scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for Staphylococcusspp.

2000 ◽  
Vol 38 (12) ◽  
pp. 4351-4355 ◽  
Author(s):  
Javier Yugueros ◽  
Alejandro Temprano ◽  
Beatriz Berzal ◽  
Marı́a Sánchez ◽  
Carmen Hernanz ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Pcr Products ◽  
Dna Fragment ◽  
Encoding Gene ◽  
Staphylococcus Hominis ◽  
Staphylococcus Simulans ◽  
Very High

The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcusspp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers.AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis,Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureusisolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureuscells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genusStaphylococcus, as it is highly specific.

scholarly journals Rapid Identification of Aspergillus Fumigatus Using Βeta-Tubulin and RodletA Genes

2017 ◽  
Vol 5 (7) ◽  
pp. 848-851
Author(s):  
Majid Zarrin ◽  
Zeinab Rashidnia ◽  
Sama Faramarzi ◽  
Lida Harooni
Keyword(s):  
Aspergillus Fumigatus ◽  
Related Species ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Pcr Products ◽  
Associated Sequences ◽  
Dna Fragment ◽  
Β Tubulin

AIM: The main purpose of the present study was to test the β-tubulin and rodletA genes for rapid identification of Aspergillus fumigatus.MATERIALS AND METHODS: Fifty-one A. fumigatus strains including environmental, clinical and reference isolates were tested in this research. PCR was carried out based on βtub and rodA partial gene sequences.RESULTS: A 198 bp DNA fragment was obtained using βtub gene. PCR amplification of the rodA gene resulted in a 313 bp band. The βtub and rodA genes PCR products exhibited a 100% homology with the associated sequences in the GenBank.CONCLUSION: In the present study, we used a PCR approach that was able to discriminate A. fumigatus from other related species within the section Fumigati.

scholarly journals Rapid Identification and Typing ofStaphylococcus aureus by PCR-Restriction Fragment Length Polymorphism Analysis of the aroA Gene

1999 ◽  
Vol 37 (3) ◽  
pp. 570-574 ◽  
Author(s):  
Javier Yugueros Marcos ◽  
Alberto Cascón Soriano ◽  
María Sánchez Salazar ◽  
Carmen Hernanz Moral ◽  
Susana Suárez Ramos ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rapid Identification ◽  
Polymorphism Analysis ◽  
Pcr Products ◽  
Dna Fragment ◽  
Restriction Fragment Length

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 102 CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.

scholarly journals Molecular Cloning and Sequencing of thearoA Gene from Actinobacillus pleuropneumoniaeand Its Use in a PCR Assay for Rapid Identification

1999 ◽  
Vol 37 (5) ◽  
pp. 1575-1578 ◽  
Author(s):  
Carmen Hernanz Moral ◽  
Alberto Cascón Soriano ◽  
María Sánchez Salazar ◽  
Javier Yugueros Marcos ◽  
Susana Suárez Ramos ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Chromosomal Dna ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Products ◽  
Dna Fragment ◽  
Specific Pcr Assay ◽  
Dna Specificity ◽  
K 12

The gene (aroA) of Actinobacillus pleuropneumoniae, serotype 2, encoding 5-enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. A pair of primers from the 5′ and 3′ termini were selected to be the basis for development of a specific PCR assay. A DNA fragment of 1,025 bp was amplified from lysed A. pleuropneumoniaeserotypes 1 to 12 of biovar 1 or from isolated DNA. No PCR products were detected when chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified whenActinobacillus equuli chromosomal DNA was used as a target, which could be easily differentiated by its NAD independence. The PCR assay developed was very sensitive, with lower detection limits of 12 CFU with A. pleuropneumoniae cells and 0.8 pg with extracted DNA. Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis of A. pleuropneumoniae infections.

scholarly journals Analysis of delta-globin gene mutations in Greek cypriots

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1647-1651
Author(s):  
P Trifillis ◽  
A Kyrri ◽  
E Kalogirou ◽  
A Kokkofitou ◽  
P Ioannou ◽  
...  
Keyword(s):  
Globin Gene ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Restriction Enzyme Digestion ◽  
Globin Genes ◽  
Pcr Products ◽  
Direct Mutation ◽  
In Cis ◽  
Greek Cypriots

We recently described four delta-globin gene mutations in Greek Cypriots studied by polymerase chain reaction (PCR) amplification and automated fluorescence-based DNA sequence analysis (Blood 78:3298, 1991). Selective restriction enzyme digestion of PCR products facilitated direct mutation detection. Twenty-eight additional samples from unrelated Cypriots with Hb A2 levels ranging from 0.6% to 3.6% were studied by PCR and showed the following: twelve had the delta 27 (ala-->ser) mutation, one was heterozygous for the delta IVS-2 AG-->GG change, and none had either the delta 116 (arg-->cys) or delta 141 (leu- ->pro) mutations. The remaining samples were divided into two groups: 11 with borderline normal Hb A2 values that were not pursued; and four with abnormal Hb A2 values. The delta-globin genes from these four samples were sequenced and the same four changes identified in each: a C-->T at -199, a C-->T at codon 4 (thr-->ile), a silent C-->T at codon 97, and an AT deletion at position 722 in IVS-2. The codon 4 change abolishes a Ple I site whereas the codon 97 creates an Nla III site, thus facilitating rapid identification. All four changes are in cis position, suggesting that the -199 C-->T, the C-->T at codon 97, and the AT deletion in IVS-2 are neutral polymorphisms present on the codon 4 (thr-->ile) chromosome. DNA haplotype analysis suggests all five delta-globin gene mutant alleles arose independently on different chromosomal backgrounds.

scholarly journals Analysis of delta-globin gene mutations in Greek cypriots

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1647-1651 ◽  
Author(s):  
P Trifillis ◽  
A Kyrri ◽  
E Kalogirou ◽  
A Kokkofitou ◽  
P Ioannou ◽  
...  
Keyword(s):  
Globin Gene ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Restriction Enzyme Digestion ◽  
Globin Genes ◽  
Pcr Products ◽  
Direct Mutation ◽  
In Cis ◽  
Greek Cypriots

Abstract We recently described four delta-globin gene mutations in Greek Cypriots studied by polymerase chain reaction (PCR) amplification and automated fluorescence-based DNA sequence analysis (Blood 78:3298, 1991). Selective restriction enzyme digestion of PCR products facilitated direct mutation detection. Twenty-eight additional samples from unrelated Cypriots with Hb A2 levels ranging from 0.6% to 3.6% were studied by PCR and showed the following: twelve had the delta 27 (ala-->ser) mutation, one was heterozygous for the delta IVS-2 AG-->GG change, and none had either the delta 116 (arg-->cys) or delta 141 (leu- ->pro) mutations. The remaining samples were divided into two groups: 11 with borderline normal Hb A2 values that were not pursued; and four with abnormal Hb A2 values. The delta-globin genes from these four samples were sequenced and the same four changes identified in each: a C-->T at -199, a C-->T at codon 4 (thr-->ile), a silent C-->T at codon 97, and an AT deletion at position 722 in IVS-2. The codon 4 change abolishes a Ple I site whereas the codon 97 creates an Nla III site, thus facilitating rapid identification. All four changes are in cis position, suggesting that the -199 C-->T, the C-->T at codon 97, and the AT deletion in IVS-2 are neutral polymorphisms present on the codon 4 (thr-->ile) chromosome. DNA haplotype analysis suggests all five delta-globin gene mutant alleles arose independently on different chromosomal backgrounds.

scholarly journals Molecular Typing of Staphylococcus aureus Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene

1998 ◽  
Vol 36 (4) ◽  
pp. 1083-1089 ◽  
Author(s):  
John V. Hookey ◽  
Judith F. Richardson ◽  
Barry D. Cookson
Keyword(s):  
Staphylococcus Aureus ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Rflp Pattern ◽  
Methicillin Resistant ◽  
Pcr Products ◽  
Restriction Fragment Length

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested withAluI and CfoI, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus(MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of S. aureus isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.

scholarly journals Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

1997 ◽  
Vol 87 (12) ◽  
pp. 1192-1196 ◽  
Author(s):  
M. Sato ◽  
K. Watanabe ◽  
M. Yazawa ◽  
Y. Takikawa ◽  
K. Nishiyama
Keyword(s):  
Pseudomonas Syringae ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enzyme Gene ◽  
Indigenous Plasmid ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Primer Sets

Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the “kudzu strain”) and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.

scholarly journals Microbiological picture of onychopathy in psoriatic patients

2020 ◽  
pp. 63-65
Author(s):  
I. S. Maximov ◽  
N. G. Kochergin ◽  
V. S. Novoselov ◽  
Z. S. Ditmarova ◽  
D. I. Ushakova
Keyword(s):  
Staphylococcus Epidermidis ◽  
Pathogenic Fungi ◽  
Klebsiella Pneumonia ◽  
Staphylococcus Lugdunensis ◽  
Opportunistic Fungi ◽  
Nail Psoriasis ◽  
Staphylococcus Capitis ◽  
Staphylococcus Hominis ◽  
Staphylococcus Simulans ◽  
Nail Infection

Objective. To evaluate the incidence of onychomycosis and bacterial contamination of onychopathy in patients with psoriasis.Material and methods. The study included 86 patients with skin psoriasis and abnormal nail plates or isolated nail psoriasis. Patients nail plates examined in laboratory using direct microscopy with 20 % KOH, mycological culture Sabourauds Dextrose Agar with chloramphenicol and сycloheximide, and bacteriological culture with indetification using the MALDI-TOF mass spectrometer.Results. Out of 86 patients, 27 (31.4 %) had onychomycosis (KOH-positive or KOH-negative with a positive result for dermatophytes in a culture study). Of the 27 patients with onychomycosis, 9 caused by pathogenic fungi, and 18 caused by opportunistic fungi. Of the 54 patients with nail psoriasis, 9 (16.7 %) had onychomycosis, 3 had dermatophytes, and 6 had opportunistic micromycetes. A total of 97 microbiological studies were conducted in 86 patients, in which the following microorganisms were detected: Staphylococcus caprae – 28 strains, Staphylococcus lugdunensis – 26, Staphylococcus epidermidis – 26, Staphylococcus haemolyticus – 15, Staphylococcus pettenkoferi – 13, Staphylococcus simulans – 11, Staphylococcus warneri – 8, Staphylococcus aureus – 5, Staphylococcus piscifermentans – 4, corinebacteria spp. – 3, Staphylococcus hominis – 3, Staphylococcus capitis – 3, Pseudomonas aeruginosa – 3, Staphylococcus pasteuri – 1, Staphylococcus cohnii – 1, Kocuria spp. – 1, Klebsiella pneumonia – 1.Conclusion. In our study, onychomycosis was detected in 31.4 % of patients with psoriasis who have onychodystrophy. In psoriatic onychia, onychomycosis occurred in 16.7 % cases. Pseudomonas nail infection was observed in two patients, one in combination with nail psoriasis.

Apicomplexan parasites of red foxes (Vulpes vulpes) in northeastern Poland

2010 ◽  
Vol 55 (3) ◽  
Author(s):  
Grzegorz Karbowiak ◽  
Viktória Majláthová ◽  
Joanna Hapunik ◽  
Branislav Pet’ko ◽  
Irena Wita
Keyword(s):  
Vulpes Vulpes ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
First Record ◽  
Hepatozoon Canis ◽  
Red Foxes ◽  
Apicomplexan Parasites ◽  
Source Of Infection ◽  
Pcr Products ◽  
Northeastern Poland

AbstractMolecular detection of apicomplexan parasites in splenic samples of red foxes collected from northeastern Poland was conducted by PCR amplification of a fragment of the 18S rRNA spanning the V4 gene region of Apicomplexa. Positive PCR products were further analysed by restriction fragment length polymorphism (RFLP) and sequencing to identify species. One hundred and eleven red foxes (Vulpes vulpes) were acquired from 15 localities in the Mazovian Province and 27 foxes were acquired from the Mazurian Lakeland. Apicomplexan 18S rDNA was detected in 15.9% of 138 fox spleens examined. Three apicomplexan species were identified: Hepatozoon canis was detected in 11.6% of the spleen samples, Toxoplasma gondii was detected in 3.6% of the spleen samples and a Babesia sp. was sequenced from 1 sample (0.7%). This data represent the first record of H. canis, T. gondii and a B. sp. from naturally infected red foxes in Poland. Infected foxes may act as sylvatic reservoirs of these apicomplexan parasites as well as serving as a source of infection for arthropod definitive hosts and vectors.

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