Reverse Transcription Loop-mediated Isothermal Amplification Primers Research Articles

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/μL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

Evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of severe fever with thrombocytopenia syndrome in clinical laboratories: A single-center study.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease prevalent in East Asia with a high mortality rate (5%-30%). Reverse transcription loop-mediated isothermal amplification (RT-LAMP), a rapid nucleic acid-based diagnostic technique, is a useful alternative for the clinical diagnosis of SFTS, particularly in resource-limited hospitals or rural clinics in SFTS virus-endemic regions. However, the actual clinical sensitivity and specificity of RT-LAMP remain unclear. This study evaluated the field application of RT-LAMP.This prospective field study included 130 patients with laboratory-confirmed SFTS from Yantai, Shandong Province, China. Two sets of RT-LAMP primers were validated, and one set of RT-LAMP assays was optimized for field detection. Nucleic acids of serially collected serum/plasma samples were identified using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and RT-LAMP.In laboratory tests, we optimized the detection time of primer set 2 for the RT-LAMP to 60 min. Notably, the onsite testing of 279 plasma samples from patients with SFTS revealed that the sensitivity and specificity of the test were 81.9% and 96.3%, respectively. We also analyzed samples with different durations of the disease, and our study showed that the sensitivity of RT-LAMP detection at the beginning of admission was 89.92%. Univariate analysis showed that the detection rate of RT-LAMP was similar to that of RT-qPCR in the first 5 days of the disease course and was lower than that of RT-qPCR on Days 6 and 14-15 of the disease course. The positive detection rate in patients aged ≥ 65 years was significantly higher than that in younger age groups.RT-LAMP is a simple, suitable, and rapid clinical detection method of SFTS onsite screening. It is more suitable for screening patients in the early stages of the disease and analyzing samples obtained from patients aged ≥ 65 years before the 6th day of the disease course.

High Sensitivity, Rapid Detection of Virus in High Traffic Environments.

The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.

Rapid detection of Potato leafroll virus and Potato virus Y by reverse transcription loop-mediated isothermal amplification method in north-east India

Potato leafroll virus (PLRV) and Potato virus Y (PVY) are two important viruses causing serious potato yield losses in the North-east region and other planting areas in India. As a consequence, it is urgent to develop an efficient and quick method for the identification and diagnosis in the field. The results presented here showed that the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was efficient and sensitive than reverse transcription-polymerase chain reaction (RT-PCR) for the detection of PLRV and PVY. The RT-LAMP primers specifically targeted PLRV and PVY (including PVYO, PVYN, and PVYNTN strains) and resulted in typical sigmoidal amplification curves. Ten-fold serial dilutions of PLRV and PVY total RNA indicated that RT-LAMP is faster and at least a hundred times more sensitive than RT-PCR in detecting both the viruses. Additionally, samples that RT-PCR could not detect at a diluted concentration of 10−3 and 10-4 ng/μl were identified by RT-LAMP. Thus, RT-LAMP offers many advantages over RT-PCR such as low cost and high accuracy, sensitivity, and specificity for the rapid diagnosis of plant virus diseases. In conclusion, the results highlighted the efficacy of the RT-LAMP method in quickly detecting PLRV and PVY in infected plants.

Development of a reverse transcription loop mediated isothermal amplification assay for the detection of Mouse reovirus type 3 in laboratory mice

Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.

“Development and comparison of reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP), RT-PCR and real time PCR for detection of Potato spindle tuber viroid in potato”

Potato spindle tuber viroid (PSTVd) is an important pathogen of potato infecting leaves, tubers as well as true potato seeds. We developed and validated a reverse transcription-loop mediated isothermal amplification (RT-LAMP) platform for visual, rapid, specific, and sensitive detection of PSTVd. The optimization of RT-LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs, and WarmStart Bst 2.0 polymerase. The assay is very simple and rapid which provide detectable results in less than 50 min by incubating all the reagents at 65 °C. The detection of RT-LAMP results could be accomplished as ladder-like bands in an agarose gel or visualized by naked eyes with inclusion of a SYBR Green I dye. The target specificity of RT-LAMP primers was assessed with an introduction of a simple restriction digestion step after the RT-LAMP assay. The RT-LAMP assay demonstrated absence of any cross-reaction with other, potato viruses. The analytical sensitivity of the assay was greater than RT-PCR either the test template was plasmid or total RNA. The detection of RT-LAMP was validated in potato samples and compared with real time PCR and RT-PCR. The RT-LAMP assay developed in this study has the potential as a beneficial tool in detection of PSTVd in a low-cost laboratory due to its simplicity, rapidness, and application in large samples.

Rapid detection of two emerging viruses associated with necrotic symptoms in Areca catechu L. by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Two reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV), respectively. These two emerging viruses both induce necrotic symptoms in areca palms. The coat protein (CP) gene of ANRSV and the 9 K gene of ANSSV were used to design the respective RT-LAMP primers for the assays. Each set of four primers designed for each of these viruses was found to be highly specific in the detection of the respective targeted virus. The optimal incubation conditions for the RT-LAMP assays were 63 °C for 40 min for ANRSV and at 61 °C for 40 min for ANSSV. The sensitivity of the RT-LAMP method for each of these viruses was 10-fold greater than that of the corresponding conventional reverse-transcription polymerase chain reaction (RT-PCR). The RT-LAMP assays may be useful for the rapid early detection of ANSSV and ANRSV in commercial areca palm production.

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the pathogen of maize rough dwarf disease in China

Maize rough dwarf disease (MRDD) is one of the most serious viral diseases of maize in China. The object of this study is to develop a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of MRDD pathogen. Four RT-LAMP primers were designed on the basis of S10. Template RNA from infected leaves were used for RT-LAMP which were carried out under isothermal conditions at 61 °C for 90 min. RT-LAMP products were analyzed by electrophoresis in agarose gels or visual inspection of color change after staining with calcein. Sensitivity of the RT-LAMP assay was 100-fold higher than RT-PCR. This method can distinguish RBSDV from SRBSDV and the vectors carrying RBSDV from Rice stripe virus (RSV). The RT-LAMP assay shows excellent potential for application in the rapid and sensitive detection of MRDD pathogens in field samples or vector.

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.

Rapid and Specific Detection of All Known Nipah virus Strains' Sequences With Reverse Transcription-Loop-Mediated Isothermal Amplification.

Nipah virus (NiV) is a zoonotic virus and can be transmitted through contaminated food or directly between people. NiV is classified as a Biosafety Level 4 agent, not only because of its relatively high case fatality rate, but also because there is no vaccine or other medical countermeasures and it appears to be transmitted by fomites/particulates. The development of rapid detection assay for NiV is of great importance because no effective field test is currently available. In this study, an isothermal (65°C) reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method was developed, targeting the nucleocapsid protein (N) gene, for the rapid detection of NiV, and was compared with conventional RT-PCR. Three pseudoviruses of NiV N gene representing all known strains were constructed to replace live NiV. A set of RT-LAMP primers, targeting a highly conserved region of the N gene in the viral genome was designed to identify all known NiV strains. Sensitivity tests indicated that the detection limit of the RT-LAMP assay was approximately 100 pg of total NiV pseudovirus RNA, which is at least 10-fold higher than that of conventional RT-PCR. Specificity tests showed that there was no cross-reactivity with nucleocapsid protein gene of Hendra virus, Newcastle disease virus, Japanese encephalitis virus, or Influenza A virus. The RT-LAMP assay provides results within 45 min, and requires no sophisticated instruments, except an isothermal water bath or metal bath with 1 μl calcein indicator. An analysis of the clinical samples showed that the assay had good stability. In conclusion, systematic experiments have shown that the RT-LAMP assay developed here effectively detects three NiV pseudoviruses representing all known strains of NiV, with high specificity, sensitivity and stability.

A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood

Isothermal nucleic acid amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are suitable for the development of a rapid, low-cost NAT that can be used at the POC. For demonstration of utility for global use, studies are needed to validate the performance of RT-LAMP for the detection of divergent subtypes. In this study, we designed and evaluated multiplexed HIV-1 integrase RT-LAMP primers to detect subtypes within group M, along with an RNase P positive internal processing and amplification control. Using a panel of 26 viral isolates representing the major circulating subtypes, we demonstrated detection of all isolates of subtypes A1, C, D, F1, F2, G, CRF01_AE, CRF02_AG, and two unique recombinant forms (URFs). A whole blood panel created with one representative isolate of each subtype was successfully amplified with the group M HIV-1 integrase and RNase P internal control primers. The group M HIV-1 RT-LAMP assay was further evaluated on 61 plasma specimens obtained from persons from Cameroon and Uganda. The sequence-conserved group M HIV-1 RT-LAMP primers, coupled to a low-cost amplification device, may improve diagnosis of acute infection at the POC and provide timely confirmation of HIV status.

The detection of ACLSV and ASPV in pear plants by RT-LAMP assays

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV), two important viruses frequently occurring in pear trees. A set of four RT-LAMP primers designed based on the highly conserved region of each CP gene of the two viruses showed high specificity and feasibility for ACLSV and ASPV detections. The RT-LAMP assays for ACLSV and ASPV in pear samples were 104 and 103 times more sensitive than that of conventional RT-PCR assays. The RT-LAMP under optimal reaction condition was subsequently utilized in the detection of the two viruses in-vitro cultures of pear and field pear samples. This study provides a rapid and sensitive tool to determine the infection statues of the two viruses in pear certification program.

Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Coxsackievirus B3 (CVB3) is a member of the genus Enterovirus within the family Picornaviridae and is an important pathogen of viral myocarditis, which accounts for more than 50% viral myocarditis cases. VP1 is major capsid protein that this region has a low homology in both amino acid and nucleotide sequences among Enteroviruses. Therefore we have chosen this region for designed a set of RT-LAMP primers for CVB3 detection. For this the total RNA was extracted from 24-h post infected-HeLa cells with complete cytopathic effect (CPE), and applied to a one-step reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using CVB3-specific primers. The optimization of RT-LAMP reaction was carried out with three variables factors including MgSO4 concentration, temperature and time of incubation. Amplification was analyzed by using 2% agarose gel electrophoresis and ethidium bromide and SYBR Green staining. Our results were shown the ladder-like pattern of the VP1 gene amplification. The LAMP reaction mix was optimized and the best result observed at 4mM MgSO4 and 60°C for 90min incubation. RT-LAMP had high sensitivity and specificity for detection of CVB3 infection. This method can be used as a rapid and easy diagnostic test for detection of CVB3 in clinical laboratories.

Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

The Feline coronavirus (FCoV) is the etiological agent of feline infectious peritonitis (FIP), a lethal disease of felids. The role of molecular methods is controversial for the diagnosis of FIP, while essential for the identification of the shedders. Thus, a fast and inexpensive method for the detection of FCoV could be beneficial, especially in multicat environments. A reverse transcription loop mediated isothermal amplification (RT-LAMP) assay was developed. RNA extraction and RT-nPCR for FCoV were performed on thirty-two samples (11 faeces, 9 blood, 8 effusions, and 4 lymph nodes) collected from 27 cats. Six RT-LAMP primers were designed from the same conserved region of RT-nPCR, and the assay was run at 63°C for one hour. Results were evaluated through both agarose gel run and hydroxynapthol blue (HNB) dye and then compared with RT-nPCR results for the assessment of sensitivity and specificity. The overall specificity was 100%, but the sensitivity was 50% and 54.5% for agarose gel and HNB respectively. Therefore, RT-LAMP seems optimal to confirm the presence of the virus, but not applicable to exclude it.

Detection of influenza A virus by reverse transcription-loop mediated isothermal amplification

Objective To detect influenza A virus by reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to established a rapid, simple and visualization nucleic acid detection method. Methods The RT-LAMP primers were designed in accordance with the hemagglutinin gene of influenza A virus. Then, the specificity of the primers was evaluated by detection of different influenza viruses, and the sensitivity was confirmed by testing multiple diluted RNA samples. Hydroxynaphthol blue (HNB) was used for visually evaluation and gel electrophoresis was used for validation. Clinical samples were detected by RT-LAMP assay. Its consistency with fluorescent quantitative polymerase chain reaction (PCR) methods was compared. Results The primers of RT-LAMP assay had high specificity. This technique could amplify influenza A virus accurately. The detection limit of RT-LAMP assay was 2.5×103 copies/mL by detection of multiple diluted RNA samples. In addition, the results of RT-LAMP assay could be visually inspected using HNB by color change, and the results was in accordance with that of gel electrophoresis. RT-LAMP assay was in consistence with fluorescent quantitative PCR when clinically applied. Conclusions RT-LAMP assay is a rapid, specific, sensitive and simple method to detect influenza A virus. Key words: Influenza A virus; Reverse transcription-loop mediated isothermal amplification; Rapid detection

Reverse transcription loop-mediated isothermal amplification to rapidly detect Rice ragged stunt virus.

Rice ragged stunt virus (RRSV) is a very important virus that infects rice and causes serious yield losses in Asian countries and other major rice planting areas. Thus, it is urgent to establish an efficient and practical approach for identification and diagnosis in the field. Our results indicated that reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions are more efficient and sensitive than RT-PCR for RRSV detection. The optimal LAMP conditions were as follows: 0.4–1.2 μM internal primers, 0.2–0.25 μM external primers, 0.8 μM loop primers, and incubation at 62 °C or 63 °C for 30 min. Furthermore, the RT-LAMP primers specifically targeted RRSV virus and resulted in typical waterfall-like bands by gel electrophoresis and sigmoidal amplification curves. The primers could not be used to amplify other common plant viruses including Papaya ringspot virus (PRSV), Rice yellow stunt virus (RYSV), Sorghum mosaic virus (SrMV), Cactus virus X (CVX), Melon yellow spot virus (MYSV) and Southern rice black-streaked dwarf virus (SRBSDV). Ten-fold serial dilutions of RRSV cDNA indicated that RT-LAMP is much faster and at least ten times more sensitive than RT-PCR in detecting the virus. The waterfall-like product bands could be observed within one hour. In the field study, about 77% samples were identified as RRSV. RT-LAMP has many benefits over RT-PCR such as low cost and high accuracy, sensitivity, and specificity. This technology meets the requirements for rapid diagnosis of plant virus diseases in the field to best guide management practices for growers.

Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification.

Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.

Reverse transcription Loop-mediated isothermal amplification을 이용한 Soybean mosaic virus의 진단

Soybean mosaic virus (SMV) is a prevalent pathogen that causes significant yield reduction in soybean production worldwide. SMV belongs to potyvirus and causes typical symptoms such as mild mosaic, mosaic and necrosis. SMV is seed-borne and also transmitted by aphid. Eleven SMV strains, G1 to G7, G5H, G6H, G7H, and G7a were reported in soybean varieties in Korea. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method allowed one-step detection of gene amplification by simple procedure and needed only a simple incubator for isothermal template. This RT-LAMP method allowed direct detection of RNA from virus-infected plants without thermal cycling and gel electrophoresis. In this study, we designed RT-LAMP primers named SML-F3/B3/FIP/BIP from coat protein gene sequence of SMV. After the reaction of RT-LAMP, products were identified by electrophoresis and with the detective fluorescent dye, SYBR Green I under daylight and UV light. Optimal reaction condition was at 58C for 60 min and the primers of RT-LAMP showed the specificity for nine SMV strains tested in this study.

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.

A One-Step, Real-Time Reverse Transcription Loopmediated Isothermal Amplification Assay to Detect Potato Virus Y

Potato viruses such as Potato virus Y (PVY) cause diseases that affect potato quality and thus damage potato production worldwide. Current tests for viral infection use double-antibody sandwich enzyme linked immunosorbent assays (DAS‑ELISA) or reverse transcription polymerase chain reaction (RT-PCR)/real-time RT-PCR. Despite many advantages, these assays have a number of drawbacks that affect cost and time of diagnosis. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) allows fast detection of target RNA. Here, we developed a closed-tube real-time RT‑LAMP assay for fluorescent detection of PVY. Specific RT-LAMP primers were designed to target the conserved region of the sequence encoding the PVY coat protein. The assay was specific and facilitated sensitive PVY detection in a single tube at 65 °C. The time-to-positive values depended on the PVY concentration in tested samples. The effectiveness of RT‑LAMP in testing field-grown plants compared favorably with DAS‑ELISA and RT-PCR; under the tested conditions, RT-LAMP was about 1000-fold more sensitive than DAS‑ELISA and lateral flow assay (LFA) and about 10-fold more sensitive than RT‑PCR. Thus, this fluorescent RT-LAMP assay has great potential for routine detection of PVY.