A universal method for direct PCR amplification of plant tissues

2017 ◽  
Vol 9 (11) ◽  
pp. 1800-1805 ◽  
Author(s):  
Yuping Li ◽  
Huanhuan Zhao ◽  
Xuefen Yan ◽  
Meng Li ◽  
Peng Chen ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Dna Purification ◽  
Plant Tissues ◽  
Universal Method ◽  
Direct Pcr ◽  
Modern Biology

PCR is a vital tool in modern biology; however, it can be costly owing to the price of commercial DNA purification kits.

scholarly journals Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in Vitis vinifera tissues

OENO One ◽  
2014 ◽  
Vol 48 (4) ◽  
pp. 261 ◽  
Author(s):  
Katia Gindro ◽  
Nicole Lecoultre ◽  
Luca Molino ◽  
Jean-Pierre De Joffrey ◽  
Sylvain Schnee ◽  
...  
Keyword(s):  
Vitis Vinifera ◽  
Downy Mildew ◽  
Fungal Pathogens ◽  
Pcr Amplification ◽  
Microscopic Analysis ◽  
Grey Mould ◽  
Dna Purification ◽  
Direct Pcr ◽  
Fungal Propagules ◽  
Pcr Method

<p style="text-align: justify;"><strong>Aims</strong>: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples.</p><p style="text-align: justify;"><strong>Methods and results</strong>: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for <em>Erysiphe necator</em>, <em>Plasmopara viticola</em>, <em>Botrytis cinerea</em> and <em>Vitis vinifera</em>. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for <em>P. viticola</em> to one conidium for <em>E. necator</em>. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of <em>E. necator</em> and<em> B. cinerea</em> in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of <em>P. viticola</em>.</p><p style="text-align: justify;"><strong>Conclusion</strong>: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations.</p>

scholarly journals COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

2013 ◽  
Vol 55 (6) ◽  
pp. 401-406 ◽  
Author(s):  
Masashi Miura ◽  
Chihiro Tanigawa ◽  
Yoshito Fujii ◽  
Satoshi Kaneko
Keyword(s):  
Dna Polymerase ◽  
Genomic Dna ◽  
Dna Polymerases ◽  
Pcr Amplification ◽  
Dna Purification ◽  
Blood Components ◽  
Direct Pcr ◽  
Taq Polymerase ◽  
Serological Studies ◽  
Original Amount

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

A rapid and universal method for isolating starch granules in plant tissues

2019 ◽  
Vol 42 (12) ◽  
pp. 3355-3371
Author(s):  
Liangjie Niu ◽  
Huiying Ding ◽  
Ruiqi Hao ◽  
Hui Liu ◽  
Xiaolin Wu ◽  
...  
Keyword(s):  
Plant Tissues ◽  
Universal Method ◽  
Starch Granules

scholarly journals Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ ® swabs

2018 ◽  
Vol 32 ◽  
pp. 80-87 ◽  
Author(s):  
Angie Ambers ◽  
Rachel Wiley ◽  
Nicole Novroski ◽  
Bruce Budowle
Keyword(s):  
Pcr Amplification ◽  
Direct Pcr

A modified direct PCR amplification method using the GlobalFiler™ PCR Amplification Kit on bloodstains collected using microFLOQ™ direct swabs

2019 ◽  
Vol 7 (1) ◽  
pp. 30-32
Author(s):  
Yongxun Wong ◽  
Boon Kiat Ng ◽  
Kevin Wai Yin Chong ◽  
Wei Siong Holden Lim ◽  
Afiqah Razanah Rosli ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Direct Pcr ◽  
Amplification Method

scholarly journals Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA

2017 ◽  
Vol 114 (36) ◽  
pp. 9623-9628 ◽  
Author(s):  
Mark Kowarsky ◽  
Joan Camunas-Soler ◽  
Michael Kertesz ◽  
Iwijn De Vlaminck ◽  
Winston Koh ◽  
...  
Keyword(s):  
Human Body ◽  
Sequence Data ◽  
Human Microbiome ◽  
Pcr Amplification ◽  
The Body ◽  
Direct Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Novel Taxa ◽  
Circulating Cell Free Dna

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

scholarly journals Direct PCR amplification of HCV RNA from human serum.

1992 ◽  
Vol 1 (4) ◽  
pp. 291-292 ◽  
Author(s):  
A Ravaggi ◽  
D Primi ◽  
E Cariani
Keyword(s):  
Human Serum ◽  
Pcr Amplification ◽  
Hcv Rna ◽  
Direct Pcr

scholarly journals Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1042-1048 ◽  
Author(s):  
C. L. Trout ◽  
J. B. Ristaino ◽  
M. Madritch ◽  
T. Wangsomboondee
Keyword(s):  
Late Blight ◽  
Phytophthora Infestans ◽  
Pcr Amplification ◽  
Accurate Method ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Direct Pcr ◽  
Field Samples ◽  
Pcr Products ◽  
Oomycete Pathogen

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

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