Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27

1995 ◽  
Vol 41 (5) ◽  
pp. 687-692 ◽  
Author(s):  
H M Steffens-Nakken ◽  
G Zwart ◽  
F A van den Bergh
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cross Reactivity ◽  
Single Step ◽  
Specific Method ◽  
Hla B27 ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.

scholarly journals Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2103-2114 ◽  
Author(s):  
SJ Thoma ◽  
CP Lamping ◽  
BL Ziegler
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cell Surface ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Rare Populations ◽  
Pcr Method ◽  
Polymerase Chain

Abstract A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

Confirmation of Presumptive Salmonella Colonies by the Polymerase Chain Reaction

1998 ◽  
Vol 61 (10) ◽  
pp. 1381-1383 ◽  
Author(s):  
A. ŠTEFANOVIČOVÁ ◽  
H. REHÁKOVÁ ◽  
A. ŠKARKOVÁ ◽  
N. RIJPENS ◽  
T. KUCHTA
Keyword(s):  
Polymerase Chain Reaction ◽  
Standard Method ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Serological Identification ◽  
Pcr Method ◽  
Selective Plating ◽  
Polymerase Chain ◽  
Time Required

The potential of a genus-specific polymerase chain reaction (PCR) for the confirmation of Salmonella colonies was evaluated on 209 presumptive Salmonella colonies obtained by the standard method ISO 6579. The PCR method employing primers STII and ST15 (S. Aabo et al., Mol. Cell. Probes 7:171–178, 1993) gave results identical (100%) to those of the biochemical and serological identification, in terms of discrimination of Salmonella from non-Salmonella strains. PCR could be used directly on the colonies from selective plating media, which allowed a reduction of the time required for confirmation to a maximum of 6 h.

scholarly journals Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2103-2114 ◽  
Author(s):  
SJ Thoma ◽  
CP Lamping ◽  
BL Ziegler
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cell Surface ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Rare Populations ◽  
Pcr Method ◽  
Polymerase Chain

A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

Development and evaluation of a real-time polymerase chain reaction for fast diagnosis of sporotrichosis caused by Sporothrix globosa

2019 ◽  
Vol 58 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Mingrui Zhang ◽  
Fuqiu Li ◽  
Jie Gong ◽  
Xin Yang ◽  
Jianzhong Zhang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Specific Method ◽  
Chain Reaction ◽  
Clinical Specimens ◽  
Sporothrix Globosa ◽  
The Real ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Sporothrix globosa is an important clinical pathogen in the Sporothrix complex, which is causing sporotrichosis. S. globosa is distributed worldwide, especially in Asia. The transmission medium of S. globosa is mainly contaminated soil or decaying vegetation, and the infection usually caused by transcutaneous trauma, through which the fungal conidia or yeast cells enter the host. Although the clinical manifestations of sporotrichosis caused by S. globosa is always benign, there have been several outbreaks worldwide. In this study, we established a novel real-time polymerase chain reaction (PCR) method based on the internal transcribed spacer (ITS) sequence for the identification of S. globosa. The assay was further evaluated by clinical specimens obtained from patients of sporotrichosis. The sensitivity and specificity of the real-time PCR method was both 100%. The detection limit was 10 fg. The positive detection rate for 30 clinical specimens, which were confirmed infected by S. globosa, was 100%. The real-time PCR method established in this paper is a rapid, sensitive and specific method for the identification of S. globosa. It can detect S. globosa in clinical specimen from patients with sporotrichosis, which is helpful for fast clinical diagnosis.

scholarly journals Development and Use of a Multiplex Polymerase Chain Reaction Assay Based on Apx Toxin Genes for Genotyping of Actinobacillus Pleuropneumoniae Isolates

2005 ◽  
Vol 17 (4) ◽  
pp. 359-362 ◽  
Author(s):  
Nabin Rayamajhi ◽  
Sung Jae Shin ◽  
Sang Gyun Kang ◽  
Deog Yong Lee ◽  
Jeong Min Ahn ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Actinobacillus Pleuropneumoniae ◽  
Cross Reactivity ◽  
Single Step ◽  
Chain Reaction ◽  
Swine Industry ◽  
Porcine Pleuropneumonia ◽  
Polymerase Chain ◽  
Primer Sets

Actinobacillus pleuropneumoniae ( A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.

Single-step method for rapid detection of Brucella spp. in soft cheese by gene-specific polymerase chain reaction

1999 ◽  
Vol 66 (2) ◽  
pp. 313-317 ◽  
Author(s):  
LUIGI SERPE ◽  
PASQUALE GALLO ◽  
NICOLETTA FIDANZA ◽  
ALFREDO SCARAMUZZO ◽  
DOMENICO FENIZIA
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Single Step ◽  
Detection Sensitivity ◽  
Chain Reaction ◽  
Step Method ◽  
Polymerase Chain

Brucellosis can be transmitted to man by direct contact with infected animals or through contaminated meat, milk and dairy products (Nicoletti, 1989). The analysis of Brucella spp. is carried out in the laboratory by microbiological or serological assays (Alton et al. 1988). The first are more specific but are also time-consuming and expose the analyst to the risk of infection (López-Merino, 1991). However, the latter can result in false positives owing to cross reactivity with other Gram-negative bacteria (Diaz-Aparicio et al. 1994). Because of these limitations, the amplification in vitro of specific DNA regions by the polymerase chain reaction (PCR) could represent a powerful tool for rapid and specific diagnostic analysis. In recent years, several PCR methods have been developed to amplify specific DNA sequences of Brucella strains (Herman & de Ridder, 1992; Romero et al. 1995; Valentino et al. 1997). In addition, direct analysis of Brucella in contaminated abortive tissues (Fekete et al. 1992), milk and blood (Leal-Klevezas et al. 1995; Rijpens et al. 1996) has been reported.In this paper we describe a method for gene-specific PCR amplification of a 443 base pair (bp) fragment of Brucella DNA that belongs to a gene encoding for a 31 kDa outer membrane protein. This protein (BCSP-31) is a membrane antigen characteristic of the Brucella genus (Mayfield et al. 1988). The PCR method was developed for the analysis of soft cheeses. We focused our attention on Mozzarella, Pecorino and ricotta samples, because such products are not subjected to the natural microbial autopurification process of maturing. They are widely consumed in Italy and a relationship between infected foods and the areas where brucellosis is a human zoonosis is a possibility.The analysis was performed without purification of DNA from bacteria. Indeed, after homogenization, the sample was subjected to thermal shock by freeze–thaw cycles that lysed bacteria and solubilized nucleic acids for subsequent PCR amplification. Amplified DNA fragments were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Several brands of soft cheeses and ricotta contaminated at different levels with Brucella cells were analysed by our procedure to evaluate the detection sensitivity and the repeatability of the method.

Routine identification of four sympatric species of calanoid copepods Pseudocalanus spp. in the Atlantic Arctic using a species-specific polymerase chain reaction

2020 ◽  
Vol 48 (1) ◽  
pp. 62-72
Author(s):  
E. A. Ershova
Keyword(s):  
Polymerase Chain Reaction ◽  
Life Cycles ◽  
The Arctic ◽  
Relative Distribution ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Routine Identification ◽  
Polymerase Chain ◽  
Species Specific ◽  
Plankton Communities

Сalanoid copepods of the genus Pseudocalanus play an important role in the plankton communities of the Arctic and boreal seas, often dominating in numbers and constituting a significant proportion of the biomass of zooplankton. Despite their high presence and significance in the shelf plankton communities, species-specific studies of the biology of these are significantly hampered by extremely small morphological differences between them, especially at the juvenile stages, at which they are virtually indistinguishable. In this paper, we describe a new, routine and low-cost molecular method for identifying all Pseudocalanus species found in the Atlantic sector of the Arctic: the Arctic P. acuspes, P. minutus and the boreal P. moultoni and P. elongatus, and apply it to describe the relative distribution of these species in four locations of the Arctic and sub-Arctic. With this method, species-specific polymerase chain reaction (ssPCR), mass identification of individuals of any developmental stage, including nauplii, is possible. This method can serve as an excellent tool for studying the species-specific biology of this group, describing their life cycles, as well as monitoring changes in Arctic marine ecosystems under the influence of changing climate.

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