Use of mitochondrial RNA genes for the differentiation of four Trichinella species by multiplex PCR amplification

2009 ◽  
Vol 83 (2) ◽  
pp. 121-128 ◽  
Author(s):  
R. Blaga ◽  
BaoQuan Fu ◽  
D. Le Rhun ◽  
E. Le Naour ◽  
A. Heckman ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Trichinella Spiralis ◽  
Pcr Amplification ◽  
Mitochondrial Rna ◽  
Mitochondrial Rrna ◽  
Polymerase Chain ◽  
Rna Genes ◽  
Rdna Amplification ◽  
Mitochondrial Rdna

AbstractUntil now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species.

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.

scholarly journals New Male Specific Markers for Hop and Application in Breeding Program

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andreja Čerenak ◽  
Zala Kolenc ◽  
Petra Sehur ◽  
Simon P. Whittock ◽  
Anthony Koutoulis ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Low Cost ◽  
Pcr Amplification ◽  
Breeding Program ◽  
Phenotypic Data ◽  
World Collection ◽  
Crude Sample ◽  
Male Sex ◽  
Male Specific

Abstract Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94–100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing

2013 ◽  
Vol 89 (1) ◽  
pp. 118-123 ◽  
Author(s):  
L. Sadaow ◽  
C. Tantrawatpan ◽  
P.M. Intapan ◽  
V. Lulitanond ◽  
T. Boonmars ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Trichinella Spiralis ◽  
Large Subunit ◽  
Pcr Amplification ◽  
Epidemiological Studies ◽  
Chain Reaction ◽  
Target Region ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Zoonotic Parasites

AbstractNematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

Detection ofTrypanosoma congolenseandTrypanosoma bruceisubspecies by DNA amplification using the polymerase chain reaction

Parasitology ◽  
1989 ◽  
Vol 99 (1) ◽  
pp. 57-66 ◽  
Author(s):  
D. R. Moser ◽  
G. A. Cook ◽  
Diane E. Ochs ◽  
Cheryl P. Bailey ◽  
Melissa R. McKane ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Large Scale ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Detection Methods ◽  
Chain Reaction ◽  
African Trypanosomes ◽  
Polymerase Chain

SUMMARYThe nuclear DNA ofTrypanosoma congolensecontains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA ofTrypanosoma brucei brucei(Sloofet al.1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite ofT. congolenseorT. bruceispp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor inLeishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected withT. congolenseand/orT. bruceispp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

scholarly journals A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (12) ◽  
pp. 2505
Author(s):  
Hiroki Hayashi ◽  
Tsutomu Kishi
Keyword(s):  
Dna Sequences ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Epitope Tagging ◽  
One Step ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The One ◽  
Step Polymerase Chain Reaction

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

Differentiation of isolates in the genus Steinernema (Nematoda: Steinernematidae).by random amplified polymorphic DNA fragments and morphological characters

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J. Liu ◽  
R. E. Berry
Keyword(s):  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Nematode Species ◽  
Morphological Characters ◽  
Dna Fragments ◽  
Undescribed Species ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Polymerase Chain

SUMMARYWe combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.

scholarly journals Our Experiences With B Cell Clonality Analysis Using Multiplex Pcr Amplification in Paraffin-Embedded Tissue

2014 ◽  
Vol 14 (1) ◽  
pp. 5-13
Author(s):  
L. Stanek ◽  
S. Llsova ◽  
D. Tvrdlk
Keyword(s):  
B Cell ◽  
Multiplex Pcr ◽  
Pcr Amplification ◽  
Heteroduplex Analysis ◽  
Clonality Analysis ◽  
Polymerase Chain ◽  
Igh Rearrangement ◽  
Formalin Fixed ◽  
Cell Clonality

Abstract The clonal determination of B-cell lymphoproliferative diseases by immunoglobulin heavy chain (IgH) rearrangement by polymerase chain reaction (PCR) is widely used. In this study we report our experiences with B cell clonality analysis using multiplex PCR amplification followed by heteroduplex analysis, which was found to be essential for the efficient resolution of monoclonal bands within polyclonal backgrounds, in formalin-fixed paraffinembedded tissue.

DNA comparisons of Trypanosoma evansi (Indonesia) and Trypanosoma brucei spp.

Parasitology ◽  
1992 ◽  
Vol 104 (1) ◽  
pp. 67-74 ◽  
Author(s):  
W. T. Artama ◽  
M. W. Agey ◽  
J. E. Donelson
Keyword(s):  
Trypanosoma Brucei ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Consensus Sequence ◽  
Pcr Amplification ◽  
Trypanosoma Evansi ◽  
Base Pairs ◽  
Spliced Leader ◽  
Polymerase Chain ◽  
Minicircle Sequence

SUMMARYTwo clones from separate isolates of Trypanosoma evansi in Indonesia were found by polymerase chain reaction (PCR) analyses to contain 3 different repeated nuclear DNA sequences of Trypanosoma brucei spp: the consensus sequence for a highly repetitive 177 base pairs and the gene repeats encoding procyclin and the spliced leader. In addition, the 994 bp minicircle sequence of one of the clones was determined, and PCR amplification primers specific for minicircles of T. evansi were identified that do not amplify minicircle sequences in the T. brucei spp. clones tested.

Identification of Microorganisms Associated to the Biodegradation of Historic Masonry Structure in San Francisco de Campeche City, México

2012 ◽  
Vol 1374 ◽  
pp. 187-194
Author(s):  
Rocío G. Escamilla Pérez ◽  
Javier Reyes Trujeque ◽  
Tezozomoc Pérez López ◽  
Víctor Monteón Padilla ◽  
Ruth López Alcántara
Keyword(s):  
Microbial Communities ◽  
San Francisco ◽  
Ribosomal Rna ◽  
Pcr Amplification ◽  
Masonry Structure ◽  
Historic Masonry ◽  
Nucleotide Database ◽  
Polymerase Chain ◽  
San Carlos ◽  
Rna Genes

ABSTRACTTropical climate create ideal conditions for the development of microbial communities associated with biodegradation of historic buildings made with stony materials. This is the case of Fort San Carlos, a historic colonial building representative of military tendencies during the XVII century in San Francisco de Campeche City. In this study the Polymerase Chain Reaction (PCR), was used to identify microorganisms related with the biodegradation of its masonry structure. Specific primers for amplification of 16S and 18S ribosomal RNA genes were used for organisms identification by PCR. Amplification products were sequenced and after that compared with GENBANK nucleotide database using-BLASTn. Results indicated that microbial communities associated to biodegradation of the Fort San Carlos are bacteria from the Phyla Cyanobacteria, Proteobacteria and Actinobacteria.

Trypanosoma (Nannomonas) congolense: molecular characterization of a new genotype from Tsavo, Kenya

Parasitology ◽  
1993 ◽  
Vol 106 (2) ◽  
pp. 151-162 ◽  
Author(s):  
P. A. O. Majiwa ◽  
M. Maina ◽  
J. N. Waitumbi ◽  
S. Mihok ◽  
E. Zweygarth
Keyword(s):  
Dna Sequences ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Molecular Level ◽  
Pcr Amplification ◽  
Kinetoplast Dna ◽  
Length Polymorphisms ◽  
New Genotype ◽  
Polymerase Chain

SUMMARYTrypanosoma (Nannomonas) congolense comprises morphologically identical but genetically heterogeneous parasites infective to livestock and other mammalian hosts; three different genotypes of this parasite have been described previously. Restriction enzyme fragment length polymorphisms (RFLPs) in both kinetoplast DNA minicircle and nuclear DNA sequences, and randomly amplified polymorphic deoxyribonucleic acid (RAPD) patterns have been used here to demonstrate the existence of another type of T. (N.) congolense that is genotypically distinct from those that have so far been characterized at the molecular level. A highly repetitive, tandemly arranged DNA sequence and oligonucleotide primers, for use in polymerase chain reaction (PCR) amplification are described, which can be used for specific identification of the trypanosome and its distinction from others within the Nannomonas subgenus.

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