scholarly journals Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles

2021 ◽  
Vol 22 (7) ◽  
Author(s):  
Muhammad Junaid ◽  
David Guest ◽  
AGUS PURWANTARA
Keyword(s):  
Dna Extraction ◽  
Standard Method ◽  
Organic Waste ◽  
Infected Plant ◽  
Primary Source ◽  
Plant Dna ◽  
Modified Method ◽  
Modified Dna ◽  
Total Plant ◽  
Plant Disease Management

Abstract. Junaid M, Purwantara A, Guest G. 2021. Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles. Biodiversitas 22: 2838-2843. Understanding the biology of the fastidious Basidiomycete Ceratobasidium theobromae occupying host-tissue is essential for plant disease management. Direct pathogen DNA extraction from infected plant tissue avoids the need for isolation in artificial media. We report a modified DNA isolation protocol to obtain total plant DNA designed to overcome DNA extraction and isolation problems caused by infected petioles rich in polysaccharides and phenolic substances as a primary source of gummosis. This study examined and compared total plant DNA isolated from petioles high in polysaccharides and polyphenolic compounds using two methods: conventional CTAB lysis buffer and Kits (the standard method), and a new modified CTAB protocol to address these PCR inhibitors. The modified method resulted in higher quality and quantity of C. theobromae crude DNA and amplified PCR product. The modified method produced large quantities of clear, transparent, aqueous DNA-containing lysate (crude DNA) with a clear separation between the upper crude DNA and organic waste layers. Mean DNA absorbance was 1.80, and the lowest DNA yield was 836.6 ng/µl. With the standard method, the blurred, viscous lysate obtained showed signs of gummosis, with poor separation between layers of crude DNA, polysaccharides, protein, and organic waste layers and low yield. Gel electrophoresis indicated poor quality DNA extract. We conclude that this modified method will be valuable for genetic diversity and disease studies in a range of previously challenging plant tissues and their pathogens.

scholarly journals Quality of DNA for PCR extracted from sunflower with different methods

2021 ◽  
Vol 185 (1) ◽  
pp. 32-42
Author(s):  
S.Z. Guchetl ◽  
◽  
M.L. Zolotavina ◽  
А.А. Grigoryan ◽  
А.V. Golovatskaya ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Standard Method ◽  
Plant Material ◽  
Green Leaves ◽  
Modified Method ◽  
Sunflower Line ◽  
Pcr Method ◽  
Method Of Extraction

Studying of plants DNA with PCR method plays an important role in the agrarian activity. Preceding stage of DNA studying is its extraction from plant material. We studied quality of DNA extracted by the different methods from seeds, seedlings, and green leaves of sunflower. We used an inbred sunflower line as a research object. For analysis we used embryos, dry and presoaked during 24 h seeds, roots, 7-day seedlings, green leaves. DNA was extracted using five methods: 1 – a standard method of extraction (1% CTAB), 2 – a modified method (2% CTAB), 3–4 – extraction with sets (Diamond DNA Plant Kit D, Russia; Lumiprobe, Germany), 5 – extraction from green leaves (2% CTAB with absorbent carbon). Amplification was conducted in thermocycler S1000тм (BioRad, USA). Spectrophotometery was done at scanning spectrophotometer LEKI SS2110UV (Russia). Analyzing all used methods we concluded they allow extracting of DNA from dry seeds, seedlings and green leaves of sunflower with sufficient reliability and repeatability, it is proved by PCR results. We couldn’t extract DNA from roots and presoaked seeds with the modified method (2% CTAB). The most economically profitable is first method (1% CTAB). Due to the results of spectrophotometery, the highest level of DNA clearance can be reached with method 3 – extraction by a set Diamond DNA Plant Kit D from seedlings and method 5 – extraction by 2% СТАВ with absorbent carbon from green leaves. The method 3 is more preferable by time necessary for DNA extraction.

A modified DNA extraction protocol and its utility in seed genetic purity assessment

2011 ◽  
Vol 39 (1) ◽  
pp. 236-242 ◽  
Author(s):  
S.N. Sharma ◽  
V. Kumar ◽  
G. Singh ◽  
R. Sharma
Keyword(s):  
Dna Extraction ◽  
Genetic Purity ◽  
Purity Assessment ◽  
Extraction Protocol ◽  
Modified Dna

Simple modification to improve reliability of copro-DNA examinations for diagnosing Echinococcus multilocularis infections in red foxes

2020 ◽  
Vol 94 ◽  
Author(s):  
T. Irie ◽  
T. Ito ◽  
H. Kouguchi ◽  
K. Uraguchi
Keyword(s):  
Dna Extraction ◽  
Echinococcus Multilocularis ◽  
Epidemiological Studies ◽  
Sequencing Analysis ◽  
Red Foxes ◽  
Simple Modification ◽  
Pcr Inhibitors ◽  
Examination Technique ◽  
Modified Method ◽  
Infection Pressure

Abstract Epidemiological studies of Echinococcus multilocularis infections in definitive hosts require a reliable and economic diagnostic method. In this study, the current copro-DNA examination technique was modified by increasing the faecal amounts tested and adding a step to neutralize the faeces before DNA extraction. Reliability of the modified method was evaluated using rectal faecal samples from red foxes and comparing them with intestinal worms detected using the sedimentation and counting technique (SCT) following necropsy. The modified copro-DNA examination method demonstrated 93.9% sensitivity (138/147) on the SCT. Its detectability increased depending on the worm burden, and the sensitivity was 100% in cases harbouring over 1000 worms. From 111 SCT-negative cases, six (5.4%) were copro-DNA-positive, and all were confirmed as E. multilocularis via sequencing analysis. Five of the remaining 105 SCT-negative cases (4.8%) retained polymerase chain reaction (PCR) inhibitors in the extracted solution, suggesting that approximately 5% of the red fox faeces retained these inhibitors after treatment with the present copro-DNA extraction method. Although further evaluation is needed for faeces deposited in the wild, the present copro-DNA examination technique will help monitor the E. multilocularis prevalence in definitive hosts. When used for detailed evaluations of endemicity (e.g. changes in infection pressure or spread in non-endemic areas), the absence of PCR inhibitors should be confirmed, and multiple trials on faecal subsamples are recommended.

scholarly journals Evaluation of distilled water as buffered water replacement during 3% Giemsa’s solution preparation in Malaria diagnosis

2019 ◽  
Vol 10 (SPL1) ◽  
Author(s):  
Lai Yan Xia ◽  
Hamidah Abu Bakar
Keyword(s):  
Standard Method ◽  
Gold Standard ◽  
Malaria Diagnosis ◽  
Cost Effective ◽  
Peripheral Blood Smear ◽  
Blood Film ◽  
Distilled Water ◽  
Malaria Parasites ◽  
Gold Standard Method ◽  
Modified Method

Malaria is a life-threatening disease which has claimed many lives. Giemsa's stain is the gold standard method in malaria diagnosis. Generally, Giemsa's stain is diluted with buffered water. However, sometimes, it produces poor staining of the blood smears, in which can create a major challenge in detecting and identifying positive malaria parasites in a peripheral blood smear. This can lead to misdiagnosis and mistreatment to a patient. The present study examined the effect of replacing the buffered water to distilled water during the preparation of 3% Giemsa's solution. Blood specimens were collected from selected positive (n=80) and negative (n=300) malaria cases in EDTA tube. The modified method employed distilled water and different concentrations of buffered water for diluting Giemsa’s solution stock. The microscopy observation was performed on each set of blood film stained by both modified and standard Giemsa staining methods by two WHO’s qualified technicians. All Giemsa solutions with different diluents were comparable in detecting malaria parasites in the blood films. There was no difference between distilled water and different concentrations of buffered water. Furthermore, distilled water produced homogeneous staining and clearer background of the blood films, which enables different species of malaria to be identified. The present study demonstrates that the modified staining using distilled water in malaria parasites identification is comparable to the gold standard method. In addition, the modified method is rapid, easily available, cost-effective, and reliable.

scholarly journals RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

BioTechniques ◽  
2020 ◽  
Vol 69 (4) ◽  
pp. 270-280 ◽  
Author(s):  
Mustafa Ahmad Munawar ◽  
Frank Martin ◽  
Anna Toljamo ◽  
Harri Kokko ◽  
Elina Oksanen
Keyword(s):  
Reaction Kinetics ◽  
Dna Extraction ◽  
Pcr Primers ◽  
Sybr Green ◽  
Recombinase Polymerase Amplification ◽  
Pcr Inhibitors ◽  
Plant Dna ◽  
Pcr Assays ◽  
Flanking Regions

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid ‘RPA-PCR couple’ concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9. We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.

scholarly journals DNA extraction technique for different rice varieties grown in Sri Lanka

2015 ◽  
Vol 3 (3) ◽  
pp. 398-401
Author(s):  
Ranganathan Kapilan
Keyword(s):  
Sri Lanka ◽  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Rice Varieties ◽  
Modified Method ◽  
Dna Extraction Methods ◽  
Very High ◽  
Different Parts

Extraction of DNA is very important nowadays in bio-molecular researches. Extracted DNA should be purified and the quality of DNA should also be very high. The objective of the study was to develop a simple efficient method to isolate DNA from the rice varieties in an open laboratory environment, and to eliminate the usage of expensive chemicals and tools. The DNA extraction methods developed by the DNeasy plant kit method supplied by QIAGEN, Cheng et al., Doyle et al. and Michiels et al. were applied to five different rice varieties grown in different parts of Sri Lanka. Based on the quantity and quality of the extracted DNA tested by measuring the absorbance of DNA at 260 nm using Nanodrop® ND-1000 spectrophotometer and measuring the ratio of A260 / A280 and gel running on agarose, the efficiency of the extraction method chosen varied among rice varieties. Among the methods used, the methods introduced by DNeasy plant kit method supplied by QIAGEN and Cheng et al, yielded good and amplifiable quality DNA with satisfactory concentration for all the rice varieties tested. Therefore the modified method of Cheng et al, 1987 could be used to extract DNA from rice varieties instead of the commercially available expensive and hazardous DNeasy plant kit method supplied by QIAGEN.Int J Appl Sci Biotechnol, Vol 3(3): 398-401

STR typing from human faeces: a modified DNA extraction method

2003 ◽  
Vol 1239 ◽  
pp. 917-920 ◽  
Author(s):  
G. Iacovacci ◽  
M. Serafini ◽  
A. Berti ◽  
G. Lago
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Str Typing ◽  
Human Faeces ◽  
Dna Extraction Method ◽  
Modified Dna
Sign in / Sign up

Export Citation Format

Share Document