scholarly journals Rapid Identification of Yersinia enterocolitica in Blood by the 5′ Nuclease PCR Assay

2000 ◽  
Vol 38 (5) ◽  
pp. 1953-1958 ◽  
Author(s):  
Keya Sen
Keyword(s):  
Yersinia Enterocolitica ◽  
Rapid Identification ◽  
Taqman Assay ◽  
Rrna Gene ◽  
Chromosomal Dna ◽  
Pcr Assay ◽  
Clinical Sepsis ◽  
Taqman Pcr ◽  
Stored Blood ◽  
The 16S Rrna Gene

Yersinia enterocolitica accounts for 50% of the clinical sepsis episodes caused by the transfusion of contaminated red blood cells. A 5′ nuclease TaqMan PCR assay was developed to detectY. enterocolitica in blood. Primers and a probe based on the nucleotide sequence of the 16S rRNA gene from Y. enterocolitica were designed. Whole-blood samples were spiked with various numbers of Y. enterocolitica cells, and total chromosomal DNA was extracted. When the TaqMan PCR assay was performed, as few as six bacteria spiked in 200 μl of blood could be detected. The assay was specific and did not detect other Yersiniaspecies. The TaqMan assay is easy to perform, takes 2 h, and has the potential for use in the rapid detection of Y. enterocolitica contamination in stored blood units.

scholarly journals Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica

2000 ◽  
Vol 38 (9) ◽  
pp. 3429-3435 ◽  
Author(s):  
J. Hoorfar ◽  
P. Ahrens ◽  
P. Rådström
Keyword(s):  
Salmonella Enterica ◽  
Relative Sensitivity ◽  
Positive Control ◽  
Taqman Assay ◽  
Diagnostic Specificity ◽  
Pcr Assay ◽  
Sample Tube ◽  
Simple Alternative ◽  
Taqman Pcr ◽  
Internal Positive Control

A simple and ready-to-go test based on a 5′ nuclease (TaqMan) PCR technique was developed for identification of presumptiveSalmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic “rough” isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonellastrains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [ΔRn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (ΔRn, ≤0.5). All 100 roughSalmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at −20°C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification ofSalmonella, particularly in large reference laboratories.

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene

2006 ◽  
Vol 66 (1) ◽  
pp. 156-164 ◽  
Author(s):  
Inge Vliegen ◽  
Jan A. Jacobs ◽  
Erik Beuken ◽  
Cathrien A. Bruggeman ◽  
Cornelis Vink
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Identification Of Bacteria ◽  
Time Amplification ◽  
The 16S Rrna Gene

scholarly journals Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum β-lactamases

2007 ◽  
Vol 56 (1) ◽  
pp. 52-55 ◽  
Author(s):  
Christopher I. Birkett ◽  
Hugo A. Ludlam ◽  
Neil Woodford ◽  
Derek F. J. Brown ◽  
Nicholas M. Brown ◽  
...  
Keyword(s):  
Real Time ◽  
Taqman Assay ◽  
Esbl Production ◽  
Pcr Assay ◽  
Extended Spectrum ◽  
Genes Encoding ◽  
Taqman Pcr ◽  
Assay Time ◽  
Phenotypic Methods ◽  
Single Reaction

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla CTX-M genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on β-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum β-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla CTX-M genes in 21 of 28 ESBL-producing isolates.

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.

scholarly journals Idiomarina xiamenensis sp. nov., isolated from surface seawater, and proposal to transfer Pseudidiomarina aestuarii to the genus Idiomarina as Idiomarina aestuarii comb. nov.

2011 ◽  
Vol 61 (4) ◽  
pp. 969-973 ◽  
Author(s):  
Liping Wang ◽  
Qiliang Lai ◽  
Yuanyuan Fu ◽  
Hua Chen ◽  
Wanpeng Wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Gene Sequence ◽  
Rrna Gene ◽  
Surface Seawater ◽  
Chromosomal Dna ◽  
16S Rrna Gene Sequences ◽  
Pr China ◽  
The 16S Rrna Gene

A taxonomic study was carried out on strain 10-D-4T, which was isolated from a crude oil-degrading consortium enriched from surface seawater collected around Xiamen Island, PR China. Strain 10-D-4T grew optimally at pH 7.0–8.0 and at 25 °C. The 16S rRNA gene sequence of strain 10-D-4T showed the highest similarity to those of Idiomarina salinarum ISL-52T (94.6 %), Idiomarina tainanensis PIN1T (94.2 %) and Idiomarina seosinensis CL-SP19T (94.1 %), and showed lower similarity (92.3–94.0 %) to other members of the genus Idiomarina. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major fatty acids were iso-C13 : 0 (5.2 %), iso-C15 : 0 (15.3 %), C16 : 0 (14.3 %), summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) (6.6 %), iso-C17 : 0 (15.4 %) and C18 : 1ω7c (13.5 %). The G+C content of the chromosomal DNA was 50.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences, together with data from phenotypic and chemotaxonomic characterization, revealed that strain 10-D-4T represents a novel species of the genus Idiomarina, for which the name Idiomarina xiamenensis sp. nov. is proposed. The type strain is 10-D-4T ( = CCTCC AB 209061T  = LMG 25227T  = MCCC 1A01370T). We also propose the transfer of Pseudidiomarina aestuarii, described recently, to the genus Idiomarina as Idiomarina aestuarii comb. nov. (type strain KYW314T  = KCTC 22740T  = JCM 16344T).

Ribosomal RNA Gene Restriction Fragment Diversity amongst Penner Serotypes of Campylobacter jejuni and Campylobacter coli

1998 ◽  
Vol 53 (1-2) ◽  
pp. 65-68
Author(s):  
S. I. Smith ◽  
D. K. Olukoya ◽  
A. J. Fox ◽  
A. O. Coker
Keyword(s):  
Campylobacter Jejuni ◽  
Ribosomal Rna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Chromosomal Dna ◽  
Ribosomal Rna Gene ◽  
Restriction Endonuclease Digest ◽  
Gene Restriction ◽  
Rna Genes ◽  
The 16S Rrna Gene

AbstractDiversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst forty-seven strains of Campylobacter made up of 38 strains of Campylobacter jejuni and 9 strains of Campylobacter coli. Restriction digests of chromosomal DNA prepared by treating with Hae III were probed with an oligonucleotide specific for Campylobacter 16S ribosomal RNA genes. Seventeen distinct hybridization patterns, each indicating the presence of 2 - 4 copies of the 16S rRNA gene are encoded in Campylobacter DNA. Differences in fragment patterns were observed not only between members of two species, but also between individual strains of the same species. Ribopattern fragments of 8.71, 7.56, 2.81 and 1.0 kb were characteristic of the majority of C. jejuni, whereas 7.59 and 4.68 kb fragments were commonly present in C. coli.In conclusion, Hae III ribotyping was even more discriminatory than the Penner serotyping of C. jejuni and C. coli, as strains of the same serotype were distinguished.

scholarly journals Nested PCR Assay for Detection of Granulocytic Ehrlichiae

1998 ◽  
Vol 36 (4) ◽  
pp. 1090-1095 ◽  
Author(s):  
Robert F. Massung ◽  
Kim Slater ◽  
Jessica H. Owens ◽  
William L. Nicholson ◽  
Thomas N. Mather ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Limit Of Detection ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Serum Samples ◽  
Blood Specimens ◽  
The 16S Rrna Gene ◽  
Edta Blood

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted fromIxodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

MULTIPLEX PCR ASSAY FOR CHLAMYDIA-LIKE BACTERIA DETECTION

2019 ◽  
Vol 72 (5) ◽  
pp. 851-855
Author(s):  
Viktoriya K. Zezekalo ◽  
Konstantin F. Pochernyaev ◽  
Vasyl M. Voloshchuk ◽  
Liudmyla V. Zasukha ◽  
Natalia S. Shcherbakova ◽  
...  
Keyword(s):  
Gc Content ◽  
Critical Parameters ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Easy Method ◽  
Melting Temperatures ◽  
Multiplex Pcr Assay ◽  
Specificity Test ◽  
Pcr Products ◽  
The 16S Rrna Gene

Introduction: Waddlia chondrophila and Parachlamydia acanthamoebae are well-known and best-studied representatives of Сhlamydia-related bacteria carrying a potential zoonotic threat. These bacteria are associated with miscarriage, ectopic pregnancy, diseases of the respiratory system in both humans and animals. Despite the importance of these Сhlamydia-like organisms for human medicine along with veterinary medicine, studies on their prevalence in Ukraine were not conducted due to the lack of available tests. The aim of our work was to create relatively cheap and easy method for detection Waddlia chondrophila and Parachlamydia acanthamoebae. Materials and methods: GenBank database was used to find nucleotide sequences of the 16S rRNA gene of bacteria Chlamydiales’ order. Alignment was performed using the MEGA7 software, in order to detect the presence of polymorphic hybridization sites specifically attributed to Waddlia chondrophila and Parachlamydia acanthamoebae. Primer- BLAST software was used to design oligonucleotide primers, to evaluate the critical parameters of the primer, in particular, the melting temperature, difference between melting temperatures for the primer pairs, the GC content, the self-complementarity, etc. Results and conclusions: The amplification of control DNA of Parachlamydia acanthamoebae and Waddlia chrondophila in single PCR using the corresponding primers and subsequent gel electrophoresis of PCR products determined the size of the amplified DNA fragments 88 b.p. and 123 b.p, respectively; the fragments were in line with the expected sizes. The analytical specificity test was performed by amplifying the control DNA of 15 species of the order Chlamydiales.

scholarly journals Detection ofYersinia enterocoliticain Retail Chicken Meat, Mashhad, Iran

2018 ◽  
Vol 2018 ◽  
pp. 1-4 ◽  
Author(s):  
Khadigeh Sirghani ◽  
Tayebeh Zeinali ◽  
Abdollah Jamshidi
Keyword(s):  
Yersinia Enterocolitica ◽  
Health Hazard ◽  
Culture Method ◽  
Good Alternative ◽  
Food Samples ◽  
Selective Medium ◽  
Chicken Meat ◽  
Poultry Meat ◽  
Rrna Gene ◽  
Pcr Assay

Poultry meat is one of the most important sources of infection ofYersiniaspp. for humans. The aim of the present study was to evaluate the incidence ofYersinia enterocoliticain chicken meat by using culture method on selective medium and confirmation by PCR assay. Also, biochemical methods were used for biotyping. A total of 100 chicken thigh meat samples were collected randomly from retail outlets in Mashhad, Iran. Samples were enriched in Peptone-Sorbitol-Bile (PSB) broth and then cultured on Cefsulodin-Irgasan-Novobiocin (CIN) agar containing antibiotics supplement. The DNA was extracted from suspected colonies ofYersiniaspp. and then PCR test using specific primers for 16S rRNA gene ofYersinia enterocoliticawas performed. In this study, 30% of chicken meat was contaminated withYersiniaspp. by culture method and 25% of chicken meat was contaminated withYersinia enterocolitica. Biotyping of isolated colonies showed that all of the isolates belonged to biotype 1A. Culture and detection ofYersiniaspp. from food samples traditionally take 4 days. Due to high accuracy and speed of PCR assay, it is a good alternative method for microbiological techniques. In conclusion, poultry meat can act as a source ofY. enterocoliticaand could be considered as a public health hazard.

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