf sap of CA-VT-AT39 was 1:320 with a maximum amplification time of 9:08 (min:sec). The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV from 40 field tree samples. A 100% agreement was observed between tests, regardless of single or mixed infections of CTV VT with other genotypes. Therefore, the IC-RT-LAMP assay can serve as a useful tool in the management of potentially severe strains of CTV.

Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme.

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

Squash Print Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detection of Potato leafroll virus in Single Aphid and in Potato

A squash print reverse transcription loop-mediated isothermal amplification amplification (SP-RT-LAMP) assay was developed for detection of Potato leafroll virus (PLRV) in a single aphid. Healthy aphids were given an acquisition feeding on potato plants infected with PLRV, and the acquired aphids were used for RNA isolation. The RT-LAMP assay was carried out using LAMP primers targeting the coat protein gene of PLRV. The amplified product was run on agarose gel; a typical ladder-like pattern in the acquired aphids indicated that the assay was able to detect PLRV from an individual aphid. The results of the RT-LAMP assay for the detection of PLRV in a single aphid were also visualized directly by adding fluorescent dye in LAMP amplified product. These results were confirmed by RT-PCR detection where an amplicon of expected size of 492 bp was observed in virus-acquired single aphids. The assay was optimized with respect to its isothermal amplification temperature and its incubation period. The technique was examined for its relative selectivity with other potato viruses transmitted by aphids where it did not show any cross-reactivity with the tested viruses indicating its specificity. With respect to its relative sensitivity, it was found to be equally sensitive as the existing RT-PCR assay. It was also found to be robust and highly reproducible as it was able to detect PLRV from known and unknown single aphid samples. The assay was successful in detection of PLRV in potato plants and tubers as well. Hence, we conclude that the assay is simple, sensitive and economical for detection of PLRV in single aphid and in potato plants and tubers.

A Mismatch-Tolerant Reverse Transcription Loop-Mediated Isothermal Amplification Method and Its Application on Simultaneous Detection of All Four Serotype of Dengue Viruses.

Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens causing infectious diseases. However, mismatches between primers (especially in the 3′-end) and templates significantly reduced the amplification efficiency of LAMP, and limited its application to genetically diverse viruses. Here, we reported a novel mismatch-tolerant LAMP assay and its application in the detection of dengue viruses (DENV). The novel method features the addition of as little as 0.15 U of high-fidelity DNA polymerase to the standard 25 μl LAMP reaction mixture. This amount was sufficient to remove the mismatched bases at the 3′-end of primers, thereby resulting in excellent tolerance for various mismatches occurring at the 3′-end of the LAMP primers during amplification. This novel LAMP assay has a markedly improved amplification efficiency especially for the mutants forming mismatches with internal primers (FIP/BIP) and loop primers (FLP/BLP). The reaction time of the novel method was about 5.6–22.6 min faster than the conventional LAMP method regardless of the presence or absence of mismatches between primers and templates. Using the novel method, we improved a previously established pan-serotype assay for DENV, and demonstrated greater sensitivity for detection of four DENV serotypes than the previous one. The limit of detection (LOD) of the novel assay was 74, 252, 78, and 35 virus RNA copies per reaction for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. Among 153 clinical samples from patients with suspected DENV infection, the novel assay detected 94.8% samples being DENV positive, higher than that detected by the commercial NS1 antigen assay (92.2%), laboratory-based RT-PCR method (78.4%), and the conventional RT-LAMP assay (86.9%). Furthermore, the novel RT-LAMP assay has been developed into a visual determination method by adding colorimetric dyes. Because of its simplicity, all LAMP-based diagnostic assays may be easily updated to the newly improved version. The novel mismatch-tolerant LAMP method represents a simple, sensitive and promising approach for molecular diagnosis of highly variable viruses, and it is especially suited for application in resource-limited settings.

Development of probe-based real-time loop-mediated isothermal amplification for detection of Brucella.

To develop a rapid, sensitive and specific diagnostic assay for the detection of Brucella. The probe-based RT-LAMP was carried out by using a set of four or six primers and different LAMP chemicals to compare its results with real-time PCR. Detection of gene amplification is done within 40min and can be seen by amplification curve, turbidity and addition of DNA-binding dye at the end of the reaction results in colour difference under normal day light and in UV. The sensitivity of probe-based real-time LAMP assay was found 10-fold higher than Taqman-based qPCR. The specificity of the developed assay was validated by the absence of any cross-reaction with other pathogenic bacteria. The developed probe-based RT-LAMP assay is extremely rapid, cost effective, highly specific and sensitive, and has potential usefulness for rapid Brucella surveillance. The developed probe-based RT-LAMP is a powerful gene amplification technique which is a specific, fast diagnostic tool for early detection and identification of Brucella.

Abstract P3-03-29: Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction (direct RT-LAMP) for the detection of lymph node metastasis in early breast cancer

Abstract Backgroud: The detection of lymph node metastasis by reverse transcription loop-mediated isothermal amplification method (RT-LAMP) had been studied previously. Even though, RT-LAMP method provides improved performance compared to intraoperative histology sentinel lymph node (SLN) evaluation, direct RT-LAMP method without RNA extraction can be more efficient and easily accessible process. Therefore, we evaluated the performance and efficacy of a direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay for visual detection of CK19, CK20, and CEA mRNAs to identify lymph node metastasis in patients with early breast cancer. Methods: A total of 92 lymph nodes dissected from 40 patients with breast cancer were collected at the breast cancer center of Kyungpook National University Chilgok Hospital between November 2015 and February 2016. All of the samples were analyzed by direct RT-LAMP assay and routine histopathology examination. Cutoff values to distinguish metastasis and nonmetasis were determined by measuring cytokerain 19 (CK19) mRNA in histopathologically positive and negative lymph node using direct RT-LAMP. Results: We set the cutoff value of direct RT-LAMP assay for CK 19 mRNA at 1ng to distinguish status of LN metastasis. The sensitivity and specificity of the RT-LAMP assay were 85.7% and 100%, respectively. The positive predictive value and negative predictive value were 100% and 94.4%. Conclusion: Direct RT-LAMP assay can allow detection of SLN metastasis in breast cancer patients intraoperatively with a good sensitivity through cost-effective and time–saving manner. Citation Format: Lee IH, Jung J, Lee S, Lee J, Lee RK, Park H, Jung J, Kang J, Chae Y. Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction (direct RT-LAMP) for the detection of lymph node metastasis in early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-29.

2021. An Ultra-Rapid Host Response Assay to Discriminate Between Bacterial and Viral Infections Using Quantitative Isothermal Gene Expression Analysis

BackgroundAccurate diagnosis and treatment of bacterial infection is critical for improving patient outcomes. However, over-prescription of antibiotics has contributed to C. difficile-infections and to the emergence of antimicrobial resistance. As assessing bacterial infection by culture is slow and molecular pathogen detection is limited in scope, an unmet need remains for a rapid test to differentiate between viral and bacterial infections. We have previously identified a set of 7 host response biomarkers demonstrating an AUROC of 0.91–0.93 for separating bacterial from viral infections across multiple independent cohorts. A clinical in-vitro diagnostic test (IVD) using these markers must be very fast to integrate with physician workflows. Loop-mediated isothermal amplification (LAMP) represents a rapid amplification solution with the potential to meet these needs. We describe an ultra-rapid LAMP assay designed to quantitate these markers for applications in point-of-care decision making.MethodsLAMP primers for gene expression analysis of selected markers with a housekeeping control were designed for mRNA specificity by targeting FIP primers to splice junctions. Assay specificity, sensitivity and linear dynamic range were evaluated using serial dilution of control material. RNA extracted from preserved patient samples was evaluated by LAMP for concordance with NanoString® nCounter® data (nCounter).ResultsIterative optimization of primer design resulted in RT-LAMP assays that selectively amplify target mRNA. Assays demonstrate a linear dynamic range spanning 6 orders of magnitude and a quantitative LOD of about 103 copies. Quantitation of relative expression levels showed good concordance with nCounter data in 10 healthy, 6 viral and 6 bacterial patient samples, with average threshold times of <15 minutes.ConclusionAccurate discrimination of bacterial and viral infection can be achieved on a true point-of-care timescale using our LAMP strategy. This assay could be run on a standard thermal cycler, or any quantitative molecular instrument that allows to measure at least eight targets. An IVD test distinguishing between bacterial and viral infections could aid in antimicrobial treatment decisions and thereby minimize over-prescription of antibiotics.Disclosures D. Rawling, Inflammatix Inc.: Employee, Salary. W. Nie, Inflammatix Inc.: Employee, Salary. M. Remmel, Inflammatix Inc.: Employee, Salary. M. Eshoo, Inflammatix Inc.: Employee, Salary. J. Romanowsky, Inflammatix Inc.: Employee, Salary. O. Liesenfeld, Inflammatix Inc.: Employee, Salary. T. Sweeney, Inflammatix Inc.: Employee, Salary.

Genetic diversity of the Cucumber green mottle mosaic virus and the development of RT-LAMP assay for its detection

To analyse the genetic diversity of the Cucumber green mottle mosaic virus (CGMMV) population in Poland and to establish the phylogenetic relationships between the Polish and other isolates described to date, 91 isolates were collected from cucumber plants. The analysis, based on coat protein (CP) gene, revealed the presence of two phylogenetic groups: one consisting of the Polish isolates collected in 2017 and those originated mainly from Asia region and the second including the Polish isolates collected in 2016 and the others from European countries. The sensitive, specific, and rapid one-step loop-mediated isothermal amplification assay was developed for the early detection of genetically diverse CGMMV isolates in seeds and plant material.

Development of a real time loop-mediated isothermal amplification method for detection of Senecavirus A

Senecavirus A (SVA), formerly known as Seneca Valley Virus (SVV), is one of causative agents of vesicular diseases in swine. Recently, the outbreaks associated with vesicular disease caused by SVA infection in pig herds have been reported in Brazil, USA, China, Thailand and Canada. Several molecular detection methods have been established to determine the infection of SVA, including real time reverse transcription PCR assay, nested PCR, a TaqMan-based qRT-PCR assay and RNA-based in situ hybridization method. In our study, an assay for the identification of SVA in pig herds using real time reverse transcription loop-mediated isothermal amplification (real time RT-LAMP) was developed. The limit of detection for the assay was 1 TCID50/ml. One hundred and eighteen field samples from pigs were used to validate the assay for clinical application. Our result demonstrated that real time RT-LAMP assay is a cost-effective and highly specific and sensitive alternative for the rapid detection of SVA in clinical samples.

Detection of Coxsackievirus B3 Using RT-LAMP Assay: Optimization by Taguchi Method and Comparison of One and Two Step RT-LAMP

Detection of<i> Coxsackievirus</i> B3 Using RT-LAMP Assay: Optimization by Taguchi Method and Comparison of One and Two Step RT-LAMP

An improved reverse transcription loop-mediated isothermal amplification assay for sensitive and specific detection of serotype O foot-and-mouth disease virus

A sensitive and specific swarm primer-based reverse transcription loop-mediated isothermal amplification (sRT-LAMP) assay for the detection of serotype O foot-and-mouth disease virus (FMDV) was developed and evaluated. The assay specifically amplified the VP3 gene of serotype O FMDV, but did not amplify the VP3 gene of other serotype FMDVs or any other viruses. The limit of detection of the assay was 102 TCID50/mL or 103 RNA copies/μL, which is 100 times lower than that of the RT-LAMP assay without swarm primers. The new assay is 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR) and is comparable to the sensitivity of real time RT-PCR (qRT-PCR). Evaluation of the assay using different serotypes of FMDV strains showed 100% agreement with the RT-PCR results. The previously reported serotype O FMDV-specific RT-LAMP assay did not detect 20 out of 22 strains of serotype O FMDVs, probably due to multiple mismatches between the primer and template sequences, showing that it is not suitable for detecting the serotype O FMDVs circulating in Pool 1 region countries, including Korea. In contrast, the developed sRT-LAMP assay with improved primers can rapidly and accurately diagnose serotype O FMDVs circulating in Pool 1 region countries and will be a useful alternative to RT-PCR and qRT-PCR.

Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay

BackgroundPorcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd.ResultsIn this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed.ConclusionsThe developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

Molecular Detection of Foot and Mouth Disease Virus (FMDV) from 2017 Outbreaks in Punjab by RT-PCR and RT-LAMP Assays

Foot and mouth disease is infectious, tranboundary, disease of cloven hoofed domesticated ruminants. In this study, ELISA confirmed positive infected and field samples were analyzed through RT-PCR using universal primer set (IF/IR and P1/P2) and RT-LAMP assays. A total of N=100 samples were collected from different livestock population areas in Punjab. Types of samples were serum, nasal secretions, tissue, saliva and swab samples. Out of N=100 infected samples, n=19 samples were serotyped into A, O and Asia 1 strains through antigen ELISA (Ag- ELISA) and these samples were further confirmed through RT-PCR and RT-LAMP assays. ELISA confirmed samples revealed 100% results with RT-PCR and RT-LAMP assays. Field samples revealed 33.33%, 27.16% and 37.03% with RT-PCR (IF/IR and P1/P2) and RT-LAMP assays respectively. It was concluded that ELISA confirmed samples were successfully analyzed with RT-PCR and RT-LAMP assays. Both the assays successfully analyzed 2017 outbreaks of FMD in Punjab.

Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of Influenza A viruses

BackgroundInfluenza A viruses (IAVs) have always remain a serious concern for the global economy and public health. A rapid, specific and sensitive detection method is always needed to control the influenza in its early stages by timely intervention of therapy and early clinical management. ObjectivesTo develop RT-LAMP assays for detection of influenza A viruses, their further subtyping into seasonal (H1N1, H3N2) and novel pandemic H1N1 viruses and to evaluate clinical applicability of optimized RT-LAMP assays on patients’ samples. Study designIn this study, we optimized RT-LAMP assay to detect IAVs by using primers against matrix gene and subtyping of IAVs was done by using primers against hemagglutinin gene. Optimized RT-LAMP assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step RT-PCR and real-time RT-PCR. ResultsRT-LAMP assays successfully detected and differentiated IAVs into H1N1, H3N2 and pdm09/H1N1 subtypes. One hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with RT-LAMP assay, detecting 30 (17.9%) samples positive for Influenza A virus. Out of 30 samples, 21, 7 and 2 were found positive for pdm09/H1N1, H3N2 and seasonal H1 respectively. Conventional one-step RT-PCR detected a total of 27 (16.2%) samples for influenza A and further subtyping showed 20 and 7 samples positive for pdm09/H1N1 and H3N2 virus respectively whereas none was found positive for seasonal H1N1. RT-LAMP assay demonstrated higher sensitivity (93.8%) than conventional RT-PCR (84.4%) for influenza A viruses detection in clinical samples. ConclusionsRT-LAMP assay is rapid, sensitive, specific and cost effective method for detection of influenza A viruses than conventional one-step RT-PCR and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries.

Development of a colorimetric reverse transcription loop-mediated isothermal amplification assay for the detection of Mirafiori lettuce big-vein virus.

An RT-LAMP assay was developed to detect Mirafiori lettuce big vein virus (MiLBVV) and was compared with DAS-ELISA and RT-PCR. All primers were designed on the basis of the coat protein gene of the virus. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection of MiLBVV was developed, and factors such as safety, simplicity, cost, user-friendliness and safety were compared with those of DAS-ELISA, RT-PCR and RT-LAMP assays. Compared with DAS-ELISA and RT-PCR, RT-LAMP and IC-RT-LAMP had higher sensitivity (100-fold) but similar specificity, with the advantage of a shorter assay time and no need for RNA extraction (in IC-RT-LAMP). As RT-LAMP requires only very basic instruments and the results can be obtained by visual inspection (using GeneFinder™ dye), this technique provides a simple and reliable tool for laboratory research.

Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus

Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings.