Rapid and inexpensive NaOH based direct PCR for amplification of nuclear and organelle DNA from ramie (Boehmeria nivea), a bast fibre crop containing complex polysaccharides

Satya, Pratik; Mitra, Sabyasachi; Ray, Deb Prasad; Mahapatra, B. S.; Karan, Maya; Jana, S.; Sharma, Amit

doi:10.1016/j.indcrop.2013.07.049

Cited by 22 publications

(17 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Despite no variation in amplification patterns between 10X and 20X diluted lysates, the latter was chosen to completely rule out any chance of presence of PCR inhibitors. Earlier also, Satya et al (2013) emphasized that the dilution factor was the most important factor for success of such direct PCR methods [15].…”

Section: Discussionmentioning

confidence: 99%

Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants

et al. 2019

View full text Add to dashboard Cite

BackgroundConventional plant DNA isolation methods are complex, time consuming and require technical expertise. These limitations were overcome using the DNA isolation kits which, however significantly add to the research costs. Hence the present study was aimed to develop a high throughput, rapid and inexpensive method of PCR ready DNA template preparation from plant materials.MethodsConcentration of SDS in lysis buffer, amount of starting material, period and temperature for lysis were optimized for obtaining PCR ready templates from plant materials. The method was tested using RAPD and ITS specific primers for different plant species like rice, wheat, mustard, pea, soybean, pigeonpea, tomato, maize, march lilly, bougainvillea, Indian blanket flower, nerium, petunia, purple pirouette petunia, moses-in-the-cradle, golden cane palm, duranta, periwinkle, chrysanthemum and two xerophytes viz. Dipterygium glaucum and Crotaleria burhia. SSR markers RM18398 and RM26108 showed successful amplification in rice varieties Improved Pusa Basmati 1 and KS Dev 12. The effectiveness of the method was tested using fresh as well as 1 year old tissues. The storability of the lysate was also tested.ResultsIn this report, we developed a novel method called rapid high throughput template preparation (rHTTP) method to prepare PCR ready DNA templates. Most striking feature of this technique is that it can be done anywhere where water can be boiled by any means. Using rHTTP method, PCR ready templates can be prepared in just 10 min. Robust and reproducible amplification for all the test plants were recorded with RAPD, plant ITS primers and SSR markers following this method. rHTTP methods works well for both fresh as well as old plant tissues. The lysates had a shelf life of 1 month when stored at 4 °C and 3 days when stored at room temperature.ConclusionsrHTTP method has several advantages over the other protocols like ease of execution, no requirement of tissue grinding/liquid nitrogen/hazardous chemicals and above all, equally effective for both fresh and old samples. Using this method, costs per prep comes down ~ 10–50 times as compared to most commercial kits. This method can be used for on-field experiments like molecular diagnostics, varietal identification etc.

show abstract

Section: Discussionmentioning

confidence: 99%

Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants

et al. 2019

View full text Add to dashboard Cite

show abstract

“…The pellet was then washed with 70 % ethanol and resuspended in 50 μl of DNA Hydration Solution. Crude extracts were prepared in 0.5 N NaOH from fresh, frozen and desiccated material using a technique modified from Satya et al 2013 [6]. Fresh or frozen leaves (10 mg) were homogenized using a plastic pestle in a 2 ml microfuge tube with 30 μl 0.5 N NaOH.…”

Section: Template Preparationmentioning

confidence: 99%

“…[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

View full text Add to dashboard Cite

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.

show abstract

“…Selain itu, serat rami juga memiliki keunggulan lain seperti resistensi terhadap bakteri dan kekuatan tarik yang lebih tinggi di bawah kondisi higroskopik (Satya et al 2013). Menurut Liu et al (2015) tinggi tanaman rami merupakan faktor penentu utama untuk hasil serat, karena serat rami diekstraksi dari kulit batang sehingga jika batang rami lebih pendek maka hasil serat juga lebih sedikit.…”

Section: Pendahuluanunclassified

Kualitas Kimia Serat beberapa Klon Rami Asal Sumatera Barat

2019

View full text Add to dashboard Cite

<p>Flax/jute is natural fiber plants that that produce fiber from the bark. Ramie fiber has a better quality than the others natural fiber, so it can meet fiber the increase needs cultivar with the best chemical composition of fiber. The aim of this study was to detect the chemical composition of fiber in several clones of ramie from West Sumatera. The study was conducted on December 2018 at the Laboratory of Technology Agriculture Product Andalas University for analyzed the content of chemical composition (water, ash, holocellulose, cellulose, hemicellulose, and lignin). The result of this study showed that the highest level of water content, ash, hemicellulose, and lignin were found in Situjuah clones those are 40.43, 6.74, 14.39, and 9.98% respectively. Whereas for the highest level of holocellulose and cellulose contents were in Ramindo 1 clone those were 70.23 and 58.46% respectively.</p>

show abstract

Rapid and inexpensive NaOH based direct PCR for amplification of nuclear and organelle DNA from ramie (Boehmeria nivea), a bast fibre crop containing complex polysaccharides

Cited by 22 publications

References 20 publications

Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants

Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants

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

Kualitas Kimia Serat beberapa Klon Rami Asal Sumatera Barat

Contact Info

Product

Resources

About