scholarly journals New PCR Primer Pairs Specific for Cryptococcus neoformans Serotype A or B Prepared on the Basis of Random Amplified Polymorphic DNA Fingerprint Pattern Analyses

1999 ◽  
Vol 37 (2) ◽  
pp. 315-320 ◽  
Author(s):  
Francisco Hideo Aoki ◽  
Tamae Imai ◽  
Reiko Tanaka ◽  
Yuzuru Mikami ◽  
Hideaki Taguchi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Primers ◽  
Dna Fingerprint ◽  
Serotype A ◽  
Specific Pcr ◽  
Fingerprint Pattern ◽  
Serotype B ◽  
Pcr Primer

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respectiveC. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

scholarly journals Random amplified polymorphic DNA (RAPD) analysis of Ornithobacterium rhinotracheale strains isolated from chickens in Turkey

2012 ◽  
Vol 50 (No. 12) ◽  
pp. 526-530 ◽  
Author(s):  
G. Ozbey ◽  
Ertas HB ◽  
A. Muz
Keyword(s):  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Random Primer ◽  
Serotype A ◽  
Dna Assay ◽  
Rapd Profile ◽  
Rapd Pattern ◽  
Serotype B ◽  
Dna Profiles ◽  
Ornithobacterium Rhinotracheale

Six field strains of Ornithobacterium rhinotracheale isolated from chickens in Elazig province located in the East of Turkey were typed by serotyping and random amplified polymorphic DNA assay using a random primer (OPG-11). Using the AGP test used for serotyping, serotype A was found to be the predominant serotype, only one strain was serotyped as serotype B. By RAPD assay, the tested ORT strains were found to have different RAPD profiles. In addition, the RAPD assay showed almost similar DNA profiles among the tested strains of the serotypes A, B, D and E. The strain of serotype C did give a different RAPD profile. Within strains of the same serotype (A), different profiles were found but the strain of serotype (B) had an identical profile as strains of serotype A. This study suggests that more genotypes of ORT strains are present within the same serotype and thus that no relationship exists between the RAPD pattern of ORT and their serotype.

scholarly journals Serotype, mating type and ploidy of Cryptococcus neoformans strains isolated from patients in Brazil

2002 ◽  
Vol 44 (6) ◽  
pp. 299-302 ◽  
Author(s):  
Misako OHKUSU ◽  
Naomi TANGONAN ◽  
Kanji TAKEO ◽  
Eriko KISHIDA ◽  
Masami OHKUBO ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mating Type ◽  
Sao Paulo ◽  
São Paulo ◽  
Aids Patients ◽  
Serotype A ◽  
Tertiary Teaching ◽  
Tertiary Teaching Hospital ◽  
Serotype B ◽  
Alpha Type

Serotype, mating type and ploidy of 84 strains of Cryptococcus neoformans isolated from 61 AIDS and 23 non-AIDS patients admitted in a tertiary teaching hospital in São Paulo, Brazil were examined. Among 61 strains isolated from AIDS patients, 60 strains were var. grubii (serotype A). Only one strain was var. gattii (serotype B). No var. neoformans (serotype D) was found. Among 23 strains isolated from non-AIDS patients, 15 were var. grubii (serotype A) and the remaining 8 were var. gattii, all of which were serotype B. Seventy-three of the 75 serotype A strains were the heterothallic alpha type (MATalpha) and the remaining 2 were untypable (asexual). Most of the MATalpha strains (69/73) were haploid and the remaining 4 strains were diploid. Similarly, both of the 2 asexual strains among the 75 serotype A strains were haploid. There were no alpha-mating type (MATalpha) strains among the 84 isolates. All of the 8 var. gattii strains were serotype B and haploid. Among a total of 84 strains tested, neither serotype AD nor serotype D were found. Neither triploid nor tetraploid were found. These results suggest that the serological, sexual and ploidy characteristics in C. neoformans strains isolated from AIDS patients in São Paulo were rather simple, whereas strains isolated from non-AIDS patients presented serotype A and B with predominance of serotype A.

scholarly journals Structural and Genetic Analyses of O Polysaccharide from Actinobacillus actinomycetemcomitans Serotype f

2001 ◽  
Vol 69 (9) ◽  
pp. 5375-5384 ◽  
Author(s):  
Jeffrey B. Kaplan ◽  
Malcolm B. Perry ◽  
Leann L. MacLean ◽  
David Furgang ◽  
Mark E. Wilson ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Pcr Primers ◽  
Cross Reactivity ◽  
Actinobacillus Actinomycetemcomitans ◽  
Molar Ratio ◽  
African American Female ◽  
Specific Pcr ◽  
Serotype B ◽  
Transposon Insertions

ABSTRACT The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed ofl-rhamnose and 2-acetamido-2-deoxy-d-galactose (molar ratio, 2:1) with the structure →2)-α-l-Rhap-(1–3)-2-O-(β-d-GalpNAc)-α-l-Rhap-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903φkan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five knownA. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitansserotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

scholarly journals Forgery Detection Beef with Mice Meat (Mus musculus) in Meatballs Using Real-Time Polymerase Chain Reaction (Real-Time PCR) Primer Specific for a Target Mitochondrial DNA ND-1 Gene

2019 ◽  
Vol 19 (1) ◽  
pp. 89
Author(s):  
Tri Joko Raharjo ◽  
Gilang Aji Pratama ◽  
Irma Nuryanti ◽  
Rarastoeti Pratiwi
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Accurate Method ◽  
Good Precision ◽  
Isolation Technique ◽  
Specific Pcr ◽  
Dna Template ◽  
Pcr Primer

The expensive beef have encouraged counterfeiting beef on processed food products such as meatballs. Mice meat is frequently reported used for adulteration of beef. The accurate method is needed to ensure the supervision of food safety. This study reports the use of DNA testing to detect the presence of mice meat in meatballs with real-time PCR primer specific. PCR primers designed based on the ND-1 gene of mice mitochondrial DNA with the sequence are 5’-CGGCATCCTACAACCATTTGC-3’ and 5’-CGGCTCGTAAAGCTCCGAA-3’, respectively, target 294 bp DNA fragment. The real-time PCR can specifically detect the presence of the mice meat in a meatball with no detection the presence of beef, mutton, chicken, pork, and horsemeat. The method showed good precision shown by the CV of repeatability test at 2%, much lower than the requirement of < 25%. Real-time PCR was able to deliver positive results for as low as 0.5 ng DNA template, equivalent to 0.08 copies of genome DNA of mice equal to 80–150 copies of mtDNA. By using standard phenol-chloroform DNA isolation technique, this method is able to detect contamination of mice meat in meatball up to 1%. Three commercial meatballs confirmed contaminated by mice meat using the method.

scholarly journals Genetic Analysis of Histoplasma capsulatum Strains Isolated from Clinical Specimens in Thailand by a PCR-Based Random Amplified Polymorphic DNA Method

1998 ◽  
Vol 36 (10) ◽  
pp. 3073-3076 ◽  
Author(s):  
Natteewan Poonwan ◽  
Tamae Imai ◽  
Nanthawan Mekha ◽  
Katsukiyo Yazawa ◽  
Yuzuru Mikami ◽  
...  
Keyword(s):  
Reference Strain ◽  
Histoplasma Capsulatum ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Primers ◽  
Dna Fingerprint ◽  
Sequence Information ◽  
Clinical Specimens ◽  
American Strain ◽  
The Difference

Thirteen strains of Histoplasma capsulatum were isolated from clinical specimens, including those from AIDS patients, in Thailand. Random amplified polymorphic DNA (RAPD) analysis with three different PCR primers showed that the DNA fingerprint patterns of the Thai isolates were very similar to each other and homogeneous, with only one exceptional strain, although the patterns were clearly different from those of a reference North American strain with all primers tested. Although the difference in the DNA fingerprinting patterns was minor, Thai isolates could be classified into two to four groups. A common PCR band (about 700 bp) in the patterns of allH. capsulatum strains was extracted, and its DNA sequence was determined. A new PCR primer set for the identification of H. capsulatum species was developed based on this sequence information. This primer set was 100% successful in the identification of the reference strain as well as all Thai isolates. The results of specificity tests of the primer set for the identification of the fungus are also discussed.

Molecular identification of some Hoplolaimus species from the USA based on duplex PCR, multiplex PCR and PCR-RFLP analysis

Nematology ◽  
2009 ◽  
Vol 11 (3) ◽  
pp. 471-480 ◽  
Author(s):  
Robert Robbins ◽  
Allen Szalanski ◽  
Chang-Hwan Bae
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Rflp Analysis ◽  
Pcr Primers ◽  
Interspecific Variation ◽  
Molecular Techniques ◽  
Specific Pcr ◽  
Pcr Multiplex ◽  
Pcr Rflp ◽  
Species Specific

AbstractTwo different molecular approaches, a multiplex PCR and PCR-RFLP of ITS-rDNA, were developed for the identification of Hoplolaimus species. DNA sequences of H. columbus, H. galeatus, H. concaudajuvencus, H. magnistylus, H. seinhorsti and three undescribed Hoplolaimus species were used to design species-specific primers. Three reverse species-specific PCR primers for H. columbus, H. galeatus and H. magnistylus were developed using the ITS1 region exhibiting interspecific variation. Three species-specific PCR primers in combination with the forward primer, Hoc-1f, produced distinct amplicons of 580 bp for H. columbus, 120 bp for H. galeatus and 340 bp for H. magnistylus. We successfully identified each of three species by multiplex PCR when all three were mixed in a single PCR reaction. Restriction enzyme digests of the PCR amplicon using HaeIII and RsaI permitted discrimination of H. columbus, H. galeatus, H. magnistylus, H. concaudajuvencus, H. sp. 1, H. sp. 2 and H. sp. 3 from each other. These results suggest that these molecular techniques allow for rapid, easy and reliable identification of Hoplolaimus species.

scholarly journals Presence of α and a Mating Types in Environmental and Clinical Collections of Cryptococcus neoformans var. gattii Strains from Australia

1999 ◽  
Vol 37 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
C. L. Halliday ◽  
T. Bui ◽  
M. Krockenberger ◽  
R. Malik ◽  
D. H. Ellis ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mating Type ◽  
Causative Agent ◽  
Pcr Primers ◽  
Mating Types ◽  
Limited Data ◽  
Environmental Isolates ◽  
Specific Pcr ◽  
Sexual Recombination ◽  
Mating Type Genes

Cryptococcus neoformans var. gattii lives in association with certain species of eucalyptus trees and is a causative agent of cryptococcosis. It exists as two mating types, MATα and MATa, which is determined by a single-locus, two-allele system. In the closely related C. neoformansvar. neoformans, the α mating type has been found to outnumber its a counterpart by at least 30:1, but there have been very limited data on the proportions of each mating type in C. neoformans var. gattii. In the present study, specific PCR primers were designed to amplify two separate α-mating-type genes from C. neoformans var.gattii strains. These were used to survey for the presence of the two mating types in clinical and environmental collections ofC. neoformans var. gattii strains from Australia. Sixty-eight of 69 clinical isolates produced both α mating type-specific bands and were assumed to be of the α mating type. The majority of environmental isolates were also of the α mating type, but the a mating type was located in two separate areas. In one area, the a mating type outnumbered the α mating type by 27:2, but in the second area, the ratio of the two mating types was close to the 50:50 ratio expected for sexual recombination.

The conversion of wheat RFLP probes into STS markers via the single-stranded conformation polymorphism technique

Genome ◽  
2003 ◽  
Vol 46 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Per-Olov Forsström ◽  
Robert Koebner ◽  
Arnulf Merker
Keyword(s):  
Powdery Mildew ◽  
Dna Sequences ◽  
Pcr Primers ◽  
Single Copy ◽  
General Strategy ◽  
Nucleotide Polymorphisms ◽  
Single Stranded Conformation Polymorphism ◽  
Specific Pcr ◽  
The Individual ◽  
Conformation Polymorphism

We describe a flexible and general strategy for converting a wheat RFLP-based assay into a PCR-based sequence-tagged site (STS), and have applied it to derive markers for a powdery mildew resistance gene present in a wheat–rye translocation. The concept is based on deriving PCR primers that amplify all of the homoeoloci defined by a single-copy cDNA sequence, and separating the resulting mixture of homoeoamplicons via single-stranded conformation polymorphism (SSCP) gels, which are able to detect minor differences between related DNA sequences. After their separation, the individual homoeoamplicons were sequenced and these were used to define nucleotide polymorphisms that could be exploited to design locus-specific PCR primers. In one case, we were able to demonstrate that the assay was allele specific.Key words: wheat–rye introgression, powdery mildew, RFLP, SSCP, STS.

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