Reverse Transcriptase Loop-mediated Isothermal Amplification Research Articles

Loop-mediated isothermal amplification (RT-LAMP): a new approach for the detection of foot-and-mouth disease virus and its sero-types in Pakistan.

Successful disease management requires a rapid and sensitive diagnosis method that can recognize early infection even before the manifestation of its clinical signs. The only available field diagnostic tests for foot-and-mouth disease (FMD) are lateral flow devices, commonly known as chromatographic strips. Low sensitivity and inability to detect FMD virus (FMDV) at the serotype level are limitations of lateral flow devices. Therefore, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was standardized using universal and sero-type specific genes in a single tube. This test does not require sophisticated equipment and can detect FMDV at serotype level in about 60 min. In addition, the sensitivity and specificity of this test is comparable to conventional reverse transcriptase PCR and real time PCR (rRT-PCR).

등온증폭법을 이용한 돼지인플루엔자바이러스 신속 진단법 개발

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at 58C and the sensitivity of the RT-LAMP (1 copy/L) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) (10 copy/L) and the same to real time RT-PCR (1 copy/L). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

Rapid Detection of Avian Infectious Bronchitis Virus by Reverse Transcriptase-Loop Mediated Isothermal Amplification.

A reverse-transcription loop mediated isothermal amplification (RT-LAMP) was developed for rapid diagnosis of infectious bronchitis (IB) in poultry by targeting the spike protein 2 gene (S2). RT-LAMP primers were designed for IBV-S2 targets and optimized to run at 60 °C for 45 min. As compared with RT-PCR, RT-LAMP was 100 times more sensitive for IBV-S2 gene. RT-LAMP showed specific amplification with IB viral genome but not with other avian respiratory pathogens due to their mismatching with IBV-S2-RT-LAMP primers. RT-LAMP reaction products were visually detected by the addition of propidium iodide stain. Out of 102 field samples tested for detection of IBV, RT-LAMP detected IBV in 12 samples for S2 gene whereas RT-PCR detected IBV in six samples for S2 gene. The sensitivity of the RT-LAMP was 100 % and the specificity was 94 % for S2 gene. Since the developed RT-LAMP to detect IBV is simple, rapid, sensitive and specific, it can be a useful diagnostic tool for detection of IB in poultry in less equipped laboratories and in field conditions.

The Comparison of RT-LAMP, RT-PCR and Dot-Blot Hybridization for Detection of Jembrana Disease Virus

Jembrana disease virus is a viral pathogen that causes Jembrana disease in Bali cattle (Bos javanicus) with high mortality rate. Infection of Jembrana Disease Virus (JDV) on Bali cattle have caused substantial economic losses for farmers in Indonesia and Australia. In order to control the spread, development of a sensitive detection method is important. In this study, we used three different detection methods based on genomic approach, i.e., reverse transcriptase-polymerase chain reaction (RT-PCR), reverse transcriptase-loop mediated isothermal amplification (RT-LAMP) and dot-blot hybridization to detect JDV. Utilization of pGEX-TM, a recombinant plasmid containing env-tm gene as a positive control showed that RT-LAMP is the most sensitive method compares the two others. It could detect template concentration as low as 10-6 ng µL-1 or equivalent to 1.52A—102 plasmid copy number, 100 and 10000 more sensitive than RT-PCR and dot-blot hybridization, respectively.

Integration of reverse transcriptase loop-mediated isothermal amplification with an immunochromatographic strip on a centrifugal microdevice for influenza A virus identification.

A novel centrifugal microdevice which could perform reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and immunochromatographic strip (ICS) based amplicon detection was demonstrated for simple and cost-effective influenza A virus identification. The proposed centrifugal microdevice consists of the sample and running buffer loading reservoirs, the RT-LAMP chamber, and the ICS for detecting gene expression. The entire process could be completed sequentially and automatically by simply controlling the rotation speed and by optimizing the microfluidic design. Monoplex and multiplex RT-LAMP reactions targeting H1 and/or M gene were executed at 66 °C for 40 min, and the resultant amplicons were successfully analysed on the ICS within 15 min. Influenza A H1N1 virus was subtyped by detecting H1 and M gene on the ICS even with 10 copies of viral RNAs. Highly specific and multiplex viral typing of the integrated RT-LAMP-ICS microdevice was also demonstrated. The combination of the rapid isothermal amplification with the simple colorimetric detection on a strip in a single centrifugal microdevice will provide an advanced genetic analysis platform in the field of on-site pathogen diagnostics.

Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection

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 47min. 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.

New method for the visual detection of human respiratory syncytial virus using reverse transcription loop-mediated amplification

Human respiratory syncytial virus (HRSV) is a seasonal respiratory pathogen that causes respiratory infection in children and the elderly. A new, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid (within 1h), simultaneous detection of A and B group HRSV. Primers specific for groups A and B were designed to amplify the N and L genes of HRSV, respectively. A fluorescent dye, calcein, was used as an indicator for the endpoint visual detection and/or real-time amplification of HRSV RNA. The detection limit of the new method was 281.17 50% tissue culture infective doses (TCID50)/ml for HRSV A and 1.58 TCID50/ml for HRSV B. To evaluate the validity of this method, a comparison with RT-PCR was performed using 77 nasopharyngeal swabs as samples. Both RT-LAMP and RT-PCR detected HRSV in 38 HRSV samples, yielding a positive rate of 49%. Of the RT-LAMP positive samples, 36 (95%) were also positive by RT-PCR, while two were negative by RT-PCR. Among the 36 RT-LAMP and RT-PCR positive samples, 11 belonged to HRSV group A, while 25 belonged to group B. The results show that the new RT-LAMP is simple, rapid and well suited for HRSV diagnosis, especially in a limited-resource setting.

Membrane-based, sedimentation-assisted plasma separator for point-of-care applications.

Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. We report on a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a "blood in-plasma out" capability, consistently extracting 275 ± 33.5 μL of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification.

Rapid Detection and Differentiation of Foot and Mouth Disease Virus Serotypes by Antigen-capture Reverse Transcriptase Loop-mediated Isothermal Amplification

Rapid Detection and Differentiation of Foot and Mouth Disease Virus Serotypes by Antigen-capture Reverse Transcriptase Loop-mediated Isothermal Amplification

Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

BackgroundTick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study.MethodsPrimers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change.ResultsThe sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV.ConclusionTBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients.

Infectious hematopoietic necrosis virus (IHNV)-검출 Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) 법의 평가

본 연구에서는 어체내 IHNV 모니터링에 LAMP법의 사용이 가능 하는지를 검토하기 위해 IHNV를 무지개송어에 인위적으로 감염시킨 후 시간 경과에 따라 LAMP법과 어류세포를 사용한 분리배양법을 이용하여 IHNV 검사를 실시하였다. IHNV를 <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish로 복강 주사한 결과, 40%, 0%, 0%의 누적폐사율이 관찰되었다. 폐사어 및 IHNV 접종 후 16일과 28일째에 각 실험구에서 채집한 생존어 5마리를 대상으로 한 IHNV 검사 결과, 폐사어에서 IHNV가 100% (8/8 마리) 분리되었고 (감염가: <TEX>$10^{4.3}-10^{6.8}\;TCID_{50}/ml$</TEX>), RT-LAMP법에서도 100% 검출되었다. 16일째 생존한 개체를 대상으로 한 IHNV 검사 결과에서는 <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish의 IHNV로 접종한 실험구에서 각각 60% (3/5 마리, 감염가: <TEX>$10^{2.8}-10^{5.05}\;TCID_{50}/ml$</TEX>), 20% (1/5 마리, <TEX>$10^{1.05}\;TCID_{50}/ml$</TEX>), 60% (3/5 마리, <TEX>$10^{1.05}-10^{4.8}\;TCID_{50}/ml$</TEX>) 의 검출율을 보였으나 LAMP법에서는 20% (1/5 마리), 0% (0/5 마리), 20% (1/5 마리) 의 검출율을 나타내었다. 28일째 생존한 개체 및 대조구의 어류에서는 IHNV가 분리 검출되지 않았다. 이상의 연구결과로 LAMP법은 IHNV-생존어에서 바이러스를 모니터링 하는데 한계가 있으나 병어로부터 IHNV를 검출하는데 유용하게 사용될 수 있을 것으로 사료되었다. A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was evaluated to monitor infectious hematopoietic necrosis virus (IHNV) from artificially infected rainbow trout Oncorhynchus mykiss. The cumulative mortalities of fish challenged with IHNV at <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish and <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish were 40%, 0% and 0%, respectively. Dead fish and survivors at 16 and 28 d post-challenge in each group were employed for IHNV detection by RT-LAMP assay and virus isolation using BF-2 cells. IHNV from <TEX>$10^{4.3}$</TEX> to <TEX>$10^{6.8}\;TCID_{50}/ml$</TEX> was isolated from all the dead fish and also detected in all of the examined dead fish by RT-LAMP assay. In survivors at 16 d, 60% (3/5 fish, <TEX>$10^{2.8}-10^{5.05}\;TCID_{50}/ml$</TEX>), 20% (1/5 fish, <TEX>$10^{1.05}\;TCID_{50}/ml$</TEX>) and 60% (3/5 fish, <TEX>$10^{1.05}-10^{4.8}\;TCID_{50}/ml$</TEX>) were found to be IHNV-positive by virus isolation in fish challenged with IHNV at <TEX>$10^{6.5}\;TCID_{50}$</TEX>/fish, <TEX>$10^{5.5}\;TCID_{50}$</TEX>/fish and <TEX>$10^{4.5}\;TCID_{50}$</TEX>/fish, respectively, while 20% (1/5 fish), 0% (0/5 fish) and 20% (1/5 fish) were IHNV-positive by RT-LAMP assay. No IHNV was detected in the survivors at 28 d and control fish. These results indicate that the RT-LAMP assay is useful for detection of IHNV in diseased fish although it is not enough to monitor virus in IHNV-survivors.

Reverse‐Transcriptase Loop‐Mediated Isothermal Amplification as a Rapid Screening/Monitoring Tool for Salmonella Enterica Detection in Liquid Whole Eggs

Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that is specific, fast, and simple. It is based on reverse transcription followed by DNA amplification using the Bst DNA polymerase large fragment requiring one temperature and a simple waterbath, without the need for any expensive equipment. Detection is by turbidity or agarose gel electrophoresis. Our objective was to apply this LAMP-based technology to rapidly and sensitively detect Salmonella enterica serovar Enteritidis in liquid whole eggs (LWEs) within 1 d. LWE were inoculated with S. Enteritidis and stomached in tetrathionate broth (TTB), and spread-plated on Xylose lysine tergitol 4 agar either immediately or after 6, 12, or 16-h enrichment. RNA was extracted from 5-mL TTB and the RT-LAMP assay was carried out using invA primers. After 16 and 12-h enrichment, improved Salmonella detection up to 10⁰ to 10¹ and 10⁴ CFU/25 mL LWE, respectively was obtained. Without enrichment, Salmonella could be detected at 10⁷ CFU/25 mL; however, after 6-h enrichment a 1-log improvement to 10⁶ CFU/25 mL was obtained. This RT-LAMP assay appears to be suitable as a potential screening/monitoring tool for Salmonella enterica from LWE products in routine settings with results obtainable within 24-h, which is significantly faster than traditional cultural assays.

Use of reverse transcriptase loop-mediated isothermal amplification assay for field detection of Newcastle disease virus using less invasive samples

A novel nucleic acid amplification method, loop-mediated isothermal amplification, was developed and recently demonstrated detection of Newcastle disease virus (NDV) in tissue samples. But slaughter of poultry for test samples is often faced with resentment by low-income farmers. This study was undertaken to determine the test properties of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) in detection of NDV in clinical cases using cloacal and oropharyngeal swabs. Samples included 46 tracheal tissues, 94 cloacal and 107 oro-pharyngeal swabs from on-station and 30 spleens, 74 cloacal and 74 oro-pharyngeal swabs from the field. Analysis was done using specific RT-LAMP targeting the fusion (F) protein. While the method detected NDV from swab samples, no RNA of other poultry disease viruses was amplified, indicating analytical specificity of 100%. RT-LAMP took ≤36 minutes in 83% (n=329) of positive reactions with all samples amplified in

Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV) within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations.

Development of loop-mediated isothermal amplification method for visualization detection of the highly virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV) in China

A novel assay method to detect the highly virulent Porcine reproductive and respiratory syndrome virus (PRRSV) termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), was reported by using hydroxynaphthol blue (HNB) as the LAMP product colorimetric judgment. By the set of special primers, targeting the sequence that belongs to Nsp2 gene containing an 87 bp deletion mutation, the LAMP-based assay could be completed within 1 h at 63°C. Final concentration of 150 mM HNB was confirmed to be appropriate in LAMP product judgment without impact on the reaction system. The detection limit of the assay was 103 copies per reaction, as determined by using a recombined plasmid. Genomes of various viruses were used to confirm the specificity of the LAMP-based assay, but only the highly virulent strains of PRRSV could be detected. All the internal organs from three stillborn piglets from a herd of pigs with PRRS-like symptoms were confirmed by LAMP-based assay to be infected not only with classical PRRSVs, but also the variant strains. These results suggest that the RT-LAMP assay with HNB provides a useful tool for the diagnosis of the highly virulent PRRSV infections in porcine rapidly. Key words: Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), highly virulent porcine reproductive and respiratory syndrome virus (PRRSV), classical porcine reproductive and respiratory syndrome virus (PRRSV), hydroxynaphthol blue (HNB).

Rapid and sensitive detection of mud crab Scylla serrata reovirus by a reverse transcription loop-mediated isothermal amplification assay

Scylla serrata reovirus (SsRV) is one of the most prevalent viral pathogens of the mud crab (S. serrata). This pathogen is widespread in east China and causes severe economic losses to the nation's mud crab industry. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In the present study, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and sensitive detection of SsRV was developed and evaluated. The LAMP reaction mix was optimized, as were the reaction temperature (62°C) and the duration of the assay (60min). The sensitivity of the RT-LAMP assay was determined to be 0.8fg SsRV dsRNA, which was 1000-fold higher than that of a one-step reverse transcription polymerase chain reaction (RT-PCR). The RT-LAMP assay also had higher sensitivity than a one-step RT-PCR, as it identified nine more positive cases from 55 mud crabs suspected of having SsRV. No cross-reactivity was found with the DNA/RNA of other tested viruses and SsRV-negative animals. Importantly, the assay can be completed within 60min and is faster than conventional RT-PCR. In summary, the RT-LAMP assay is a simple, cost-effective, sensitive, and specific tool for the rapid detection of SsRV infection.

Development of rapid, simple and sensitive real-time reverse transcriptase loop-mediated isothermal amplification method (RT-LAMP) to detect viral diseases (PRDV, YHV, IHHNV and TSV) of penaeid shrimp

A one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as penaeid rod shaped DNA virus (PRDV), hypodermal and haematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV) and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR, RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid, where specific amplification is obtained for PRDV, IHHNV, TSV and YHV in 60 min under isothermal conditions at 63 o C employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed that all of these viruses can be detected up to 100 copies of template DNA, rendering it ten-fold more sensitive than conventional LAMP assay.

Reverse transcriptase loop-mediated isothermal amplification assay for infectious hematopoietic necrosis virus in Oncorhynchus keta

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting infectious hematopoietic necrosis virus (IHNV) from chum salmon Oncorhynchus keta in South Korea with high specificity, sensitivity and rapidity. A set of 6 IHNV-specific primers was designed, based on the G-protein sequence of IHNV (PRT strain), recognizing 8 distinct sequences of the target RNA. The assay was optimized to detect IHNV at 63 degrees C for 30 min. The limit of detection was 0.01 fg of RNA extracted from IHNV-infected CHSE-214 cells, compared with 1.0 fg for nested RT-PCR. The applicability of this RT-LAMP assay was further tested by comparison with nested RT-PCR using field samples. Of 473 samples tested, 191 samples (40.38%) were IHNV-positive by RT-LAMP, whereas 162 samples (34.25%) were IHNV-positive by nested RT-PCR. These results indicate that, because of its high sensitivity and rapidity, the RT-LAMP assay is useful for early diagnosis of IHN.

Mobile and accurate detection system for infection by the 2009 pandemic influenza A (H1N1) virus with a pocket-warmer reverse-transcriptase loop-mediated isothermal amplification

The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse‐transcriptase loop‐mediated isothermal amplification (RT‐LAMP) kit with a disposable pocket‐warmer as a heating device (designated as pwRT‐LAMP). The pwRT‐LAMP can detect as few as 100 copies of the virus—which is nearly as sensitive as real‐time reverse‐transcription polymerase chain reaction (RT‐PCR)—and does not cross‐react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT‐LAMP system, nasal swab samples were collected from 56 patients with flu‐like symptoms and were tested. Real‐time RT‐PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A + B immunochromatography detected the virus in only 11 samples (11/27; 40.7%), whereas the pwRT‐LAMP system detected the virus in 26 of the 56 samples (26/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT‐LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics. J. Med. Virol. 83:568–573, 2011. © 2011 Wiley‐Liss, Inc.

An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses.

We designed a new set of primers for reverse transcriptase loop-mediated isothermal amplification (RTLAMP) to specifically amplify the HA gene of avian influenza viruses subtype H5N1. By testing nine H5N1 virus strains and 41 clinical samples collected in Northern Vietnam, we found that the new primers showed higher sensitivity and specificity than the previously published RT-LAMP primers and were comparable to the RT-PCR method currently recommended by WHO. These results suggest that our RT-LAMP assay may be a better choice as a diagnostic tool for current H5N1 influenza virus infection.