A rapid and cheap protocol for preparation of PCR templates in peanut

Wang, Chuan Tang; Wang, Xiu Zhen; Tang, Yu Lan; Zhang, Jian Cheng; Yu, Shan; Xu, Jian; Bao, Zhen Min

doi:10.2225/vol12-issue2-fulltext-11

Cited by 11 publications

(11 citation statements)

References 3 publications

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“…PCR template was prepared from leaflets by using a simple protocol for groundnut developed recently in our laboratory with minor modification (Wang et al 2009). Briefly, small leaflet discs (6.5 mm 2 diam.)…”

Section: Preparation Of Pcr Templatesmentioning

confidence: 99%

Phylogeny of Arachis based on internal transcribed spacer sequences

Wang

Wang²,

Tang³

et al. 2010

Genet Resour Crop Evol

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Phylogenetic analysis, based on nuclear rDNA internal transcribed spacers (ITS) of Arachis species, corroborated a broad sub-classification of the genus. Three clustering algorithms were used to generate dendrograms which showed that Arachis Sections Extranervosae, Heteranthae and Triseminata were most primitive, and Section Arachis was most advanced, with Sections Caulorrhizae, Erectoides, Procumbentes, Rhizomatosae, and Trierectoides intermediate in evolutionary terms, in relation to the genus Stylosanthes, when it was used as the outgroup.

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Section: Preparation Of Pcr Templatesmentioning

confidence: 99%

Phylogeny of Arachis based on internal transcribed spacer sequences

Wang

Wang²,

Tang³

et al. 2010

Genet Resour Crop Evol

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“…[2][3][4]. The disadvantages of PCR/RT-PCR for pathogen detection are the dependence on a thermocycler, inhibitory effects of co-extracted host plant inhibitors on amplification, and the time investment per sample [5][6][7]. While PCR/RT-PCR assays are used in the detection of plant viruses in research, the cost and time required are too great for many plant disease clinics.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics

Londoño

Harmon

Polston

2016

Virol J

119

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BackgroundPlant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. An isothermal amplification assay, Recombinase polymerase amplification (RPA), was evaluated for its ability to detect three begomoviruses and for its suitability for use in plant diagnostic clinics. Methods for DNA extraction and separation of amplicons from proteins used in the assay were modified and compared to RPA manufacturer’s protocols. The modified RPA assays were compared to PCR assays for sensitivity, use in downstream applications, cost, and speed.ResultsRecombinase polymerase amplification (RPA) assays for the detection of Bean golden yellow mosaic virus, Tomato mottle virus and Tomato yellow leaf curl virus (TYLCV) were specific, only amplifying the target viruses in three different host species. RPA was able to detect the target virus when the template was in a crude extract generated using a simple inexpensive extraction method, while PCR was not. Separation of RPA-generated amplicons from DNA-binding proteins could be accomplished by several methods, all of which were faster and less expensive than that recommended by the manufacturer. Use of these modifications resulted in an RPA assay that was faster than PCR but with a similar reagent cost. This modified RPA was the more cost effective assay when labor is added to the cost since RPA can be performed much faster than PCR. RPA had a sensitivity approximate to that of ELISA when crude extract was used as template. RPA-generated amplicons could be used in downstream applications (TA cloning, digestion with a restriction endonuclease, direct sequencing) similar to PCR but unlike some other isothermal reactions.ConclusionsRPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0504-8) contains supplementary material, which is available to authorized users.

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“…Bacterial species were determined by sequencing and analysis of 16S Ribosomal Ribonucleic Acid (16S rRNA) as previously described (16), with modification as follows: The genomic DNA was extracted from the 50 mL liquid Brain Heart Infusion (BHI) broth medium according to previous protocols (17,18). The 16S rRNA fragment was amplified using selected universal primers (both forward and reverse) in a GeneAmp Polymerase Chain Reaction (PCR) System 9700 thermal cycler with the following programs: 30 sec at 94°C, 30 sec at 55°C, and 1.5 min at 72°C for 35 cycles.…”

Section: Identification Of Bacteriamentioning

confidence: 99%