Real-time PCR assay for the discrimination and quantification of wheat and barley strains of Wheat dwarf virus

Gadiou, S.; Ripl, Jan; Jaňourová, B.; Jarošová, Jana; Kundu, Jiban Kumar

doi:10.1007/s11262-011-0699-0

Cited by 26 publications

(17 citation statements)

References 28 publications

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“…Hence, it further confirms the validation for duplex RT-qPCR set up in this study. Also these results support the necessity of the correct choice of reference genes for valid experimental data as reported elsewhere [28,29]. …”

Section: Resultssupporting

confidence: 89%

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Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR

Zhang

Mar

Liu

et al. 2013

Virol J

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BackgroundThe diseases caused by Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) have been occurring epidemically in China and southeastern Asia in recent years. A sensitive, reliable and quantitative method is required to detect and distinguish for RBSDV and SRBSDV in rice and vector insects.ResultsWe developed a sensitive and lineage-specific duplex real time RT-qPCR for detection of RBSDV and SRBSDV in a single or/and double infection in rice samples. The duplex RT-qPCR was optimized using standard samples transcribed by T7 Large Scale RNA Production System in vitro. We developed a reliable system for duplex RT-qPCR, in which its co-efficiency of RBSDV and SRBSDV, were 91.6% and 90.7%, respectively. The coefficient of determination was more than 0.990; the slope of linear equation was −3.542, and −3.567, respectively. Out of 30 samples collected in North and Central China, which were suspected to be infected with these two viruses, 10 samples were detected RBSDV positive by RT-PCR and 12 samples by RT-qPCR. No mixed infections were found. Simultaneously, out of total 60 samples collected from Southern China, which were also suspected to be infected with these two viruses, 41 samples were determined SRBSDV positive by RT-PCR and 47 samples by RT-qPCR. Also in this case no mixed infections were found. The rice genes eEF-1a and UBQ5 were selected as internal controls for quantification assay also performed as good expression stability.ConclusionThe duplex RT-qPCR assay provided as a sufficiently sensitive, specific, accurate, reproducible and rapid tool for the detection and differentiation of RBSDV and SRBSDV. The RT-qPCR assay can be used in routine diagnostic of these two viruses in order to study the disease epidemiology in rice crops.

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Section: Resultssupporting

confidence: 89%

“…In RT-qPCR used in discrimination and quantification of several viruses [20,24,29], the Ct value is a parameter reflecting the quantity of template present in the reaction. Usually, lower Ct values indicate a high concentration of template and higher Ct values indicate a low concentration of template [30].…”

Section: Discussionmentioning

confidence: 99%

Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR

Zhang

Mar

Liu

et al. 2013

Virol J

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show abstract

“…In RT-qPCR assay, the Ct value is a parameter reflecting the quantity of template presented in the reaction [18,22,26]. Usually, lower Ct values indicate a high concentration of template and higher Ct values indicate a low concentration of template [27].…”

Section: Discussionmentioning

confidence: 99%

A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus(WYMV)

Liu

Zhao

Zhang

et al. 2013

Virol J

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BackgroundWheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases.MethodsIn this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and β-actin were selected in order to adjust the veracity of the real-time RT-PCR assay.ResultsWe developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: β-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV.ConclusionsThe Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods.

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“…Existing strand-specific PCR protocols developed for circular ssDNA viruses are not quantitative717 and established quantitative PCR (qPCR) protocols do not discriminate between the two viral strands181920212223. We anticipate that quantitative determination of VS and CS strands during infection will provide important insights into circular ssDNA viral replication and infection dynamics.…”

mentioning

confidence: 99%

A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

Rodríguez-Negrete

Sánchez-Campos

Cañizares

et al. 2014

Sci Rep

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Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules.

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Real-time PCR assay for the discrimination and quantification of wheat and barley strains of Wheat dwarf virus

Cited by 26 publications

References 28 publications

Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR

Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR

A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus(WYMV)

A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

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