The Adaptation of Silica Capture RT-PCR for the Detection of Potato Virus Y

Zacharzewska, Bogumiła; Przewodowska, Agnieszka; Treder, Krzysztof

doi:10.1007/s12230-014-9383-y

Cited by 9 publications

(12 citation statements)

References 39 publications

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“…Thus this method has the potential to be applied as point-of-care method De Boer and Lopez 2012). However, PVY detection by RT-LAMP requires prior isolation of RNA from tested samples, which extends the time required for the assay by at least half an hour, depending on the method of RNA isolation (Zacharzewska et al 2014) and makes the assay difficult under field conditions. RT-LAMP was reported to be less sensitive to inhibitory compounds than RT-PCR (Kaneko et al 2007); thus it is possible that in the RT-LAMP workflow, RNA isolation may be omitted or significantly simplified.…”

Section: Discussionmentioning

confidence: 99%

“…RT-PCR was carried out according to the conditions optimized previously (Zacharzewska et al 2014), except that the primer set Y5 was replaced with the RT-LAMP outer primers (F3 and B3, Table 1) to target the PVY genome segment amplified in the RT-LAMP assay. Reverse transcription reactions were carried out with the Reverse Super Verte KIT with random primers (Novazym Poland) following the manufacturer's protocol.…”

Section: Rt-pcrmentioning

confidence: 99%

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

et al. 2015

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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-topositive 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. 1000 veces más sensible que DAS ELISA y que el ensayo de flujo lateral, y como 10 veces más sensible que RT-PCR. La efectividad de RT-LAMP al probarla en plantas en el campo se comparó favorablemente con DAS ELISA y RT-PCR. En consecuencia, presenta un gran potencial para detección de rutina del PVY.

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Section: Discussionmentioning

confidence: 99%

Section: Rt-pcrmentioning

confidence: 99%

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

et al. 2015

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“…For the silicon dioxide-based isolation, silica suspension, as well as buffers L2 and L6, was prepared according to the recipe (Boom et al 1990) with some modifications (Zacharzewska et al 2014). For the silica suspension, silica powder (Honeywell/Fluka S5631) was used.…”

Section: Dna Isolation Methodsmentioning

confidence: 99%

“…In this paper, we would like to present a developed and optimized simple DNA purification method for fungi, using silica powder (Boom et al 1990) and later adapted for other research (Li et al 2010;Malinowski 1997;Zacharzewska et al 2014). In general, the method based on silica particles relies on nucleic acids binding to the silica surface in the presence of chaotropic salts, like guanidinium thiocyanate (GuSCN) or guanidinium hydrochloride (GuHCl) (Boom et al 1990;Li et al 2010).…”

Section: Introductionmentioning

confidence: 99%

“…In general, the method based on silica particles relies on nucleic acids binding to the silica surface in the presence of chaotropic salts, like guanidinium thiocyanate (GuSCN) or guanidinium hydrochloride (GuHCl) (Boom et al 1990;Li et al 2010). This approach proves to be versatile because it was used to purify plasmid DNA from E. coli, for detection of the potato virus Y or in isolating DNA from blood consumed by mosquitoes (Guelbéogo et al 2018;Li et al 2010;Zacharzewska et al 2014). In contrast to highly optimized and efficient but expensive commercial kits, the silica-based method can be easily adapted to individual sub-protocols.…”

Section: Introductionmentioning

confidence: 99%

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Isolation of nucleic acids using silicon dioxide powder as a tool for environmental monitoring

Urbaniak¹,

Janowski

Jacewski³

2019

Environ Monit Assess

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Isolation and purification of nucleic acids are basic laboratory procedures used in molecular analysis supporting determination of organisms in environmental monitoring. However, many different methods of isolation are commonly used, often being designed for a particular type of DNA extraction. While researchers commonly decide on commercial isolation kits for their ease of use and efficiency, they require large amounts of studied tissue, and the cost of purchasing such kits over a long run can be high. To provide an alternative to using commercial kits, we have developed a simple, rapid, cost-effective, and reliable protocol for DNA isolation from cultured fungi on slants and from dried fungal samples using silica particles (silicon dioxide powder) in chaotropic conditions. With the presented method, it is possible to isolate good-quality DNA from fungi in less than 1.5 h, using easily accessible chemicals. Compared with other methods employing CTAB or commercial kits, it allows fast, easy, and cheap DNA purification from two main sources of fungi routinely used for research. In addition to the method protocol, we also provide advice for further optimization of the isolation process to account for specific conditions, making the procedure more useful.

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Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes

et al. 2017

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Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

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The Adaptation of Silica Capture RT-PCR for the Detection of Potato Virus Y

Cited by 9 publications

References 39 publications

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

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

Isolation of nucleic acids using silicon dioxide powder as a tool for environmental monitoring

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes

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