Detection of Coat Protein Gene of the Potato Leafroll Virus by Reverse Transcription Loop-Mediated Isothermal Amplification (original) (raw)
Related papers
Journal of Phytopathology, 2013
Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll virus. One-step RT-LAMP was performed using RNA of PLRV-infected potato leaves and a set of primers (F3, B3, FIP, BIP, LF and LB) designed for RT-LAMP reaction of the coat protein (CP) gene of PLRV. Positive effects of RT-LAMP were detected by agarose gel electrophoresis and hydroxynaphthol blue (HNB) dye and were shown by a colour change from violet to sky blue. RT-LAMP with HNB dye proved to be a simple assay for the rapid detection of PLRV.
Journal of Virological Methods, 2013
The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3 h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock.
Using different methods to confirm of RT-LAMP reaction for detection of Potato Leafroll Virus (PLRV)
Potato leafroll virus is an important virus in potato that causes economic loss in the yield and quality of potato tubers. One of the primary methods of managing infection in potato crops is to use of certified virus-free tuber as ‘seed’ for planting. Early and efficient detection of virus is essential for production PLRV-infection free tubers. There are several techniques to detect the virus including serological test and molecular methods. LAMP method is a new method to identify pathogens that in this study used it to detection of potato leafroll virus. Potato plants with symptoms similar to PLRV were collected from Zanjan provinces were subjected for a serological test. For doing molecular works, total RNA were extracted and RT-LAMP reactions were carried out. Then were used different methods to confirm of performance this reaction and positive reaction was confirmed with see the resulting turbidity, load product on agaros gel and use of SYBR and ethidium bromide florescence days...
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.
Academic research, 2024
ABSTRACT Sweet potato is one of the most important food crops (root crops) in most if not all parts of the world due to its commercial, nutritional, medical, and aesthetic values. However, this importance is being constrained by many viruses among many biotic and abiotic factors with Sweet Potato Feathery Mottle Virus (SPFMV) being the most abundant, widespread, and the most devastating causing over 50% yield loss. Early detection of these viruses is critical for controlling the spread and the devastating effects on sweetpotatoes. Currently, serological and several molecular and biological diagnostic techniques are being used but they are either not sensitive enough to reliably detect viruses directly from sweetpotatoes or require expensive laboratory equipment to perform and a high level of experienced personnel rendering them ineffective. This work therefore developed a simple loop-mediated isothermal amplification (LAMP) assay for rapid on-site detection of Sweet Potato Feathery Mottle Virus. The gene sequence of SPFMV was accessed from NCBI and the specific LAMP primers were designed based on the conserved sequence of the gene by the use of Primer Explorer V6 software (hhp://primerexplorer.jp/e/). The primers were then synthesized in South Africa and then optimized, and standardized. The new LAMP was compared with PCR test. The results demonstrate that LAMP has high sensitivity of 96.1%, meaning its effective at correctly identifying plants with SPFMV infection. Similarly, the specificity was found to be 95%, implying its ability to accurately identify plants without the disease signs. Field performance of the LAMP was also tested in six districts to find out whether the developed LAMP is ready to be deployed in a non-laboratory setting such as in the field for use during inspections, and seed certification or virus incidence surveys. These results were compared to that of PCR. The results showed 90% similarity implying that the LAMP is just as effective detective tool as the PCR indicating its readiness for deployment at the point of care.
'Torrado' disease caused by tomato torrado virus (ToTV) is responsible for considerable losses in tomato production. Therefore, a one-step reverse transcription loop-mediated isothermal amplification protocol for early and fast detection of ToTV isolates has been developed. The RNA extracted from ToTV-infected plants was tested using this protocol with a set of six primers specific for the Vp35 coat protein gene sequence. The amplified products were analyzed using amplification curves, electrophoresis, and direct staining of DNA. The sensitivity of the protocol was tenfold higher than that of conventional RT-PCR. This new protocol is inexpensive, rapid, simple, and very sensitive.
Journal of Plant Pathology & Microbiology, 2013
Loop-Mediated Isothermal Amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. To diminish the time required for some diagnostic assays including Reverse Transcription PCR (RT-PCR), Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) and also DAS-ELISA into a minimum level, an innovative colorimetric IC-RT-LAMP and IC-RT-PCR protocol on the basis of Potato Virus Y (PVY) genome were used and optimized. Firstly, DAS-ELISA assay was performed to detect of the virus in a collection containing 95 suspicious samples. Lastly, five samples were detected as the positive samples. Then, the positive samples were verified by molecular methods. In this regard, all four RT-LAMP primers (i.e. F3, B3, FIP and BIP) together with RT-PCR primers (F and B) were selected on the basis of coat protein gene (CP) of PVY genome. Even though DAS-ELISA, RT-PCR and RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Furthermore, the results demonstrated that the RT-LAMP assay was 100 times sensitive and 3 time faster compared to RT-PCR. LAMP assay was accomplished in the water bath either frees from any thermal cycler machine or sophisticated laboratories facility. Meanwhile, among six different visual dyes to accurately detect IC-RT-LAMP products, Hydroxynaphthol blue, GeneFinder TM and SYBR Green I could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. We accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PVY recognition and probably other viral-based diseases.
2021
Cotton leafroll dwarf disease caused by Cotton leafroll dwarf virus (CLRDV) is an emerging viral disease in cotton in the US. Infection with the virus shows varying degrees of symptoms making proper early diagnosis difficult. Conventional reverse transcription-polymerase chain reaction (RT-PCR) from symptomatic tissues is the most common diagnostic method for CLRDV but it is relatively complex and time consuming. To overcome these shortcomings, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of CLRDV and its specificity and sensitivity were compared with conventional RT-PCR assay. For this assay, a set of six primers to amplify the partial P1 (ORF1) and P0 (ORF0) region of the viral genome were designed. The primers were used for amplification at isothermal temperature with total RNA extracted from CLRDV infected cotton leaf tissues, and the optimum primer concentrations, reaction temperature, and assay time were determi...
Frontiers in Microbiology, 2021
Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/μl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-po...
Journal of Plant Pathology & Microbiology, 2012
Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied a rapid detection protocol, for the first time, which utilizes colorimetric loop-mediated isothermal amplification for detection of Tomato Yellow Leaf Curl virus. In this regard, all four LAMP primers (i.e. F3, B3, FIP and BIP,) together with PCR primers (F and R) were designed on the basis of the SF gene of the DNA sequences of TYLCV. Even though DAS-ELISA, PCR and LAMP assays could successfully detect positive infected samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect LAMP products, both hydroxynaphthol blue and GeneFinderTM could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. All the results, overall, indicated that the LAMP offers an interesting novel and convenient assay format for the rapid, sensitive, cost-effective, and fairly user friendly diagnostic tool of recognition of Tomato Yellow Leaf Curl virus and therefore presents an alternative to PCR-based assays.