scholarly journals SHV-1 β-Lactamase Is Mainly a Chromosomally Encoded Species-Specific Enzyme in Klebsiella pneumoniae

2001 ◽  
Vol 45 (10) ◽  
pp. 2856-2861 ◽  
Author(s):  
José Chaves ◽  
Margarita G. Ladona ◽  
Concepción Segura ◽  
Amparo Coira ◽  
Roser Reig ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Chromosomal Dna ◽  
Promoter Regions ◽  
Specific Pcr ◽  
Positive Hybridization ◽  
Species Specific

ABSTRACT The nature of the SHV-1 β-lactamase gene was analyzed in 97 epidemiologically unrelated Klebsiella pneumoniae strains isolated from clinical samples. β-Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of β-lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples withK. pneumoniae TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with EcoRI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 β-lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 β-lactamase originated from the K. pneumoniaechromosome.

scholarly journals Molecular Characterization of Yarrowia lipolytica and Candida zeylanoides Isolated from Poultry

2000 ◽  
Vol 66 (10) ◽  
pp. 4340-4344 ◽  
Author(s):  
T. Deak ◽  
J. Chen ◽  
L. R. Beuchat
Keyword(s):  
Yarrowia Lipolytica ◽  
Rapd Analysis ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
Intergenic Sequences ◽  
Species Specific ◽  
Candida Zeylanoides ◽  
Its Pcr

ABSTRACT Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI andHaeIII produced two fragments of 200 and 150 bp fromY. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.

scholarly journals Efficiency of PCR-RFLP and species-specific PCR for the identification of meat origin in dry sausages

2017 ◽  
Vol 35 (No. 5) ◽  
pp. 386-391
Author(s):  
Kušec Ivona Djurkin ◽  
Samac Danijela ◽  
Margeta Vladimir ◽  
Radišić Žarko ◽  
Vincek Dragutin ◽  
...  
Keyword(s):  
Rflp Analysis ◽  
Chicken Meat ◽  
Specific Primers ◽  
Specific Pcr ◽  
Sheep Meat ◽  
Pcr Rflp ◽  
Pork Sausages ◽  
Species Specific ◽  
Dry Sausages

The purpose of this investigation was the identification of chicken, beef and sheep meat in pork sausages using PCR-RFLP and PCR with pecies-specific primers. Six dry fermented pork sausages were produced by adding beef, sheep and chicken meat to each in the amount of 1 and 5%. DNA was extracted from five regions of each sausage and PCR-RFLP together with PCR using species-specific primers was performed. PCR-RFLP analysis was successful only for chicken meat, while species-specific PCR was effective for identification of chicken, eef and sheep meat in all ratios and from all regions of the sausages. The results of our study show that discovering adulteration using PCR-RFLP is suitable only for chicken meat in the investigated products, while for detection of beef and sheep meat use of species-specific oligonucleotides is more effective.

scholarly journals Outer Membrane Profiles of Clonally Related Klebsiella pneumoniae Isolates from Clinical Samples and Activities of Cephalosporins and Carbapenems

1998 ◽  
Vol 42 (7) ◽  
pp. 1636-1640 ◽  
Author(s):  
Carmen Ardanuy ◽  
Josefina Liñares ◽  
María Angeles Domínguez ◽  
Santiago Hernández-Allés ◽  
Vicente J. Benedí ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Parental Strain ◽  
Electrophoretic Analysis ◽  
Clinical Samples ◽  
Nosocomial Outbreak ◽  
Chromosomal Dna ◽  
Clonal Type ◽  
Extended Spectrum ◽  
Gene Coding ◽  
High Level

ABSTRACT Fifteen isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) isolated during a nosocomial outbreak were studied. The strains belonged to the same clonal type, as shown by pulsed-field gel electrophoretic analysis of chromosomal DNA. All the isolates were resistant to extended-spectrum cephalosporins, aztreonam, gentamicin, and fluoroquinolones and were susceptible to carbapenems, tobramycin, netilmicin, and amikacin. None of the isolates expressed the OmpK36 porin. Eight isolates, for which the MICs of cefoxitin were ≥64 μg/ml, showed a diminished level or no expression of a 35-kDa porin. The MICs of meropenem, cefotaxime, and cefpirome were three to eight times higher for porin-deficient isolates than for isolates expressing the 35-kDa porin, but the MICs of imipenem increased two times for porin-deficient isolates compared to those for isolates expressing the porin. This MIC increase reverted to a level similar to that for the parental strain when porin-deficient isolates were transformed with the gene coding for theK. pneumoniae porin OmpK36. It is concluded that the high level of resistance to cefoxitin and the increase in the MICs of meropenem, cefotaxime, and cefpirome for the ESBL-producingK. pneumoniae isolates studied are associated with porin deficiency.

scholarly journals Isolation and Characterization of Mycoplasma mycoides Subspecies capri from Milk of Natural Goat Mastitis Cases

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Vijay Kumar ◽  
Rajneesh Rana ◽  
Somya Mehra ◽  
Pramod Kumar Rout
Keyword(s):  
Restriction Analysis ◽  
Clinical Signs ◽  
Strain Variation ◽  
Specific Pcr ◽  
Isolation And Characterization ◽  
Mycoplasma Mycoides ◽  
Species Specific ◽  
Amplified Rdna Restriction Analysis ◽  
Indian Goat

Association of Mycoplasma mycoides subspecies capri (Mmc) with natural goat mastitis has been studied earlier largely by detecting the Mmc DNA using molecular methods. However, report on detection of cultivable Mmc isolates from natural goat-mastitis milk is still very rare. In this study, Mmc was isolated from milk samples () of goats with or without clinical signs of mastitis. Mmc isolates were further characterized by biochemical and species-specific PCR methods. Intra species strain variation was also studied by 16S amplified rDNA restriction analysis (16S ARDRA). The study recovered a total of 6 Mmc isolates (3.5%). Three types of intraspecies variants among the recovered Mmc isolates were found by 16S ARDRA. The study concluded that Mmc may be an etiological agent of mycoplasmal mastitis in Indian goat herds.

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 ASarcoptes scabieispecific isothermal amplification assay for detection of this important ectoparasite of wombats and other animals

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5291 ◽  
Author(s):  
Tamieka A. Fraser ◽  
Scott Carver ◽  
Alynn M. Martin ◽  
Kate Mounsey ◽  
Adam Polkinghorne ◽  
...  
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Sarcoptes Scabiei ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Specific Pcr ◽  
Animal Populations ◽  
Thermal Cycler ◽  
Low Sensitivity ◽  
Species Specific

BackgroundThe globally distributed epidermal ectoparasite,Sarcoptes scabiei,is a serious health and welfare burden to at-risk human and animal populations. Rapid and sensitive detection ofS. scabieiinfestation is critical for intervention strategies. While direct microscopy of skin scrapings is a widely utilised diagnostic method, it has low sensitivity. PCR, alternatively, has been shown to readily detect mite DNA even in microscopy-negative skin scrapings. However, a limitation to the latter method is the requirements for specialised equipment and reagents. Such resources may not be readily available in regional or remote clinical settings and are an important consideration in diagnosis of this parasitic disease.MethodologyA Loop Mediated Isothermal Amplification (LAMP) assay targeting the ITS-2 gene forS. scabieiwas developed and evaluated on clinical samples from various hosts, previously screened with conventionalS. scabies-specific PCR. Species specificity of the newly developed LAMP assay was tested against a range of DNA samples from other arthropods. The LAMP assays were performed on a real-time fluorometer as well as thermal cycler to evaluate an end-point of detection. Using skin scrapings, a rapid sample processing method was assessed to eliminate extensive processing times involved with DNA extractions prior to diagnostic assays, including LAMP.ResultsTheS. scabieiLAMP assay was demonstrated to be species-specific and able to detect DNA extracted from a single mite within a skin scraping in under 30 minutes. Application of this assay to DNA extracts from skin scrapings taken from a range of hosts revealed 92.3% congruence (with 92.50% specificity and 100% sensitivity) to the conventional PCR detection ofS. scabiei. Preliminary results have indicated that diagnostic outcome from rapidly processed dry skin scrapings using our newly developed LAMP is possible in approximately 40 minutes.DiscussionWe have developed a novel, rapid and robust molecular assay for detectingS. scabieiinfesting humans and animals. Based on these findings, we anticipate that this assay will serve an important role as an ancillary diagnostic tool at the point-of-care, complementing existing diagnostic protocols forS. scabiei.

ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species

Nematology ◽  
2009 ◽  
Vol 11 (5) ◽  
pp. 649-668 ◽  
Author(s):  
Wolfgang Burgermeister ◽  
Helen Braasch ◽  
Kai Metge ◽  
Jianfeng Gu ◽  
Thomas Schröder ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Restriction Analysis ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Its Sequence ◽  
Additional Species ◽  
New Information ◽  
Specific Fragment ◽  
Species Specific ◽  
Different Origins

Abstract Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.

Absolute Quantitative Detection of Abl Tyrosine Kinase Domain Mutations Using a Novel Microfluidic Platform and Allele-Specific PCR.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2859-2859
Author(s):  
Vivian Oehler ◽  
Suchitra Ananthnarayan ◽  
Frank McCormick ◽  
Antoine Daridon ◽  
Geoff Facer ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Point Mutations ◽  
Kinase Domain ◽  
Clinical Samples ◽  
Tyrosine Kinase Domain ◽  
Abl Tyrosine Kinase ◽  
Specific Pcr ◽  
Allele Specific ◽  
T315i Mutation ◽  
Acid Sample

Abstract Point mutations in the Abl tyrosine kinase domain are the main mechanism of secondary resistance to imatinib mesylate (IM) therapy in chronic myeloid leukemia (CML) patients. Current mutation detection approaches including direct sequencing, DHPLC, and SSCP range in sensitivity from 5% to 20%. Detection of mutations in early treatment and possibly pre-treatment samples could allow patient therapy to be altered before resistance is detected cytogenetically or disease progression. A sensitive assay using a novel nanofluidic chip designed for digital isolation and detection (DID) by mutant allele-specific amplification was used to detect rare mutant copies of Abl in a very high background of wild-type (Wt) Abl cDNA. The DID chip partitions a nucleic acid sample into thousands of isolated reaction chambers, thereby enriching rare targets and enabling absolute quantitative, specific and sensitive detection of rare sequences and mutations. Each sample mix of 7.2 mcl containing the necessary reagents for a quantitative PCR 5′-nuclease assay is filled into a nanofluidic network. The sample is partitioned into 1,200 chambers using microfluidic valves fabricated by multilayer-soft-lithography. Each 6 nl reaction chamber contains a fraction of the original nucleic acid sample and reagents for detecting the target of interest. Since the mutant target is rare, the majority of partitions will not contain the mutant, while a small number of partitions will contain just one mutant copy. Single copy detection using the DID chip is reproducible and reliable. For initial proof of concept experiments a set of assays able to detect a group of p-loop mutations and the T315I mutation were optimized using model mixtures. Studies by French centers and Australian groups suggest that these mutations confer worse prognosis and the T315I mutation is refractory to other targeted therapies. Mutation-specific PCR assays were developed for the following mutations: G250E, Q252H (2 substitutions), Y253F, E255K, E255V, and T315I. Initial experiments utilized mutated Bcr-Abl cDNA and plasmids serially diluted into the background of 105 equivalents of Wt Bcr-Abl cDNA from a transformed BaF3 cell line. As few as 9-88 copies were specifically and reliably detected in each of the mutation-specific assays in the background of 105 Wt copies. A non-extendable blocker primer for the Wt and streptavidin beads are also being tested to enrich for mutants prior to PCR. Wt Abl is also quantified to allow for the determination of percent Abl mutated in the assays applied to clinical samples. Conclusion: This method utilizes a novel platform to provide highly sensitive and specific detection of known point mutations and can be applied to early clinical samples. The assay is unique in that it can be extended to a large group of mutations as detection is achieved in very small amounts of rare samples partitioned into thousands of wells prior to PCR. Figure 1 Figure 1.

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