Rapid visual detection method for barley yellow mosaic virus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Plant Disease ◽  
2020 ◽  
Author(s):  
Zhiwei Chen ◽  
Shuihua Mao ◽  
Wan Zhang ◽  
Xaorui Fan ◽  
Wenjing Wu ◽  
...  
Keyword(s):  
Food Security ◽  
Mosaic Virus ◽  
Reverse Transcription ◽  
Mosaic Disease ◽  
Isothermal Amplification ◽  
Yellow Mosaic Virus ◽  
Lamp Method ◽  
Yellow Mosaic Disease ◽  
Loop Mediated Isothermal Amplification ◽  
Barley Yellow Mosaic Virus

Barley yellow mosaic disease, caused mainly by barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), is a devastating disease of barley and is a threat to Eurasian barley production. Early detection is essential for effective management of the pathogens and to assure food security. In the present study, a simple, rapid, specific, sensitive, and visual method was developed to detect BaYMV using loop-mediated isothermal amplification (LAMP). Two pairs of oligonucleotide primers (inner and outer primers) were designed to amplify the gene encoding the coat protein of BaYMV. The optimal conditions for the LAMP method were determined, and a one-step reverse transcription (RT)-LAMP method was also developed. Subsequently, the fastest processing time for RT-LAMP was determined. Among eight plant viruses examined using the LAMP method, only BaYMV was detectable, suggesting that the assay was highly specific. The RT-LAMP method was ten times more sensitive than the RT-PCR method in the sensitivity test. To further shorten the virus detection process, a dye was added to the RT-LAMP products, and positive reactions were simply read by the naked eye via a color change (from orange to light green) under visible light. Barley samples from the middle and lower reaches of the Yangtze River basin, where barley yellow mosaic disease broke out very seriously in 1970s, were detected by the newly established RT-LAMP method. The results showed that all samples were positive for BaYMV, indicating that the potential risk of the virus in these areas. This newly established LAMP/RT-LAMP method could be a promising tool for barley protection and food security control.

​RCA-based Detection of Begomoviruses in Weed Ggenera Associated with Legumes in Southern Karnataka

2021 ◽  
Author(s):  
Sudeep Pandey ◽  
T.R. Girish ◽  
S. Basavaraj ◽  
A.S. Padmaja ◽  
N. Nagaraju
Keyword(s):  
Mosaic Virus ◽  
Rolling Circle Amplification ◽  
Mosaic Disease ◽  
Yellow Mosaic Virus ◽  
Insect Vector ◽  
Weed Species ◽  
Ageratum Conyzoides ◽  
Yellow Mosaic Disease ◽  
Mungbean Yellow Mosaic Virus ◽  
Legume Crops

Background: Yellow mosaic disease (YMD) caused by begomoviruses transmitted through the insect vector Bemisia tabaci poses a serious threat to the production of legume crops. Methods: Season-long surveys were carried out for YMD occurrence in six different legume crops and associated natural weeds both symptomatic and asymptomatic across the districts of southern Karnataka, India. The samples were analyzed through RCA PCR using specific primer pairs. Result: Up to 94.1 per cent YMD incidence was recorded and nine weed species were commonly found associated with legume crops. The weeds viz., Ageratum conyzoides, Alternanthera sessilis, Commelina benghalensis and Euphorbia geniculata were abundantly found in the surveyed regions. The weeds were both symptomatic and asymptomatic. Rolling circle amplification coupled polymerase chain reaction method was employed to detect yellow mosaic virus in asymptomatic weeds. Phylogenetic analysis based on the sequences of PCR amplified products of weeds and symptomatic legumes revealed a close clustering of the weed samples with horsegram yellow mosaic virus, legume yellow mosaic virus and mungbean yellow mosaic virus. Overall, our data suggests the role of weed species associated with legume crops as alternative/collateral hosts of begomoviruses and their role in the epidemiology of yellow mosaic disease.

scholarly journals Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

2020 ◽  
Vol 48 (1) ◽  
Author(s):  
Hiroka Aonuma ◽  
Itoe Iizuka-Shiota ◽  
Tokio Hoshina ◽  
Shigeru Tajima ◽  
Fumihiro Kato ◽  
...  
Keyword(s):  
Virus Strain ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Virus Disease ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Simple Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Virus Strains

Abstract Background Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries. Results Common primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present. Conclusions The RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

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