scholarly journals Qualitative Polymerase Chain Reaction Methods for Detecting Major Food Allergens (Peanut, Soybean, and Wheat) by Using Internal Transcribed Spacer Region

2009 ◽  
Vol 92 (5) ◽  
pp. 1464-1471 ◽  
Author(s):  
Takashi Hirao ◽  
Satoshi Watanabe ◽  
Yusuke Temmei ◽  
Masayuki Hiramoto ◽  
Hisanori Kato
Keyword(s):  
Internal Transcribed Spacer ◽  
Its Region ◽  
Detection Methods ◽  
Target Species ◽  
Internal Transcribed Spacer Region ◽  
Qualitative Polymerase Chain Reaction ◽  
Target Dna ◽  
Allergen Detection ◽  
Specific Amplification ◽  
Polymerase Chain

Abstract Allergen detection methods for peanut, soybean, and wheat were developed by designing PCR primer pairs for specific amplification of a fragment of the internal transcribed spacer (ITS) region reported for Arachis spp. for peanut, Glycine spp. for soybean, and Triticum and Aegilops spp. for wheat. The target species for detection included not only cultivated, but also wild and ancestor species, which were thought to be potentially allergenic. The ability of the resultant primer pairs to detect the target species was verified using genomic DNA extracted from A. hypogaea for peanut and G. max for soybean; T. aestivum, T. turgidum, T. durum, T. aestivum-rye amphidiploid, T. monococcum, T. timopheevi, Ae. speltoides, and Ae. squarrosa for wheat. The LODs were 50500 fg of target DNA, which were comparable to those of the most sensitive PCR methods previously reported. The results from the present work, as well as those from our previous work on buckwheat and kiwifruit, prove that the ITS region, for its high copy number and interspecific diversity, is particularly useful as the target of allergen detection methods.

Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush)

Genome ◽  
1994 ◽  
Vol 37 (4) ◽  
pp. 664-671 ◽  
Author(s):  
Lang Zhuo ◽  
S. L. Sajdak ◽  
R. B. Phillips
Keyword(s):  
Polymerase Chain Reaction ◽  
Intraspecific Variation ◽  
Ribosomal Dna ◽  
Internal Transcribed Spacer ◽  
Lake Trout ◽  
Intergenic Spacer ◽  
Coding Region ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain

Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5′ external spacer region (5′ ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5′ ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3–6 kb from the 18S. Sequencing of a 609-b segment of the 5′ ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5′ ETS using the polymerase chain reaction. No intraspecific variation in this region in lake trout was found when the DNA amplified from this region in 12 individuals from four populations was digested with eight restriction enzymes.Key words: ribosomal DNA, internal transcribed spacer regions, 5′ external spacer region, transcribed spacer, lake trout.

Russulaceous ectomycorrhizae of Abies lasiocarpa and Picea engelmannii

1997 ◽  
Vol 75 (11) ◽  
pp. 1843-1850 ◽  
Author(s):  
G. Kernaghan ◽  
R. S. Currah ◽  
R. J. Bayer
Keyword(s):  
Internal Transcribed Spacer ◽  
Fragment Length Polymorphism ◽  
Picea Engelmannii ◽  
Polymorphism Analysis ◽  
Abies Lasiocarpa ◽  
Tree Line ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Lactarius Deliciosus ◽  
Polymerase Chain

During a 3-year study of the ectomycorrhizal fungi of subalpine forests in the Front Ranges of the Canadian Rockies, species of Russula and Lactarius were conspicuous mycobionts of both erect and krummholz forms of Abies lasiocarpa (Hook.) Nutt. and Picea engelmannii Parry. Morphological identifications of Russulaceous mycorrhizae were confirmed by comparing polymerase chain reaction amplified ribosomal DNA (internal transcribed spacer region) with that of sporocarp tissue. Restriction fragment length polymorphism analysis using AluI, HhaI, HinfI, and RsaI gave a distinctive profile for each of 14 Russulaceous sporocarps and facilitated the identification of five mycorrhizae. Mantles formed by Lactarii (Lactarius alnicola, Lactarins caespitosus, and Lactarius deliciosus var. areolatus) exhibit characteristic laticifers and pigments comparable to the associated sporocarp. Those formed by species of Russula (R. brevipes and R. silvicola) bear distinctive cystidia or sulphovanillin-reactive cells. Key words: ITS, Lactarius, RFLP, Russula, subalpine, tree line.

scholarly journals Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
K. Kageyama ◽  
A. Ohyama ◽  
M. Hyakumachi
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Pcr Amplification ◽  
Its Region ◽  
Pythium Ultimum ◽  
Pythium Species ◽  
Chain Reaction ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

scholarly journals PCR-Based Detection of Cephalosporium gramineum in Winter Wheat

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 437-442 ◽  
Author(s):  
K. L. E. Klos ◽  
L. M. Vásquez-Siller ◽  
H. C. Wetzel ◽  
T. D. Murray
Keyword(s):  
Winter Wheat ◽  
Internal Transcribed Spacer ◽  
Seed Infection ◽  
Wheat Seed ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain ◽  
Cephalosporium Gramineum ◽  
Field Plots

A polymerase chain reaction (PCR) assay was developed amplifying a 496-bp fragment of the internal transcribed spacer region of Cephalosporium gramineum genomic DNA at concentrations of 100 fg/μl. Winter wheat seed and seedlings were collected from field plots where C. gramineum was present. Seed was tested by PCR using 20-seed samples bulked for DNA extraction. Estimates of seed infection, based on isolation of the pathogen on semiselective medium and PCR, were comparable at 0.18 and 0.13% of winter wheat ‘Stephens’ (P = 0.6042), and 0.45 and 0.58% of experimental line WA7970 (P = 0.5636), respectively. PCR differentiated between plants with well-developed symptoms of Cephalosporium stripe and noninoculated plants. Positive PCR was obtained from 22% of asymptomatic leaf blades from inoculated plants. We found no false positives when PCR and C. gramineum isolation on a semiselective medium were performed using tissue from the same leaf. The PCR assay has potential to diagnose Cephalosporium stripe disease prior to the appearance of symptoms. Negative PCR for some samples from which C. gramineum was isolated suggests that C. gramineum may be present below the level of detection in some asymptomatic leaves. This PCR assay may be useful for investigations into C. gramineum infection of wheat.

Phylogenetic Abnalysis of Malaysian Pineapples Cultivars Based on the DNA Sequence of the Internal Transcribed Spacer Region

2013 ◽  
Vol 62 (2) ◽  
Author(s):  
Topik Hidayat ◽  
K. Chandrika ◽  
A. Farah Izana ◽  
S. A. Azman ◽  
W. Alina
Keyword(s):  
Internal Transcribed Spacer ◽  
Genomic Dna ◽  
Its Region ◽  
Biochemical Properties ◽  
Phylogenetic Study ◽  
Evolutionary Relationships ◽  
Parsimony Analysis ◽  
Internal Transcribed Spacer Region ◽  
Phylogenetic Status ◽  
Genetic Pattern

The phylogenetic study was conducted to determine the phylogenetic status and evolutionary relationships among the nine commercial pineapple cultivars using sequences of the internal transcribed spacer (ITS) region. Genomic DNA was extracted, and the ITS region was amplified and sequenced. Parsimony analysis revealed that Malaysian cultivars could be classified into two major groups based on the ITS region. The first group comprised of the cultivars Sarawak Green Local, Gandul, and N36 whereas the second group consisted of the cultivars Josapine, Yankee, Morris Bentanggur, Morris Gajah, MD2 and MD2/T. Several combinations of synapomorphic characters (leaf and fruit) support this classification system, suggesting the ITS region has the ability to determine the phylogenetic status and relationships of pineapple cultivars. Since each group has its own similar genetic pattern and presumably certain specific biochemical properties, the relationships of pineapple cultivars revealed in the phylogenetic tree can be used as a basis for successful hybridizations to generate new pineapple cultivars.

scholarly journals Diagnosing bovine parafilariosis: utility of the cytochrome c oxidase subunit 1 gene and internal transcribed spacer region for PCR detection of Parafilaria bovicola in skin biopsies and serohemorrhagic exudates of cattle

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Andreas W. Oehm ◽  
Alexander Stoll ◽  
Cornelia Silaghi ◽  
Annette Pfitzner-Friedrich ◽  
Gabriela Knubben-Schweizer ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Serological Test ◽  
Its Region ◽  
Effective Control ◽  
Intermediate Hosts ◽  
Internal Transcribed Spacer Region ◽  
Oxidase Subunit ◽  
Skin Biopsies ◽  
Pcr Targeting ◽  
Its Pcr

Abstract Background Parafilaria bovicola (Nematoda: Filariidae) causes cutaneous bleedings in bovine species. Flies serve as intermediate hosts. In recent years, reports on bovine parafilariosis have become more frequent, corroborating the necessity of reliable diagnostic interventions especially since no molecular or serological test has been available. We aimed to establish a polymerase chain reaction assay to detect DNA of P. bovicola in flies, skin biopsies and serohemorraghic exudates of bleeding spots. Methods PCRs targeting the cytochrome c oxidase subunit 1 (cox1) gene and the internal transcribed spacer region (ITS) of the ribosomal RNA gene cluster were evaluated for their diagnostic sensitivity as well as performance and specificity on biopsy and serohemorrhagic exudate samples from P. bovicola-infected cattle. Results Using serohemorrhagic exudates (n = 6), biopsies (n = 2) and flies (n = 1), the PCR targeting the cox1 gene resulted in a gel band of almost 700 bp. Cloning, sequencing, and removal of primer sequences yielded a 649-bp fragment of the P. bovicola cox1 gene. The PCR targeting the ITS region showed a band of about 1100 bp. Cloning, sequencing, and removal of primer sequences resulted in a 1083 bp stretch of the P. bovicola ITS region. Testing samples from presumably affected animals, the cox1-PCR resulted in bands with the expected size and they were all confirmed as P. bovicola by sequencing. In contrast, the ITS-PCR proved to be less sensitive and less specific and additionally amplified the ITS region of Musca domestica or buttercup DNA. When analysing for sensitivity, the cox1-PCR yielded visible bands up to 2 ng of genomic DNA, whereas the ITS-PCR produced bands up to 3 ng. In a plasmid dilution series, the minimum number of target DNA copies was 102 for the cox1-PCR and 101 in the ITS-PCR. Conclusions The evaluated cox1-PCR enables reliable detection of P. bovicola DNA in skin biopsies and serohemorrhagic exudates. This PCR and, to a limited extent, the ITS-PCR, may help evaluate different therapeutic approaches. Furthermore, the cox1-PCR may be useful for epidemiological studies on the geographical distribution of P. bovicola. Further understanding of the epidemiology of this parasite will help develop and implement effective control strategies.

scholarly journals Molecular systematics of Keratinophyton: the inclusion of species formerly referred to Chrysosporium and description of four new species

IMA Fungus ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Roman Labuda ◽  
Andreas Bernreiter ◽  
Doris Hochenauer ◽  
Alena Kubátová ◽  
Hazal Kandemir ◽  
...  
Keyword(s):  
New Species ◽  
Internal Transcribed Spacer ◽  
Large Subunit ◽  
Its Region ◽  
Soil Samples ◽  
Lsu Rdna ◽  
Internal Transcribed Spacer Region ◽  
Its Phylogeny ◽  
Combined Data ◽  
Terminal Cluster

AbstractFour new Keratinophyton species (Ascomycota, Pezizomycotina, Onygenales), K. gollerae, K. lemmensii, K. straussii, and K. wagneri, isolated from soil samples originating from Europe (Austria, Italy, and Slovakia) are described and illustrated. The new taxa are well supported by phylogenetic analysis of the internal transcribed spacer region (ITS) region, the combined data analysis of ITS and the nuclear large subunit (LSU) rDNA, and their phenotype. Based on ITS phylogeny, within the Keratinophyton clade, K. lemmensii is clustered with K. durum, K. hubeiense, K. submersum, and K. siglerae, while K. gollerae, K. straussii and K. wagneri are resolved in a separate terminal cluster. All four new species can be well distinguished from other species in the genus based on phenotype characteristics alone. Ten new combinations are proposed for Chrysosporium species which are resolved in the monophyletic Keratinophyton clade. A new key to the recognized species is provided herein.

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