scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

Plant Disease ◽  
2005 ◽  
Vol 89 (6) ◽  
pp. 605-610 ◽  
Author(s):  
Xianzhou Nie
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Time Saving ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

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

2015 ◽  
Vol 92 (3) ◽  
pp. 303-311 ◽  
Author(s):  
Agnieszka Przewodowska ◽  
Bogumiła Zacharzewska ◽  
Joanna Chołuj ◽  
Krzysztof Treder
Keyword(s):  
Real Time ◽  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Isothermal Amplification ◽  
Amplification Assay ◽  
One Step

scholarly journals Variability of Potato virus Y in Tomato Crops in Poland and Development of a Reverse-Transcription Loop-Mediated Isothermal Amplification Method for Virus Detection

2015 ◽  
Vol 105 (9) ◽  
pp. 1270-1276 ◽  
Author(s):  
Beata Hasiów-Jaroszewska ◽  
Joanna Stachecka ◽  
Julia Minicka ◽  
Mateusz Sowiński ◽  
Natasza Borodynko
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Protein Gene ◽  
Virus Detection ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Polymerase Chain

A collection of 147 Potato virus Y (PVY) isolates from tomato, originating from several commercial fields and greenhouses in different regions of Poland, was tested for the presence of PVY by reverse-transcription polymerase chain reaction. However, in some cases, the results obtained were ambiguous. Therefore, a sensitive reverse-transcription loop-mediated isothermal amplification method was developed for rapid detection of PVY isolates. Phylogenetic and recombination analyses were performed based on sequences of the coat protein gene. In comparison with results obtained in 2008, the presence of other strains besides PVYNWi-P was confirmed. A novel recombinant between PVYNTN and PVYNWi-P strains was detected. Our results indicate an increasing distribution and variability of the PVY population on tomato in Poland.

scholarly journals Comparison of reverse-transcription loop-mediated isothermal amplification and reverse-transcription polymerase chain reaction for grass carp reovirus

2015 ◽  
Vol 84 (3) ◽  
pp. 215-223
Author(s):  
Shuixian Yang ◽  
Kai-Yu Wang ◽  
Zexiao Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Grass Carp ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Time Saving ◽  
Grass Carp Reovirus ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain

Grass carp reovirus (GCRV) has been assigned to a newly established Aquareovirus genus in the family of Reoviridae which leads to haemorrhagic disease and extremely high mortality rate in grass carp. In this study, comparison was made between the novel one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the reverse transcription polymerase chain reaction (RT-PCR) for detection of grass carp reovirus. The result indicated that RT-LAMP had × 10 higher sensitivity comparable to RT-PCR. The specificity of the two methods for GCRV detection were both developed successfully by other three aquatic viruses. In the field trial, both RT-PCR and RT-LAMP methods were applied to detect the samples from different infected organs and tissues. The result demonstrated that RT-LAMP had a high accuracy to confirm the diagnosis as well as the RT-PCR. This study showed that the RT-LAMP, compared to the RT-PCR, was simple, time-saving, convenient, but required specificity primers and possibly generated false positive product. Its products, unlike RT-PCR, could not be direcly used in further molecular research after purification. Thus RT-LAMP might be an optimal diagnostic method for rapid and preliminary diagnosis of GCRV infection in resource-limited setting situation.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

Prevalence and strain composition of potato virus Y circulating in potato fields in China’s north-central province Shanxi

Plant Disease ◽  
2021 ◽  
Author(s):  
Pengcheng Ding ◽  
Dexin Chen ◽  
Haixu Feng ◽  
Jiao Li ◽  
Hui Cao ◽  
...  
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Potato Production ◽  
Central China ◽  
Shanxi Province ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Single Strain ◽  
North Central ◽  
Production Areas

Potato is an important crop in Shanxi province located in north-central China. During 2019-2020, 319 potato leaf samples were collected from eight locations distributed in three major potato production areas in Shanxi. Bio-chip detection kit revealed the presence of several potato viruses, and among them potato virus Y (PVY) was the most common one, reaching the incidence of 87.8% of all symptomatic samples. The immuno-captured multiplex reverse transcription (RT)-PCR was used to identify strains for all 280 PVY-positive samples, unveiling 242 samples infected with a single strain of PVY (86.4%) and 38 (13.6%) with a mixed infection. Of samples with a single-strain infection, PVY -SYR-II accounted for 102 (42.1%), followed by PVYN-Wi (33, 13.6%) , PVY -SYR-I (28, 11.6%), 261-4 (22, 9.1%), PVYNTNa (20, 8.3%), PVYNTNb (19, 7.9%), and PVY -SYR-III (18, 7.4%). Seven isolates representing different recombinants were selected for whole genome sequencing. Phylogenetic and recombination analyses confirmed the RT-PCR based strain typing for all seven strains of PVY found in Shanxi. SXKL-12 is the first SYR-III strain from potato reported from China. However, unlike that in other known SYR-III isolates, the region positioned from 1,764 to1,902 nt in SXKL-12 shared the highest sequence identity of 82.2% with an uncharacterized PVY isolate, JL-23, from China. Interestingly, the PVYN-Wi isolate SXZY-40 also possessed a more divergent sequence for the region positioned from 6,156 to 6,276 nt than other N-Wi isolates known to date, sharing the highest identity of 86.6% with an uncharacterized Chinese PVY isolate, JL-11. Pathogenicity analysis of dominant strains PVY -SYR-II and PVYN-Wi in six local popular potato cultivars revealed that Kexin 13, Helan 15 and Jizhangshu 12 were susceptible to these two strains with mild mottling or mosaic symptoms expression, while three cultivars, Jinshu 16, Qingshu 9, Xisen 6 were found fully resistant.

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