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 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 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 Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

2005 ◽  
Vol 71 (9) ◽  
pp. 5494-5500 ◽  
Author(s):  
Susan M. Barns ◽  
Christy C. Grow ◽  
Richard T. Okinaka ◽  
Paul Keim ◽  
Cheryl R. Kuske
Keyword(s):  
Related Species ◽  
Environmental Samples ◽  
Pcr Amplification ◽  
Soil Samples ◽  
Rrna Gene ◽  
New Subspecies ◽  
Pcr Products ◽  
Primer Sets ◽  
Initial Survey ◽  
Close Relatives

ABSTRACT Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

scholarly journals Identification of Tinomiscium petiolare from Vietnam using the DNA barcode

2021 ◽  
Vol 182 (2) ◽  
pp. 114-122
Author(s):  
B. B. Thinh ◽  
R. V. Doudkin ◽  
L. D. Chac ◽  
H. V. Chinh ◽  
Q. V. Hoi ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Medicinal Species ◽  
Cycle Sequencing ◽  
Stage Of Development ◽  
Pcr Products ◽  
Genetic Sequences ◽  
Total Dna

Background. Tinomiscium petiolare Hook.f. & Thomson is a medicinal species of the family Menispermaceae. This species is currently being intensively exploited for therapeutic purposes. Precise and rapid identification of T. petiolare is critical and essential for the classification, propagation, use and conservation of its genetic resources. In recent years, DNA barcoding has been known to be a fast and sensitive method for identifying species at any stage of development, using short DNA sequences. In this study we have performed the identification of T. petiolare specimens in Vietnam based on the sequence analysis of 4 DNA barcode loci: ITS, matK, rbcL and rpoC.Materials and methods. Total DNA was extracted from leaf samples using DNeasy Plant Mini Kit. PCR amplification of the ITS, matK, rbcL and rpoC regions was carried out on the GeneAmp PCR System 9700 with specific primers. The purified PCR products were sequenced on the ABI 3500 Genetic Analyzer system, using BigDye®Terminator v3.1 Cycle Sequencing Kit. These genetic sequences were analyzed and compared, and a phylogenetic tree was constructed using BioEdit, BLAST, and MEGA 6 programs.Results and conclusion. The success rate of amplification and sequencing was 100% for all 4 DNA barcode loci (ITS, matK, rbcL and rpoC) in the studied specimens. The produced sequence sizes of ITS, matK, rbcL and rpoC in the specimens were 574 bp, 810 bp, 527 bp and 488 bp, respectively. Further, we identified that all studied specimens were genetically related to each other and associated with the same species T. petiolare. Overall, the results of the study generated the most complete DNA barcode database of T. petiolare collected in Vietnam, contributing to the taxonomy and identification of this species. 

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 Gaeumannomyces graminis vars. avenae, graminis, and tritici Identified Using PCR Amplification of Avenacinase-like Genes

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 652-660 ◽  
Author(s):  
Sansanalak Rachdawong ◽  
Carole L. Cramer ◽  
Elizabeth A. Grabau ◽  
Verlyn K. Stromberg ◽  
George H. Lacy ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rapid Identification ◽  
Gaeumannomyces Graminis ◽  
Specific Primers ◽  
Single Tube ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Cereal Fields ◽  
Take All

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiating among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers. Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5′ primers specific for Gga, Ggt, and Ggg and a single 3′ common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5′ primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.

scholarly journals Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sohyun Lee ◽  
Nanjoo Park ◽  
Sujung Yun ◽  
Eunseon Hur ◽  
Jiwon Song ◽  
...  
Keyword(s):  
South Korea ◽  
Pcr Amplification ◽  
Public Health Problem ◽  
Test Method ◽  
Quinolone Resistance ◽  
Human Salmonellosis ◽  
Pcr Products ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

Sign in / Sign up

Export Citation Format

Share Document