scholarly journals PCR Assay for Species-Specific Identification ofBacteroides thetaiotaomicron

2000 ◽  
Vol 38 (4) ◽  
pp. 1672-1675 ◽  
Author(s):  
Lee-Jene Teng ◽  
Po-Ren Hsueh ◽  
Jui-Chang Tsai ◽  
Feng-Lin Chiang ◽  
Ching-Yi Chen ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Accurate Method ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Restriction Enzyme Digestion ◽  
Amplification Product ◽  
Dot Blot ◽  
Pcr Assay

Bacteroides thetaiotaomicron is the second most frequently encountered species of the anaerobes isolated from clinical specimens. We developed a PCR-based assay for the rapid identification of B. thetaiotaomicron. Specific primers were based on shared amplicons of about 1.2 kb generated from B. thetaiotaomicron by randomly amplified polymorphic DNA. This 1.2-kb fragment was sequenced and then used to design a set of PCR amplification primers. This PCR generated an amplification product of 721 bp, which was unique to all 65 isolates of B. thetaiotaomicron tested. There was no amplification with isolates of other bacterial species. Restriction enzyme digestion of the amplification product and dot blot hybridization further verified the specificity of the assay. These results suggest that this PCR assay targets a nucleotide sequence that is strongly conserved in B. thetaiotaomicron. This simple and rapid PCR assay provides a rapid and accurate method for identification of B. thetaiotaomicron and shows promise for the detection of B. thetaiotaomicron in clinical samples.

scholarly journals Distribution and Diversity of Geminiviruses in Trinidad and Tobago

1998 ◽  
Vol 88 (12) ◽  
pp. 1262-1268 ◽  
Author(s):  
Pathmanathan Umaharan ◽  
Malla Padidam ◽  
Ralph H. Phelps ◽  
Roger N. Beachy ◽  
Claude M. Fauquet
Keyword(s):  
Trinidad And Tobago ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Sweet Pepper ◽  
Yellow Mosaic Virus ◽  
Restriction Enzyme Digestion ◽  
Weed Species ◽  
Dot Blot ◽  
Degenerate Primers

Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.

Isolation and PCR amplification of a species-specific oxidoreductase-coding gene region in Listeria grayi

2005 ◽  
Vol 51 (1) ◽  
pp. 95-98 ◽  
Author(s):  
Dongyou Liu ◽  
Mark L Lawrence ◽  
A Jerald Ainsworth ◽  
Frank W Austin
Keyword(s):  
Blot Hybridization ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Distinct Region ◽  
Dot Blot ◽  
Pcr Assay ◽  
Dna Library ◽  
Listeria Species ◽  
Listeria Ivanovii ◽  
Species Specific

Listeria grayi is a nonpathogenic Gram-positive bacterium that demonstrates considerable similarities to other members in the genus Listeria, including the foodborne human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. A rapid diagnostic test to identify and diagnose listeriosis would be valuable, especially in cases where the presence of L. grayi may complicate diagnosis. This test would be based on a unique gene present in L. grayi. In this study, after comparative screening of a recombinant L. grayi DNA library by dot blot hybridization, an L. grayi specific clone (lgr20-246) with an insert of 722 bp was isolated. By applying PCR primers derived from a distinct region of the clone not shared by other bacteria, a specific band of 420 bp was amplified from the genomic DNA of L. grayi only and not of other Listeria species or common bacteria. These results suggest that the PCR assay employing primers lgr20-246F and lgr20-246R provides an independent and precise means of distinguishing L. grayi from other Listeria species and common bacteria. Therefore, it would be another useful technique for laboratory differentiation of Listeria bacteria.Key words: Listeria grayi, PCR, identification, diagnosis.

scholarly journals Rapid identification of health care–associated infections with an integrated fluorescence anisotropy system

2016 ◽  
Vol 2 (5) ◽  
pp. e1600300 ◽  
Author(s):  
Ki Soo Park ◽  
Chen-Han Huang ◽  
Kyungheon Lee ◽  
Yeong-Eun Yoo ◽  
Cesar M. Castro ◽  
...  
Keyword(s):  
Health Care ◽  
Fluorescence Anisotropy ◽  
Bacterial Species ◽  
Rapid Identification ◽  
Integrated System ◽  
Clinical Samples ◽  
Drug Resistant ◽  
Polarization Anisotropy ◽  
Major Health ◽  
Health Care Associated Infections

Health care–associated infections (HAIs) and drug-resistant pathogens have become a major health care issue with millions of reported cases every year. Advanced diagnostics would allow clinicians to more quickly determine the most effective treatment, reduce the nonspecific use of broad-spectrum antimicrobials, and facilitate enrollment in new antibiotic treatments. We present a new integrated system, polarization anisotropy diagnostics (PAD), for rapid detection of HAI pathogens. The PAD uses changes of fluorescence anisotropy when detection probes recognize target bacterial nucleic acids. The technology is inherently robust against environmental noise and economically scalable for parallel measurements. The assay is fast (2 hours) and performed on-site in a single-tube format. When applied to clinical samples obtained from interventional procedures, the PAD determined the overall bacterial burden, differentiated HAI bacterial species, and identified drug resistance and virulence status. The PAD system holds promise as a powerful tool for near-patient, rapid HAI testing.

scholarly journals SCA Medium: A New Culture Medium for the Isolation of All Candida auris Clades

2021 ◽  
Vol 7 (6) ◽  
pp. 433
Author(s):  
Ahmad Ibrahim ◽  
Lucie Peyclit ◽  
Rim Abdallah ◽  
Saber Khelaifia ◽  
Amanda Chamieh ◽  
...  
Keyword(s):  
Culture Medium ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Candida Auris ◽  
Phenotypic Profile ◽  
Stool Samples ◽  
Selective Culture Medium

Candida auris is an emerging multidrug-resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation are necessary for diagnosis and containing its spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection ranging between 101 and 102 CFU/mL. The 100% specificity of SCA (specific C. auris) medium is confirmed on a set of 135 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, allowing the latter to study its phenotypic profile.

scholarly journals Species-Specific and Ubiquitous-DNA-Based Assays for Rapid Identification of Staphylococcus aureus

1998 ◽  
Vol 36 (3) ◽  
pp. 618-623 ◽  
Author(s):  
Francis Martineau ◽  
François J. Picard ◽  
Paul H. Roy ◽  
Marc Ouellette ◽  
Michel G. Bergeron
Keyword(s):  
Staphylococcus Aureus ◽  
Genomic Library ◽  
Antibiotic Resistance Genes ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Clinical Microbiology Laboratory ◽  
Chromosomal Dna ◽  
Pcr Assay ◽  
Serious Infections ◽  
Species Specific

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

Rapid identification of Medicago nodulating strains by using two oligonucleotide probes complementary to 16S rDNA sequences

1997 ◽  
Vol 43 (9) ◽  
pp. 854-861 ◽  
Author(s):  
Sophie Rome ◽  
Jean-Claude Cleyet-Marel ◽  
Luis A. Materon ◽  
Philippe Normand ◽  
Brigitte Brunel
Keyword(s):  
16S Rdna ◽  
Sinorhizobium Meliloti ◽  
Bacterial Species ◽  
Blot Hybridization ◽  
Dot Blot ◽  
Plant Host ◽  
Rdna Sequences ◽  
Sinorhizobium Medicae ◽  
16S Rdna Sequences ◽  
Annual Medicago

Symbiotic bacteria associated with the Medicago genus are separated into two closely related species named Sinorhizobium meliloti and Sinorhizobium medicae. To discriminate rapidly between these two bacterial species, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiate S. meliloti from S. medicae. Their specificities were evaluated by dot-blot hybridization experiments on 25 reference strains representing 13 species of Rhizobium and Sinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific for S. medicae. By contrast, as expected according to the 16S rDNA sequence alignment, the labeled 16Smfs probe cross-hybridized with the DNAs of S. meliloti, Sinorhizobium fredii, and Sinorhizobium saheli but not with the DNA of S. medicae. Since S. saheli and S. fredii do not nodulate Medicago, 16Smed and 16Smfs can be routinely used to characterize the two Sinorhizobium species nodulating Medicago from pure cultures or from Medicago root nodules. Fifty strains isolated from eight annual Medicago species were then characterized by using colony hybridizations. Sinorhizobium meliloti was more frequently obtained (>80% isolates) than was S. medicae. Both Sinorhizobium species seemed to be trapped by annual Medicago and no plant-host specificity was detected.Key words: Sinorhizobium meliloti, Sinorhizobium medicae, Medicago, oligonucleotide probe, 16S rDNA gene.

Regeneration of vgb-transgenic poplar (Populus alba×P. glandulosa) and the primary observation of growth

2006 ◽  
Vol 3 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Zhang Bing-Yu ◽  
Su Xiao-Hua ◽  
Li Yi-Liang ◽  
Huang Qin-Jun ◽  
Zhang Xiang-Hua ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
First Year ◽  
Populus Alba ◽  
Rt Pcr ◽  
Dot Blot ◽  
Transgenic Lines ◽  
Polymerase Chain ◽  
Morphological Differences

AbstractIncreasing the growth rate is especially important for low-quality wood applications, so this has become an important goal in poplar breeding. The present study describes the transfer of Vitreoscilla haemoglobin (VHb) gene (vgb) driven by constitutive promoters, by Agrobacterium tumefaciens into poplar (Populus alba×P. glandulosa). From about 450 leaf discs used for transformation, 60 Kan-resistant plants were obtained, and 52 proved to be true transgenic plants. The transgenic nature of these plants was confirmed by polymerase chain reaction (PCR) amplification and Southern dot blot hybridization. The expression of vgb gene in transgenic plants was confirmed by reverse transcriptase-PCR (RT-PCR). The performance of the transgenic lines was evaluated during the first year of growth in a greenhouse. These plants showed no significant stable morphological differences from the untransformed plants. Among them, three vgb-transgenic lines exhibited noticeably higher growth rates in terms of height and diameter.

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.

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