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Results for “Polymerase Chain Reaction”

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At least 181 records · Page 10Linked to original sources

PubMed1992

Detection of clonality in childhood B-lineage acute lymphoblastic leukaemia by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to study clonality in a group of children with B-lineage acute lymphoblastic leukaemia (ALL). Rearrangement of the immunoglobulin heavy chain gene (IgH) results in a hypervariable sequence known as the complementarity determining region III. This can be amplified by the PCR using one pair of consensus primers. The PCR product is highly clone-specific in both size and sequence. Successful amplification was achieved in 50 of 62 cases of B-lineage ALL studied (81%). Both DNA and RNA gave almost identical results. In contrast amplification was only achieved in 2 of 42 control cases (non-B-lineage leukaemias, normal and reactive marrows); these were both cases of T-ALL with IgH rearrangement on Southern blotting. The main advantages of this technique over Southern blot assessment of clonality are the short time to result and requirement for much less DNA allowing study of small samples eg cerebrospinal fluid and testicular biopsies. It is also generally more sensitive for the detection of a malignant clone in a polyclonal marrow cell population and forms the basis of techniques to study minimal residual disease (MRD).

Adolescent↗

PubMed1996

Detection of Salmonella DNA in chicken embryos and environmental samples by polymerase chain reaction.

By polymerase chain reaction (PCR) using a pair of primers specific for Salmonella phoE gene a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. Out of 45 dead-in-shell embryo samples, 20 (44.4%) were found positive for Salmonella DNA by PCR compared to 11 (24.4%) by bacteria isolation. Salmonella DNA could also be detected from infertile eggs, chicken faeces, floor litter and chick fluff, which incidence was higher than that by bacteria isolation.

Animals↗

PubMed1990

[The polymerase chain reaction].

The polymerase chain reaction (PCR) is a technique that enables us to prepare in a test tube billions of copies of a specific piece of DNA. The principles and practical aspects of the PCR are exposed. Within veterinary medicine the most important applications are expected to be i) the diagnostics of infectious diseases and ii) the detection of genetic disorders or desired genetic traits. The PCR can be carried out on a multitude of samples ranging from mummies to paraffin-embedded tissue specimens. However, inhibitors present in e.g. serum, urine and faeces have to be removed.

Animals↗

PubMed1991

Recent advances in the polymerase chain reaction.

The polymerase chain reaction (PCR) has dramatically altered how molecular studies are conducted as well as what questions can be asked. In addition to simplifying molecular tasks typically carried out with the use of recombinant DNA technology, PCR has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences. PCR can provide insights into the intricacies of single cells as well as the evolution of species. Some recent developments in instrumentation, methodology, and applications of the PCR are presented in this review.

Binding Sites↗

PubMed1995

[The detection of Toxoplasma gondii in abortion tissues of sheep using the polymerase chain reaction].

A polymerase chain reaction was applied to detect Toxoplasma gondii DNA in placental and fetal tissue samples of 47 unselected ovine abortions of the lambing season 1990/91 (Baden-Württemberg, Rhineland, Hesse). For the amplification a 190 bp or 223 bp sequence of the B1-gene of T. gondii was selected as the target sequence. Both sequences were detected in five abortions. All positive results were immunohistochemically confirmed using the peroxidase antiperoxidase technique (PAP-staining). Thus, in Germany, too, T. gondii infection in sheep during pregnancy should be considered as a possible cause of abortions, particularly in case of abortions of unknown genesis.

Abortion, Veterinary↗

PubMed1991

Bovine viral diarrhea virus infection: rapid diagnosis by the polymerase chain reaction.

The polymerase chain reaction (PCR) was applied to detect bovine viral diarrhea virus (BVDV) by amplification of its nucleic acid sequences in cell cultures, in serum samples of persistently infected cattle, and in organ specimens of acutely diseased calves. The primers and the probes were selected from the gp48 region of the cytopathic NADL strain. The products of single PCR or double PCR were identified by electrophoresis as well as by hybridization with biotinylated probes. The results thus obtained correlated with those of conventional diagnostic procedures, i.e., virus isolation and serology. The detection assay of the BVDV genome by the PCR amplification proved to be both specific and sensitive.

Animals↗

PubMed1992

Diagnosis of equid herpesviruses -1 and -4 by polymerase chain reaction.

The polymerase chain reaction (PCR) is a sensitive technique used to detect DNA of viral pathogens. We have applied the technique to the detection of Equid herpesviruses-1 and -4 (EHV-1 and EHV-4) DNA within nasopharyngeal swab samples from horses. Ninety-eight samples from suspected field cases and in-contact horses were analysed. The assays were conducted blind and later decoded and compared with virus isolation data. Our results indicate that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infection.

Animals↗

PubMed1994

Detection of DNA of causative agent of spotted fever group rickettsiosis in Japan from the patient's blood sample by polymerase chain reaction.

The polymerase chain reaction (PCR) was applied for the etiological diagnosis of spotted fever group (SFG) rickettsiosis in Japan. Nucleotide primers derived from the 17-kDa antigen gene of Rickettsia rickettsii primed a rickettsia-specific 246-base-pair product for all of the Katayama, Abe, Misaka and Kojima strains, which we had isolated previously. Moreover, we were able to detect the same product by PCR amplification from the peripheral blood of a patient in the acute stage of the illness. The PCR method is considered to be useful for rapid etiological diagnosis of SFG rickettsiosis in Japan.

Animals↗

PubMed1998

Detection of Brucella species in organs of naturally infected cattle by polymerase chain reaction.

A polymerase chain reaction (PCR) assay was used to detect Brucella species in the uterus, udder, spleen, lymph nodes, kidney and liver of three cows which had been naturally infected in an outbreak of brucellosis, and the results were compared with the results of bacteriological investigations. All 18 samples reacted positively in the PCR, but five samples had weak bands after the electrophoretic separation of PCR mixtures. No Brucella strains could be detected in these five samples by bacterial cultivation, but all the other samples gave positive results. A pre-enrichment procedure was necessary for the PCR. A PCR with DNA from eight Yersinia strains gave no amplification product.

Animals↗

PubMed1994

Solid-phase polymerase chain reaction.

The polymerase chain reaction (PCR) has facilitated the diagnosis of infectious diseases and genetic disorders, because of its ability to amplify minute amounts of nucleic acids. To distinguish genes of interest from nonspecifically amplified DNA, PCR products commonly are fractionated by electrophoresis, transferred to membranes, and then probed with a labeled internal sequence-specific oligonucleotide. Alternatively, the PCR products have been labeled directly, and hybridized to immobilized oligonucleotide probes. These methods require the tedious physical transfer of the PCR products. In order to amplify and immobilize genes simultaneously, we have developed a simple solid-phase PCR method. The technique enabled us to detect the HIV envelope gene readily without any transfer of amplified DNA.

Base Sequence↗

PubMed1992

Detection of spores of Bacillus anthracis using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to identify spores of Bacillus anthracis. By using an assay capable of amplifying a 1247-bp fragment from the gene that encodes the edema factor of B. anthracis, as few as 10(3) copies of a plasmid containing the edema factor gene and as few as 2 x 10(4) spores were detected. Subjecting the product of this PCR to a second PCR designed to amplify a 208-bp fragment nested within the 1247-bp product improved detection to a single plasmid copy per PCR and to two spores of B. anthracis per PCR.

Bacillus anthracis↗

PubMed1992

Variation in human T cell receptor V beta and J beta repertoire: analysis using anchor polymerase chain reaction.

Anchor polymerase chain reaction has been applied to the study of human T cell receptor beta chain repertoire in peripheral blood. The use of this technique has demonstrated that considerable variation in V beta and J beta usage exists, both within and between individuals. Particular V beta families, including V beta 6, V beta 4 and V beta 12 are commonly utilized, while families such as V beta 10, V beta 11 and V beta 15 are rare in all individuals studied. Marked interindividual variation in V beta usage was detected for V beta 12 and V beta 4. Biased usage of J beta elements is a prominent feature of peripheral repertoire, while there is no evidence for preferential V beta-J beta recombination events. Biased J beta usage in expressed V beta-D beta-J beta-C beta transcripts, subject to selection, was the same as that in aberrant, unselected D beta-J beta-C beta transcripts, implying that bias resulted from events relating to rearrangement itself, in the absence of selection. N-region diversity showed some evidence for preferential insertion of deoxyguanosine, consistent with the action of terminal deoxytransferase. No P-nucleotide incorporation was seen in association with intact J beta elements. These data provide evidence of some of the variation in human T cell receptor beta chain repertoire and provide a basis for comparisons with sequences which may be obtained in autoimmune and superantigen-mediated diseases.

Amino Acid Sequence↗

PubMed1994

The detection of tomato spotted wilt virus using the polymerase chain reaction.

A polymerase chain reaction (PCR) based assay was used to detected a number of UK tomato spotted wilt virus (TSWV) isolates in total RNA extractions made from infected plant material. Extracts were reverse transcribed and the resultant cDNA amplified by PCR, using oligonucleotide primers specific for a 276 base pair fragment of the L RNA segment. Assay products were electrophoresed on agarose gels and visualised by ethidium bromide staining. The viral origin of the product produced was confirmed by sequencing, with the data obtained having very high homology with previously published L RNA sequence data. The specificity and sensitivity of the RT-PCR assay, in comparison with existing tests, is discussed.

Base Sequence↗

PubMed1996

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction.

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.

Animals↗

PubMed1997

Rapid and sensitive detection of Campylobacter spp. from chicken using the polymerase chain reaction.

The polymerase chain reaction (PCR) using a target region in the flaA gene of C. coli VC167 flagellin was used to detect Campylobacter spp. in chicken without an enrichment culture. DNA extracted from 79 cloacal swabs from broiler chickens gave an amplification signal in the 450-bp region upon PCR. DNA extracted from 9 enteric and 6 non-enteric organisms included in the assay as negative controls failed to hybridize with the probe. Direct plating of all cloacal specimens on Campylobacter blood agar plates did not yield any growth. The PCR assay was sensitive enough to detect between 35-120 bacteria per PCR and thus provide a basis for detecting Campylobacter spp. in poultry.

Animals↗

PubMed1994

Bypassing successive cloning of two fragments by one step cloning using polymerase chain reaction.

The polymerase chain reaction (PCR) was used for bringing together two DNA fragments present in two different plasmids. This helped avoiding the difficulties of two successive subcloning in the same plasmid and uncertainities of obtaining rightly oriented constructs. Two different fragments of DNA present in different plasmid were digested with the same enzyme and then ligated. The ligation mixture was used for the PCR using two oligo primers; one was specific for the 5' end of the other fragment and the other one was for the 3' end of the other fragment. The desired amplified fragment was separated by gel electrophoresis, eluted and was cloned in plasmid pBluescript KS(+). The same procedure is also applicable for one step cloning of more than two fragments in desired orientation.

Cloning, Molecular↗

PubMed1995

Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction.

The polymerase chain reaction (PCR) can be used to amplify a DNA fragment with the concomitant creation of numerous mutations provided that one dNTP substrate is in excess over the three others. Advantage was taken of this behavior to systematically mutagenize a 291-bp-long DNA fragment and to define the rules relating the frequencies of each possible bp substitution to the set of the dNTP concentrations in the PCR experiment. Sets of parameters governing the rules were determined under various mutagenic conditions including the addition of MnCl2. Finally, validity of the rules was assessed in several mutagenesis experiments showing that a wide range of substitution frequencies including AT-->GC and GC-->AT transitions as well as AT-->TA transversions can be obtained at will.

Base Sequence↗

PubMed1995

Monitoring for engraftment following rat orthotopic liver transplantation by in vitro amplification of Y-chromosome gene using polymerase chain reaction.

The polymerase chain reaction (PCR) using primers specific for the rat Y-chromosome gene made it possible to distinguish a very small number of male rat cells from a large excess of female rat cells. In nonimmunosuppressed LEW recipients of ACI liver allografts, the donor cells in the bloodstream disappeared rapidly by day 3, earlier than the biochemical changes indicative of liver dysfunction. In immunosuppressed LEW recipients of ACI liver allografts, the donor cells were detected for a longer time. Moreover, in LEW recipients surviving for long period, the PCR revealed mixed-microchimerism. Our results indicated that this Y chromosomal gene-specific PCR method is useful for assessing engraftment following rat liver transplantation.

Animals↗