scholarly journals Identification of Eutypa lata by PCR-RFLP

Plant Disease ◽  
2004 ◽  
Vol 88 (9) ◽  
pp. 925-929 ◽  
Author(s):  
P. E. Rolshausen ◽  
F. Trouillas ◽  
W. D. Gubler
Keyword(s):  
Universal Primers ◽  
Phaeomoniella Chlamydospora ◽  
Phomopsis Viticola ◽  
Eutypa Lata ◽  
Incorrect Diagnosis ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Rflp Assay

Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.

scholarly journals RAPID PCR–BASED DETECTION OPTIMIZATION OF PORCINE DNA IN GELATIN CAPSULE SHELL

2018 ◽  
Vol 10 (6) ◽  
pp. 217
Author(s):  
Amalia Sitti Khayyira ◽  
Viktoria Mardhika Estepane ◽  
Amarila Malik
Keyword(s):  
Molecular Detection ◽  
Universal Primers ◽  
Gelatin Capsule ◽  
Duplex Pcr ◽  
Rapid Pcr ◽  
Capsule Shell ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Bovine Species

Objective: This study was conducted to optimize the genomic deoxyribonucleic acid (DNA) based molecular detection of gelatin derived from porcine by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and duplex PCR method employing cyt B gene.Methods: Optimization was carried out for DNA extraction, PCR conditions, and the sensitivity of the PCR-RFLP method. Due to the very low DNA trace in gelatin after the various manufacturing process, the extraction was optimized to obtain sufficient DNA which was visible on the agarose gel. PCR-RFLP was carried out using universal primers and BsaJI restriction enzyme, and duplex PCR was carried out using two sets of porcine-specific primers. Porcine and bovine DNA were mixed in various concentration to confirm sensitivity of both methods, i.e. 100%, 50%, 10%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%Results: Both methods, PCR-RFLP, and Duplex PCR, were able to detect as low as 0.01% porcine DNA, indicated by the presence of porcine DNA amplicon bands (131 bp and 228 bp for PCR-RFLP, 212 bp and 398 bp for duplex PCR). Although DNA bands presented in low intensity, identification of porcine and bovine species and estimation of DNA quantities were possible.Conclusion: Both conventional PCR methods, i.e. PCR-RFLP and Duplex PCR, were sensitive, specific, and suitable as a rapid initial detection method for molecular detection of porcine in gelatin capsule shells.

Levels of Ancylostoma infections and phylogenetic analysis of cox 1 gene of A. ceylanicum in stray cat faecal samples from Guangzhou, China

2015 ◽  
Vol 90 (4) ◽  
pp. 392-397 ◽  
Author(s):  
W. Hu ◽  
X.G. Yu ◽  
S. Wu ◽  
L.P. Tan ◽  
M.R. Song ◽  
...  
Keyword(s):  
South China ◽  
Potential Risk ◽  
Stray Cats ◽  
Faecal Samples ◽  
High Prevalence ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Stray Cat ◽  
A Minor

AbstractAncylostoma ceylanicum is a common zoonotic nematode. Cats act as natural reservoirs of the hookworm and are involved in transmitting infection to humans, thus posing a potential risk to public health. The prevalence of feline A. ceylanicum in Guangzhou (South China) was surveyed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). In total, 112 faecal samples were examined; 34.8% (39/112) and 43.8% (49/112) samples were positive with hookworms by microscopy and PCR method, respectively. Among them, 40.8% of samples harboured A. ceylanicum. Twelve positive A. ceylanicum samples were selected randomly and used for cox 1 sequence analysis. Sequencing results revealed that they had 97–99% similarity with A. ceylanicumcox 1 gene sequences deposited in GenBank. A phylogenetic tree showed that A. ceylanicum isolates were divided into two groups: one comprising four isolates from Guangzhou (South China), and the other comprising those from Malaysia, Cambodia and Guangzhou. In the latter group, all A. ceylanicum isolates from Guangzhou were clustered into a minor group again. The results indicate that the high prevalence of A. ceylanicum in stray cats in South China poses a potential risk of hookworm transmission from pet cats to humans, and that A. ceylanicum may be a species complex worldwide.

scholarly journals Generation of a whole chromosome painting probe from monochromosomal hybrid cells by the alu-polymerase chain reaction

2007 ◽  
Vol 59 (2) ◽  
pp. 89-95 ◽  
Author(s):  
Danijela Drakulic ◽  
Gordana Nikcevic ◽  
Vesna Djordjevic ◽  
Milena Stevanovic
Keyword(s):  
Chromosome Painting ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Rearrangements ◽  
Painting Probe ◽  
Complex Chromosomal Rearrangements ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Complex Chromosome ◽  
Whole Chromosome Painting

Fluorescent in situ hybridization (FISH) has become a widespread technique applicable in basic science and diagnostics. Chromosome painting represents a special application of FISH that has found increasing use in identification of complex chromosome rearrangements. Here we present a version of the Alu-PCR method modified to generate a whole chromosome painting probe (WCP) for human chromosome 19 using monochromosomal cell hybrids. In setting up conditions for this method, we established a cheap and fast approach to generation of WCPs for other human chromosomes that could be particularly useful for unambiguous identification of complex chromosomal rearrangements associated with cancer. .

scholarly journals A PCR Method That Can Be Further Developed into PCR-RFLP Assay for Eight Animal Species Identification

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Feng Guan ◽  
Yu-Ting Jin ◽  
Jin Zhao ◽  
Ai-Chun Xu ◽  
Yuan-Yuan Luo
Keyword(s):  
Species Identification ◽  
Animal Species ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Universal Primers ◽  
Universal Primer ◽  
Routine Identification ◽  
Pcr Rflp ◽  
Fast Processing ◽  
Rflp Assay

There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20 µl PCR mixture. Each PCR product could be further digested into fragments with variable sizes and qualitative analysis by SspI restriction enzyme. This developed PCR-RFLP assay was sufficient to distinguish all targeted species. Compared with the previous published related methods, this approach is simple, with high throughput, fast processing rates, and more cost-effective for routine identification of meat in foodstuffs.

Comparison of SNP-Based Detection Assays for Food Analysis: Coffee Authentication

2014 ◽  
Vol 97 (4) ◽  
pp. 1114-1120 ◽  
Author(s):  
Stelios Spaniolas ◽  
Christos Bazakos ◽  
Gregory A Tucker ◽  
Malcolm J Bennett
Keyword(s):  
Detection Methods ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Advantages And Disadvantages ◽  
Coffee Beans ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Analytical Approaches ◽  
Rflp Assay ◽  
Food Model

Abstract Recently, DNA-based authentication methods were developed to serve as complementary approaches to analytical chemistry techniques. The single nucleotide polymorphism (SNP)-based reaction chemistries, when combined with the existing detection methods, could result in numerous analytical approaches, all with particular advantages and disadvantages. The dual aim of this study was (a) to develop SNP-based analytical assays such as the single-base primer extension (SNaPShotTM) and pyrosequencing in order to differentiate Arabica and Robusta varieties for the authentication of coffee beans and (b) to compare the performances of SNaPshot, pyrosequencing and the previously developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using an Agilent 2100 Bioanalyzer on the basis of linearity (R2) and LOD, expressed as percentage of the adulterant species, using green coffee beans (Arabica and Robusta) as a food model. The results showed that SNaPshot analysis exhibited the best LOD, whereas pyrosequencing revealed the best linearity (R2 = 0.997). The PCR-RFLP assay using the Agilent 2100 Bioanalyzer could prove to be a very useful method for a laboratory that lacks sequencing facilities but it can be used only if a SNP creates/deletes a restriction site.

scholarly journals Susceptibility of Cultivated and Wild Vitis spp. to Wood Infection by Fungal Trunk Pathogens

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1529-1536 ◽  
Author(s):  
Renaud Travadon ◽  
Philippe E. Rolshausen ◽  
Walter D. Gubler ◽  
Lance Cadle-Davidson ◽  
Kendra Baumgartner
Keyword(s):  
Production Systems ◽  
Post Inoculation ◽  
Thompson Seedless ◽  
Phaeomoniella Chlamydospora ◽  
Phomopsis Viticola ◽  
Eutypa Lata ◽  
Eutypa Dieback ◽  
Botryosphaeria Dieback ◽  
Cultivar Susceptibility ◽  
Vitis Spp

Cultivars of European grapevine, Vitis vinifera, show varying levels of susceptibility to Eutypa dieback and Esca, in terms of foliar symptoms. However, little is known regarding cultivar susceptibility of their woody tissues to canker formation. Accordingly, we evaluated the relative susceptibility of V. vinifera cultivars (‘Cabernet Franc’, ‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Merlot’, ‘Riesling’, ‘Petite Syrah’, and ‘Thompson Seedless’) and species or interspecific hybrids of North American Vitis (Vitis hybrid ‘Concord’, V. arizonica ‘b42-26’, V. rupestris × V. cinerea ‘Ill547-1’, and Fennell 6 [V. aestivalis] × Malaga [V. vinifera] ‘DVIT0166’) to canker formation by seven trunk pathogens (Neofusicoccum parvum, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Togninia minima, Phomopsis viticola, Eutypa lata, and an undescribed Eutypa sp.). Susceptibility was based on the length of wood discoloration (LWD) in the woody stems of rooted plants in duplicate greenhouse experiments. Cultivars of V. vinifera and Concord did not vary significantly in susceptibility to N. parvum or L. theobromae (LWD of 21 to 88 mm at 14 weeks post inoculation; P > 0.16), suggesting that they are similarly susceptible to Botryosphaeria dieback. The table-grape Thompson Seedless was most susceptible to P. viticola (mean LWD of 61 mm at 11 months post inoculation; P < 0.0001). V. vinifera cultivars and Concord showed similar susceptibility to the Esca pathogens, Phaeomoniella chlamydospora and T. minima. Susceptibility to E. lata was greatest in V. arizonica b42-26 (mean LWD of 96 mm at 11 months post inoculation; P < 0.03). In fact, all four American Vitis spp. were more susceptible to Eutypa dieback than the V. vinifera cultivars. Our findings suggest that no one cultivar is likely to provide resistance to the range of trunk pathogens but that certain cultivars may be promising candidates for commercially relevant host resistance in grape-production systems where the dominant cultivars are very susceptible.

Application on in situ PCR methods in histochemistry

1995 ◽  
Vol 53 ◽  
pp. 754-755
Author(s):  
Omar Bagasra
Keyword(s):  
Thermostable Enzyme ◽  
Small Sample ◽  
Marker Genes ◽  
Infected Cell Line ◽  
Gene Sequences ◽  
Minute Amount ◽  
In Situ Pcr ◽  
Pcr Method ◽  
Polymerase Chain

Since the publication of the first report regarding the in situ amplification of HIV-1 gag gene in a HIV-l infected cell line in 1990, there has been an explosion of research in the area of in situ PCR. There are over 200 publications describing various forms of in situ gene, identifying various infectious agents, tumor marker genes and other genetic elements of interest, in peer reviewed journals (reviewed in 2-15). The polymerase chain reaction (PCR) method for amplification of defined gene sequences has proved a valuable tool not only for basic researchers but also for clinical scientists. Using even a minute amount of DNA or RNA and choosing a thermostable enzyme from a large variety of sources, one can enlarge the amount of the gene of interest, which can be analyzed and/or sequenced. Thus genes or portions of gene sequences present only in a small sample of cells or small fraction of mixed cellular populations can be examined.

scholarly journals Restriction fragment length polymorphism analysis of isolates of infectious bursal disease viruses from Turkey

2012 ◽  
Vol 48 (No. 12) ◽  
pp. 359-362 ◽  
Author(s):  
G. Ozbes ◽  
Ertas HB ◽  
A. Muzo
Keyword(s):  
Rflp Analysis ◽  
Infectious Bursal Disease ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Vp2 Gene ◽  
Infectious Bursal Disease Viruses ◽  
Pcr Rflp ◽  
Bursal Disease ◽  
Polymerase Chain ◽  
Rflp Assay

Infectious bursal disease Virus (IBDV) specific reverse transcriptase/polymerase chain recation (RT/PCR) positive 40 broiler bursa fabricius samples obtained from a commercially reared flock were investigated for genetic diversity by PCR-RFLP assay. The assay amplifies a 743 bp fragment of the IBDV VP2 gene. The RFLP profiles of 40 of these positive samples were determined using the enzyme MboI. Most of the viruses had the same RFLP with the MboI enzyme. RFLP analysis of the isolates produced two different band profiles. The results of this study showed that little genetic heterogeneity exists among IBDV strains in a infected flock.

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