Real-time Reverse Transcription Loop-mediated Isothermal Amplification Research Articles

Rapid detection of tomato spotted wilt virus by real-time RT-LAMP and in-field application

Tomato spotted wilt virus (TSWV) is considered one of the most threatening viruses worldwide for different economically important agricultural crops. In this scenario, it is important to perform an early detection by laboratory tests to prevent TSWV spread. A rapid and sensitive TSWV detection protocol based on real time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed in this work, also using cost-effective and simplified sample preparation procedure, to assess the suitability of the RT-LAMP assay in field conditions on tomato and pepper samples. A set of six primers was designed within the nucleotide sequence region coding for the nucleocapsid protein (N) of segment S, targeting a 220-nucleotide sequence. Sensitivity, specificity, accuracy, and in-field application of the real-time RT-LAMP assay were evaluated. The developed real-time RT-LAMP assay proved to be one thousand and one hundred times more sensitive than end-point RT-PCR and real-time RT-PCR methods, respectively, detecting a total of 9.191 × 101 genome copies as minimum target, and no cross-reactivity were detected with other viruses belonging to Tospoviridae and Bromoviridae families used as outgroup. In addition, the in-field application of the assay using the rapid sample preparation gave adequate and reliable results within 60 minutes, with an acceptable reaction delay when compared to canonical RNA extraction. The in-field analyses showed an increase of TSWV-positive samples (37%) detection compared with end-point RT-PCR and real-time RT-PCR (32% and 29%, respectively), particularly on asymptomatic samples, confirming that the real-time RT-LAMP assay can be implemented as a routine test both in-field and laboratory conditions as a rapid and sensitive technique for TSWV detection.

Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/μL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

Development and evaluation of an easy to use real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus

West Nile Virus (WNV) causes a serious public health concern in many countries around the world. Virus detection in pathological samples is a key component of WNV infection diagnostic, classically performed by real-time PCR. In outbreak situation, rapid detection of the virus, in peripheral laboratories or at point of care, is crucial to guide decision makers and for the establishment of adequate action plans to prevent virus dissemination. Here, we evaluate a Loop-mediated isothermal amplification (LAMP) tool for WNV detection. Amplifications were performed comparatively on extracted viral RNA and on crude samples using a classical thermal cycler and a portable device (pebble device). qRT-PCR was used as gold standard and two sets of urine samples (n = 62 and n = 74) were used to evaluate the retained amplification protocols and assess their sensitivity and specificity. RT-LAMP on RNA extracts and crude samples showed a sensitivity of 90 % and 87 %, respectively. The specificity was 100 % for extracts and 97 % for crude samples. Using the device, the RT-LAMP on extracted RNA was comparable to the gold standard results (100 % sensitivity and specificity) and it was a bit lower on crude samples (65 % sensitivity and 94 % specificity). These results show that RT-LAMP is an efficient technique to detect WNV. RT-LAMP provides a rapid, sensitive, high-throughput and portable tool for accurate WNV detection and has potentials to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, especially in developing countries.

Compact Point-of-Care Device for Self-Administered HIV Viral Load Tests from Whole Blood.

Human immunodeficiency virus (HIV) is a significant problem to consider as it can lead to acquired immune deficiency syndrome (AIDS). Fortunately, AIDS is manageable through antiretroviral therapy (ART). However, frequent viral load monitoring is needed to monitor the effectiveness of the therapy. The current reverse transcription-polymerase chain reaction (RT-PCR) viral load monitoring is highly effective, but is challenged by being resource-intensive and inaccessible, and its turnaround time does not meet demand. An unmet need exists for an affordable, rapid, and user-friendly point-of-care device that could revolutionize and ensure therapeutic effectiveness, particularly in resource-limited settings. In this work, we explored a point-of-care HIV viral load device to address this need. This device can perform streamlined plasma separation, viral RNA extraction, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) semiquantitative testing in an ultracompact device. We developed an absorption-based membrane plasma separation method suitable for finger-prick blood samples, achieving an efficiency of 80%. We also designed a syringe-based RNA extraction method for on-site plasma processing with a viral recovery efficiency of 86%. We created a portable device with a smartphone interface for real-time semiquantitative RT-LAMP, which is useful for monitoring viral load. The device uses lyophilized reagents, processed with our lyophilization method, which remain stable for 16 weeks. The device can accurately categorize viral load into low, medium, and high categories with 95% accuracy. We believe this point-of-care HIV self-test device, offering convenience and long-term storage, could aid patients in home-based ART treatment monitoring.

Detection by Sensitive Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification of Olive Leaf Yellowing Associated Virus and Its Incidence in Italy and Spain

Olive trees (Olea europea L.) are constantly threatened by many viruses, such as the olive leaf yellowing-associated virus (OLYaV), that belong to the Olivavirus genus, family Closteroviridae. In this work, the OLYaV incidence in different regions of Italy and Spain, which represent the two most important European areas for olive production, was evaluated through the development of a real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for reliable and sensitive OLYaV detection. The specificity and accuracy of the developed real-time RT-LAMP assay were determined; the assay showed that potential cross-reactivity with other viruses belonging to the Closteroviridae family was excluded. The LAMP assay detected OLYaV with a higher sensitivity than conventional end-point RT-PCR, detecting a total of 1.34 × 10−2 genome copies. A total of 80 and 120 plants of different olive cultivars from Spain (Comunitat Valenciana, Andalusia) and Italy (Sicily, Calabria, Apulia, Lazio, and Umbria) regions were tested, respectively. The percentage of infected plants was 46.25% and 30% for Spain and Italy, respectively, while the most susceptible cultivars were “Serrana Espadán” and “Villalonga” from Comunitat Valenciana and Andalusia regions (Spain) and “Ogliarola barese” from Apulia region (Italy). In addition, the survey demonstrated that the real-time RT-LAMP showed good sensitivity for OLYaV-positive sample detection, especially on asymptomatic olive trees. For this reason, the developed assay could be very suitable for phytopathological laboratories as a reliable and efficient method for a rapid and sensitive routine test on olive samples.

PATHPOD – A loop-mediated isothermal amplification (LAMP)-based point-of-care system for rapid clinical detection of SARS-CoV-2 in hospitals in Denmark

Sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a vital goal in the ongoing COVID-19 pandemic. We present in this comprehensive work, for the first time, detailed fabrication and clinical validation of a point of care (PoC) device for rapid, onsite detection of SARS-CoV-2 using a real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction on a polymer cartridge. The PoC system, namely PATHPOD, consisting of a standalone device (weight less than 1.2 kg) and a cartridge, can perform the detection of 10 different samples and two controls in less than 50 min, which is much more rapid than the golden standard real-time reverse-transcription Polymerase Chain Reaction (RT-PCR), typically taking 16–48 h. The novel total internal reflection (TIR) scheme and the reactions inside the cartridge in the PoC device allow monitoring of the diagnostic results in real-time and onsite. The analytical sensitivity and specificity of the PoC test are comparable with the current RT-PCR, with a limit of detection (LOD) down to 30–50 viral genome copies. The robustness of the PATHPOD PoC system has been confirmed by analyzing 398 clinical samples initially examined in two hospitals in Denmark. The clinical sensitivity and specificity of these tests are discussed.

Development of colloidal gold immunochromatography and reverse transcription loop-mediated isothermal amplification assays to detect lychnis mottle virus.

Lychnis mottle virus (LycMoV; genus Unassigned, family Secoviridae) infection of Angelica sinensis produces mottle and mosaic symptoms, damaging the host. Early detection of relevant pathogens is the most critical step in preventing the potential transmission of infectious disease. Polyclonal antibodies (pAbs) with high potency and high specificity were prepared using the recombinant LycMoV capsid protein as an antigen. Here, we developed and optimized a rapid colloidal gold immunochromatography assay (GICA) detection system for LycMoV using this antibody. Under optimum conditions, GICA specifically detected (up to 10,000-fold) positive LycMoV samples. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was also established by selecting the primers with high sensitivity and specificity to LycMoV. The RT-LAMP detection threshold was 1.42 fg/μl (291 copies/μl). A GICA-RT-LAMP assay system was further established and optimized. The minimum GICA detection line was calculated at 1.52x10-2 ng/μl. Although GICA did not detect positive samples after capturing 2.53x10-3 ng/μl of virus, GICA-LAMP and GICA-RT-PCR did, whose sensitivity was comparatively greater than six-fold. This is the first report showing that GICA-RT-LAMP is a cost effective approach for use in detecting LycMoV without extracting nucleic acids. These sensitive assays will help improve virus disease management in A. sinensis crops.

Rapid detection of apple latent spherical virus infecting Angelica sinensis by gold immunochromatography reverse transcription loop-mediated isothermal amplification assay

Apple latent spherical virus (ALSV; genus: Cheravirus, family: Secoviridae) is capable of infecting Angelica sinensis under natural conditions and causing symptoms of greenish discoloration and mottling, affecting plant growth and causing a reduction in root quality and yield. Virus detection of ALSV is an urgent need for the continued development of angelica industry. Consequently, it is necessary to develop simple, sensitive, and reliable ALSV detection methods. We describe the preparation of polyclonal antibodies with high titer and high specificity using recombinant ALSV coat protein (CP) as the antigen. Using these antibodies, we established and optimized a rapid detection system using colloidal gold immunochromatography assay (GICA) to detect ALSV. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system with a minimum detection line of 2.72 × 103copys/μl was also developed to detect ALSV. This assay was further enhanced using a combination GICA-RT-LAMP assay. This GICA assay successfully detected ALSV in infected plants without cross reactivity recorded from three other plant viruses. The sensitivity of GICA was the same as that of GICA-RT-PCR, whereas the sensitivity of GICA-RT-LAMP was at least 10-fold higher than that of GICA and GICA-RT-PCR. These methods can detect and identify ALSV under field conditions and accurately measure virus concentration in the laboratory.

Development of real time reverse transcription loop-mediated isothermal amplification assay for rapid detection of genotype VII of Newcastle disease viruses

ABSTRACT 1. This paper details the establishment of a diagnostic system based on the real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid, simple and sensitive detection of genotype VII of Newcastle disease virus (NDV) directly from field samples. One specific set of six primers were designed which targeted the fusion protein gene of G-VII viruses. The target gene can be amplified and the results recorded within 40 min. 2. The merit of this technique was the feasibility of reading results either by examining turbidity by the naked eye or via the amplification curve generated by real-time PCR. This study tested the sensitivity and specificity of this system against NDV-G-VII and other avian viruses. The real-time RT-LAMP has been found to be more sensitive than real-time RT-PCR. Moreover, 24 out of 35 suspected field samples were positive for genotype VII by real-time RT-LAMP within 30 min in comparison to the real-time RT-PCR for detection of universal NDV. 3. Accordingly, real-time RT-LAMP revealed higher sensitivity than real-time RT-PCR and had specificity only for the NDV-G-VII genotype. Additionally, this system was more rapid and had lower cost than real-time RT-PCR. Based on the results, the RT-LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV-G-VII infection.

Fingerpick Blood-Based Nucleic Acid Testing on A USB Interfaced Device towards HIV self-testing

HIV self-testing is an emerging innovative approach that allows individuals who want to know their HIV status to collect their own specimen, perform a test, and interpret the results privately. Existing HIV self-testing methods rely on rapid diagnostic tests (RDTs) to detect the presence of HIV-1/2 antibodies, which could miss a significant portion of asymptomatic carriers during the window period. In this work, we present a fully integrated nucleic acid testing (NAT) device towards streamlined HIV self-testing using 100 μL finger-prick whole blood. The device consists of a ready-to-use microfluidic reagent cartridge and an ultra-compact NAT-on-USB analyzer. The test requires simple steps from the user to drop the finger-prick blood sample into a collection tube with lysis buffer and load the lysate onto the microfluidic cartridge, and the testing result can be easily read out by a custom-built graphical user interface (GUI). The microfluidic cartridge and the analyzer automatically handle the complexity of sample preparation, purification, and real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP). With a turnaround time of ∼60 min, we achieved a limit of detection (LoD) of 214 viral RNA copies/mL of whole blood at a 95% confidence level. Due to its ease of use and high sensitivity, we anticipate the HIV NAT-on-USB device would be particularly useful for the high-risk populations seeking private self-testing at the early stages of exposure.

An integrated microfluidic platform featuring real-time reverse transcription loop-mediated isothermal amplification for detection of COVID-19

An integrated microfluidic platform (IMP) utilizing real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed here for detection and quantification of three genes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., coronavirus diseases 2019 (COVID-19)): RNA-dependent RNA polymerase, the envelope gene, and the nucleocapsid gene for molecular diagnosis. The IMP comprised a microfluidic chip, a temperature control module, a fluidic control module that collectively carried out viral lysis, RNA extraction, RT-LAMP, and the real-time detection within 90 min in an automatic format. A limit of detection of 5 × 103 copies/reaction for each gene was determined with three samples including synthesized RNAs, inactive viruses, and RNAs extracted from clinical samples; this compact platform could be a useful tool for COVID-19 diagnostics.

Multiplexed detection of respiratory pathogens with a portable analyzer in a \u201craw-sample-in and answer-out\u201d manner

Coronavirus disease 2019 (COVID-19) has emerged, rapidly spread and caused significant morbidity and mortality worldwide. There is an urgent public health need for rapid, sensitive, specific, and on-site diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, a fully integrated and portable analyzer was developed to detect SARS-CoV-2 from swab samples based on solid-phase nucleic acid extraction and reverse transcription loop-mediated isothermal amplification (RT-LAMP). The swab can be directly inserted into a cassette for multiplexed detection of respiratory pathogens without pre-preparation. The overall detection process, including swab rinsing, magnetic bead-based nucleic acid extraction, and 8-plex real-time RT-LAMP, can be automatically performed in the cassette within 80 min. The functionality of the cassette was validated by detecting the presence of a SARS-CoV-2 pseudovirus and three other respiratory pathogens, i.e., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The limit of detection (LoD) for the SARS-CoV-2 pseudovirus was 2.5 copies/μL with both primer sets (N gene and ORF1ab gene), and the three bacterial species were successfully detected with an LoD of 2.5 colony-forming units (CFU)/μL in 800 μL of swab rinse. Thus, the analyzer developed in this study has the potential to rapidly detect SARS-CoV-2 and other respiratory pathogens on site in a “raw-sample-in and answer-out” manner.

Simultaneous detection of SARS-CoV-2 and pandemic (H1N1) 2009 virus with real-time isothermal platform

The recent ongoing outbreak of novel coronavirus SARS-CoV-2 (known as COVID-19) is a severe threat to human health worldwide. By press time, more than 3.3 million people have died from COVID-19, with many countries experiencing peaks in infections and hospitalizations. The main symptoms of infection with SARS-CoV-2 include fever, chills, coughing, shortness of breath or difficulty breathing, fatigue, muscle or body aches and pains. While the symptoms of the pandemic (H1N1) 2009 virus have many similarities to the signs and transmission routes of the novel coronavirus, e.g., fever, cough, sore throat, body aches, headache, chills and fatigue. And a few cases of serious illness, rapid progress, can appear viral pneumonia, combined with respiratory failure, multiple organ function damage, serious people can die. Therefore, there is an urgent need to develop a rapid and accurate field diagnostic method to effectively identify the two viruses and treat these early infections on time, thus helping to control the spread of the disease. Among molecular detection methods, RT-LAMP (real-time reverse transcription-loop-mediated isothermal amplification) has some advantages in pathogen detection due to its rapid, accurate and effective detection characteristics. Here, we combined the primers of the two viruses with the fluorescent probes on the RT-LAMP detection platform to detect the two viruses simultaneously. Firstly, RT-LAMP method was used respectively to detect the two viruses at different concentrations to determine the effectiveness and sensitivity of probe primers to the RNA samples. And then, the two virus samples were detected simultaneously in the same reaction tube to validate if testing for the two viruses together had an impact on the results compared to detecting alone.We verified the detection efficiency of three highly active BST variants during RT-LAMP assay. We expect that this assay can effectively and accurately distinguish COVID-19 from the pandemic (H1N1) 2009, so that these two diseases with similar symptoms can be appropriately differentiated and treated.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and On-Site Detection of Avian Influenza Virus.

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.

A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP Assay to Detect SARS-CoV-2.

The SARS-CoV-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). reverse-transcription (RT)-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high-technological, infrastructure-dependent, and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as an alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step), affordable (~1.7 €/sample), and rapid detection of SARS-CoV-2 targeting both ORF1ab and N genes of the novel virus genome. We demonstrate the assay on RT-qPCR-positive clinical samples, obtaining most positive results under 25 min. In addition, a novel 30-min one-step drying protocol has been developed to stabilize the RT-LAMP reaction mixtures, allowing them to be stored at room temperature functionally for up to two months, as predicted by the Q10. This Dry-RT-LAMP methodology is suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, it could be easily applied both in developed and in low-income countries yielding rapid and reliable results.

Comparison of clinical diagnostic performance between commercial RRT-LAMP and RT-qPCR assays for SARS-CoV-2 detection

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in isolating infected patients and preventing further viral transmission. In this study, we evaluated the clinical diagnostic performances of a commercial real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay (Isopollo® COVID-2 assay, M-monitor, Daegu, Korea) using eighty COVID-19 suspected clinical samples and compared these with the results of a commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) assay (AllplexTM 2019-nCoV rRT-QPCR Assay, SeeGene, Seoul, Korea). The results of the RRT-LAMP assay targeting the N or RdRp gene of SARS-CoV-2 showed perfect agreement with the RT-qPCR assay results in terms of detection. Furthermore, the RRT-LAMP assay was completed in just within a 20-min reaction time, which is significantly faster than about the 2 h currently required for the RT- qPCR assay, thus enabling prompt decision making regarding the isolation of infected patients. The RRT-LAMP assay will be a valuable tool for rapid, sensitive, and specific detection of SARS-CoV-2 in human or unexpected animal clinical cases.

C4COVID-19 Human: A Rapid SARS-Cov-2 Salivary Test Compared to the Gold Standard – Clinical Results

The SARS-CoV-2 detection campaign is a key element in the viral pandemic response. Here we present a monocentric study to evaluate a simplified molecular amplification technology alternative to classical RT-qPCR platforms. The test, C4Covid-19 HumanTM makes use of saliva as a starting sample allowing for self, painless collection, avoiding risk of viral transmission for the operator who performs the nasopharyngeal (NP) procedure. Total time to result is 30 minutes. The principle relies on an extraction-free detection of two targets within the viral genomic RNA, RdRp and N genes, by real-time reverse transcription loop mediated isothermal amplification (RT-LAMP). Saliva and NP swabs were collected simultaneously from 1491 symptomatic or contact cases and analyzed in parallel by RT-LAMP and RT-qPCR respectively. The 249 positives RT-qPCR samples had a Ct <37. Sensitivity and specificity of RT-LAMP were 85.7% (95% CI 80.4 to 89.7) and 97.3% (95% CI 96.1 to 98.1) respectively. Among the total cohort, the 56.9% of asymptomatic subjects showed higher sensitivity (90.6% with a 95% CI of 81.0 to 95.6) while specificity remained unchanged (97.4% with a 95% CI of 95.6 to 98.4). C4Covid-19 HumanTM is a rapid, simple and reliable SARS-CoV-2 detection test for population screening.

Development of a Real-Time Loop-Mediated Isothermal Amplification Method for the Detection of West Nile Virus

Background: The West Nile Virus (WNV), discovered in New York, USA in 1999 after it was first isolated in Uganda in 1937, has since spread not only in the United States but also around the world. Africa, Eurasia, Australia, and the Middle East have sporadic cases of the disease. Objectives: We aimed to find real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to be more sensitive than conventional RT-PCR, and more rapid and efficient than conventional RT-PCR and real-time RT-PCR for WNV detection. Methods: A total of 32 genomic sequences from different strains of WNV were analyzed to identify conserved nucleotide sequence regions. Six WNV specific RT-LAMP primers targeting the E gene were designed. Results: The novel primer for the real-time RT-LAMP assay can detect WNV with high specificity. The efficiency of the real-time RT-LAMP assay is higher than the conventional RT-PCR and real-time RT-PCR. Real-time RT-PCR and conventional PCR require at least 30 – 40 min and 2 h, respectively, to yield results, whereas real-time RT-LAMP provides positive results in only 10 – 20 min. Conclusions: The novel primers were developed by analyzing of 32 genomic sequences of WNV strains. The primers were designed from the most conserved region of the E gene for real-time RT-LAMP. The LAMP assay is a rapid, efficient, highly sensitive, and specific tool for the identification of WNV.

Probe-based real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay for rapid and specific detection of foot-and-mouth disease virus.

Rapid and specific detection of foot-and-mouth disease virus (FMDV) is a key factor for promoting prompt control of FMD outbreaks. In this study, a real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay with high sensitivity, rapidity and reliability was developed using a targeted gene-specific assimilating probe for real-time detection of seven FMDV serotypes. Positive assay signals were generated within 15min for the lowest concentration of a standard RNA sample at 62°C; this was substantially faster than that achieved by the OIE (World Organisation for Animal Health)-recommended real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The new assay specifically amplified the 3D gene of all seven FMDV serotypes and did not amplify other viral nucleic acids. The detection limit of the assay was 102 copies/µl which is comparable to that achieved by qRT-PCR. Furthermore, using clinical samples, the results of the RRT-LAMP assay were largely in agreement with those from the qRT-PCR assay with a kappa value (95% confidence interval [CI]) of 0.94 (0.86-1.02). The established RRT-LAMP assay that features assimilating probes is an advanced molecular diagnostic tool that is easily applicable to a wide range of circumstances and has high potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of FMDVs in clinical samples.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2.

BackgroundHighly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.MethodsA rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.ResultsThis assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.