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Results for “Polymerase Chain Reaction”

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PubMed1999

Detection of human herpesvirus 8 in Korean Kaposi's sarcoma cases by polymerase chain reaction and in situ polymerase chain reaction.

Several infectious agents, including herpesvirus-like particles, had been suggested as possible candidates for the development of Kaposi's sarcoma (KS), and a new herpesvirus, human herpesvirus 8 (HHV-8), was recently identified in the vast majority of KS lesions, irrespective of their association with human immunodeficiency virus (HIV) infection. However, the etiologic role of HHV-8 in KS remains controversial. We undertook this study to screen for and localize the presence of HHV-8 in KS in Korea. A total of 46 paraffin-embedded specimens were studied, including KS, hemangioproliferative disorders, and 10 non-KS lesions from HIV-positive patients. We performed nested polymerase chain reaction (PCR) and in situ PCR with HHV-8 specific primers. HHV-8 DNA sequences were detected in 8 of 11 KS specimens. All specimens of hemangioproliferative disorders, non-KS lesions from HIV-positive patients, and other skin samples were negative for HHV-8. When sequencing PCR products, the sequences were almost identical with the prototypic sequence for HHV-8. In PCR-positive tissues, in situ PCR staining of HHV-8 localized to nuclei of endothelial cells and perivascular spindle-shaped tumor cells. The results of this study suggest that HHV-8 is not widespread and has a certain causative role in the development of KS. Further studies, including serological and animal studies, will be helpful to appreciate an epidermiological link and pathogenetic mechanism between HHV-8 and KS.

Adult↗

PubMed1996

Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA.

BACKGROUND/AIMS: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). METHODS: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. RESULTS: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV, both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. CONCLUSIONS: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

Hepacivirus↗

PubMed1992

Identification of meningococcal serosubtypes by polymerase chain reaction.

The polymerase chain reaction was used as the basis of a novel typing method for Neisseria meningitidis. Southern hybridization experiments demonstrated that it was possible to identify genes encoding different serological variants of the meningococcal class 1 outer membrane protein by probing with polymerase chain reaction products corresponding to known epitopes. A set of 14 defined variable regions was prepared in bacteriophage M13mp19 by the cloning of polymerase chain reaction products. The phage were dot blotted onto membrane filters, which were used as targets for hybridization of radiolabeled amplified class 1 outer membrane protein genes. Thus, the presence of many different subtype-specific epitopes could be investigated in one experiment. This technique was evaluated with a set of serological reference strains, mainly of serogroup B organisms, and provided an alternative, rapid, and comprehensive typing system that was capable of distinguishing known serosubtypes and also of defining currently untypeable strains independently of sodium dodecyl sulfate-polyacrylamide gel electrophoresis or serological analysis. An additional advantage of this technique was that in the case of an unknown serosubtype (i.e., one that did not hybridize with any of the known samples), the DNA amplified from the original sample could be used to determine the nucleotide sequence of the novel serosubtype and to clone the corresponding variable region into bacteriophage M13. It may be possible to develop this procedure for the diagnostic detection and typing of meningococci directly from clinical samples even when culture is not possible because of antibiotic treatment of an acute case.

Bacterial Outer Membrane Proteins↗

PubMed1993

Rapid comparison of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris using polymerase chain reaction amplification.

Polymerase chain reaction amplification was used to compare different regions of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris, to examine homology within the species. Six 30-base polymerase chain reaction primers and three probes were synthesized on the basis of the published nucleic acid sequence of the cytochrome c3 gene from D. vulgaris, NCIMB 8303. Amplifications were performed on genomic DNA isolated from NCIMB 8303 as well as eight other strains. Six strains, NCIMB 8302, 8305, 8306, 8311, 11779, and DSM 2119, showed amplification products of equal size and quantity to those of strain 8303. Two other strains, NCIMB 8456 and DSM 1744, either showed reduced levels or no detectable amplification products. These results were confirmed by hybridization of amplified DNA to radiolabeled probes specific for each product. DNA sequencing of a 145-bp polymerase chain reaction fragment from strains NCIMB 8302, 8303, 11779, and DSM 2119 revealed complete sequence homology between these strains, whereas slight differences were seen with strain NCIMB 8456. Amino acid sequencing of the first 20 residues of cytochrome c3 purified from strains NCIMB 8456 and 8303 also showed differences in the two proteins. In contrast to the results obtained with strain NCIMB 8456, limited homology was observed between the first 20 amino acid residues of cytochrome c3 from strain DSM 1744 and strain NCIMB 8303.

Amino Acid Sequence↗

PubMed1991

Detection of human papillomaviruses in paraffin-embedded condylomata acuminata--comparison of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

The polymerase chain reaction (PCR) is used for detection of human papillomavirus (HPV) DNA in paraffin-embedded tissue sections of condylomata acuminata. Incorporation of biotinylated nucleotides during the amplification process allows a highly sensitive, fast, and non-isotopic detection of viral DNA in a subsequent Southern dot blot. In 100% (13 of 13) of histologically confirmed condylomata, HPV-6 or -11 could be detected by polymerase chain reaction. By in situ hybridization 77% (10 of 13) and by immunohistochemistry (IHC) 69% (nine of 13) positive results were obtained. Because HPV genital infection is linked to penile and cervical dysplasia, polymerase chain reaction provides a powerful and highly sensitive tool for epidemiologic studies on sexual transmission of HPV.

Adult↗

PubMed1991

Rapid detection of bovine viral diarrhea virus by polymerase chain reaction.

The polymerase chain reaction was used to detect genomic sequences of the positive-stranded RNA of bovine viral diarrhea virus (BVDV), a member of the family Togaviridae. Using a set of 20-bp primers located within the conserved 3' region of the BVDV genome, we were able to consistently amplify a 205-bp target sequence from BVDV cDNA. BVDV RNAs from cell culture-propagated BVDV reference strains, diverse unrelated cytopathic and noncytopathic field isolates, and clinical serum samples were transcribed to cDNA by using avian myeloblastosis virus reverse transcriptase and further specifically amplified by using the polymerase chain reaction assay. The amplification assay was sensitive enough to detect one molecule of cloned BVDV cDNA. Reconstitution experiments conducted by adding decreasing amounts of BVDV (NADL strain) to BVDV-free serum indicated that the threshold of sensitivity of the assay was less than or equal to 1 50% tissue culture infective dose. These results show that the polymerase chain reaction may be used for the rapid detection of diverse strains of BVDV in cell cultures, biological products, and clinical specimens from cattle.

Animals↗

PubMed1990

Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction.

The polymerase chain reaction was used to measure the DNA copy number of human immunodeficiency virus (HIV). Differences in polymerase chain reaction amplification efficiency were controlled by amplifying known amounts of HIV DNA in parallel with samples. This technique is a sensitive, accurate, and reproducible method for the quantitation of HIV DNA.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction.

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.

Base Sequence↗

PubMed2000

[The method of DNA isolation can affect the rate of preferential amplification of alleles by the polymerase chain reaction].

Today, polymerase chain reaction is a common part of approaches serving for identification of individuals in legal medicine. This method is easily practicable, however attention must be paid to the optimization of reaction conditions and to the interpretation of results. From the literature, such cases are known, in which during amplification of extremely small amount of DNA (e.g. from one cell) the polymerase chain reaction preferably amplifies only one of two in the template DNA present alleles. If the amplified fragments differ in length, the shorter one is amplified preferably, and it may be cause of false results. In the presented study, DNA from 23 stains of male blood on different fabrics was isolated by two different methods (by treatment with proteinase K and boiling and by treatment with Chelex 100). The obtained DNA samples were amplified using primers, they are complementary to the amelogenin gene sequences. The system is suitable for sex determination, because amplification of the X-chromosomal sequence provides a fragment in length of 632 bp, amplification of the Y-chromosomal one a fragment in length of 443 bp. The isolation method based on proteinase K led in 17.38% of samples to the very intensive preferential amplification of the longer allele, and therefore to a false result. The isolation method based on Chelex 100 provided in all cases correct results with clearly recognizable preferential amplification of the shorter allele. The reported results accentuate the meaning of choice of the appropriate isolation method, the need of accurate PCR optimization, and the careful interpretations of its outputs.

Alleles↗

PubMed1996

Genomic instability in ovarian cancer: a reassessment using an arbitrarily primed polymerase chain reaction.

Traditional polymerase chain reaction (PCR) amplification of multiple microsatellite markers to detect microsatellite instability (MI) is labor intensive and may be marker dependent. An arbitrarily primed polymerase chain reaction (AP-PCR) is a modification of PCR that generates a genomic DNA fingerprint using a single, arbitrarily chosen primer which is useful in the detection of somatic genetic alterations. We hypothesize that AP-PCR of an AluI DNA restriction digest, which we term Alu/AP-PCR, identifies genomic instability as well. In order to test this hypothesis, we correlated Alu/AP-PCR fingerprints with analyses of traditional PCR amplified microsatellite markers using paired germline and tumor DNA samples obtained from 68 patients with ovarian cancer. The microsatellite markers tested included dinucleotide, trinucleotide, and tetranucleotide repeats. We found that MI is more common in ovarian cancer than previously thought. We estimate a minimum incidence of MI at 37% based on the 10 traditional markers we tested, to an incidence of 53% based upon our Alu/AP-PCR analysis. All cases of MI were associated with an abnormal Alu/AP-PCR banding pattern. Both MI, detected by polymorphic markers (P=0.03), and an abnormal Alu/AP-PCR pattern (P=0.01) were significantly associated with the occurrence of a second primary malignancy in the same patient. In addition, abnormal Alu/AP-PCR patterns were associated with higher grade lesions (P=0.02), and family history of cancer (P=0.009). These findings suggest: (1) MI may play an important role in ovarian carcinogenesis, and (2) Alu/AP-PCR is a novel technique for identification of genomic instability.

Adult↗

PubMed1992

Development of oligonucleotide primers and probes against structural and regulatory genes of human immunodeficiency virus type 1 (HIV-1) and their use for amplification of HIV-1 provirus by using polymerase chain reaction.

The polymerase chain reaction is a powerful technique for amplifying a few copies of double-stranded genetic material to millions of copies in a few hours. The sensitivity and specificity of the polymerase chain reaction technique depend to some extent on the nucleotide sequences of the oligonucleotide primer pair used in the amplification. We report new oligonucleotide primers and probes which can be used for the amplification and detection of human immunodeficiency virus type 1 provirus sequences of not only structural but also regulatory genes. These primers are very sensitive and specific and can be used for the detection of African and North American strains of human immunodeficiency virus type 1.

Adult↗

PubMed1992

Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification.

Polymerase chain reaction amplification of DNA from 112 Pseudomonas solanacearum strains with the tRNA consensus primers T3A and T5A divided the species into three fingerprint groups. These groups correspond well with previous divisions made by restriction fragment length polymorphism analysis. This polymerase chain reaction test is a facile method for rapidly classifying P. solanacearum strains.

Base Sequence↗

PubMed1996

Identification of serotypes of Rickettsia tsutsugamushi by nested polymerase chain reaction.

Nested polymerase chain reaction (PCR) was applied to identify the serotypes of Rickettsia tsutsugamushi isolated from patients. The primers used for PCR were based on the nucleotide sequences encoding a 56 kDa antigen of rickettsiae. Comparing to the conventional immunofluorescence assay (IFA), which displays a considerable degree of cross-reactivity among different species, the result obtained suggests that the polymerase chain reaction method is much more reliable than IFA.

Animals↗

PubMed1990

Target amplification for DNA analysis by the polymerase chain reaction.

The polymerase chain reaction (PCR) has become a standard laboratory technique. An enzymatic reaction, as simple to perform as it is satisfying to contemplate, the PCR solves two of the more universal problems in the chemistry of natural nucleic acids. It allows for the physical separation any particular sequence of interest from its context; and then provides for an in vitro amplification of this sequence which is virtually without limit. The surprising robustness of PCR derives from its fortuitous combination of three phenomena, each of which is intrinsically powerful. The first of these is the impressive ability of almost all oligodeoxynucleotides to bind tightly and specifically to their complementary nucleic acid sequences, discriminating easily between hundreds of thousands of sites. The second familiar phenomenon is illustrated by the notion that the probability for the occurrence of a compound action is the product of the individual probabilities for the occurrence of each of its components. The third phenomenon embodied in the polymerase chain reaction relates to the branching structure of its propagation and the inherent robustness attached to such a form. Consideration of the above leads to certain generalities regarding the relative utility of various protocols for carrying out the PCR. Specific conditions of time, temperatures, concentrations, etc. will be described, as well as sample preparation and analytical methods.

DNA Probes↗

PubMed1991

Detection of varicella-zoster virus (VZV) DNA in clinical samples from patients with VZV by the polymerase chain reaction.

A polymerase chain reaction system for the detection of varicella-zoster virus was established. Of 25 nucleotides, 4 oligonucleotide pairs (regions of thymidine kinase, thymidylate synthetase, glycoprotein I, and immediate early gene) were synthesized. The first three oligonucleotide pairs could be used as primers on the basis of specific DNA amplification. Varicella-zoster virus DNA was amplified by this polymerase chain reaction system in 20 of 20 vesicle samples, 5 of 6 crusts, and 12 of 13 throat swabs collected from patients with clinical varicella.

Base Sequence↗

PubMed1991

Prenatal diagnosis for dystrophia myotonica using the polymerase chain reaction.

The polymerase chain reaction has been used to detect an abundant class of short repeat DNA families of the form (dC-dA)n.(dG-dT)n, known as microsatellites. These units are found throughout the human genome and have been characterized for several loci including APOC2 on chromosome 19q12-q13.2. The locus APOC2 is linked to the gene for dystrophia myotonica and a microsatellite within this locus was used to derive polymorphisms in a family to predict the inheritance of the disease. Chorionic villus sampling (CVS) was performed at 15 1/2 weeks' gestation. Following DNA extraction from the CVS material and parental blood samples, microsatellite analysis was carried out by the polymerase chain reaction.

Adult↗

PubMed2004

MTA obturation of pulpless teeth with open apices: bacterial leakage as detected by polymerase chain reaction assay.

Polymerase chain reaction (PCR) followed by reverse dot blot was used to detect Enterococcus faecalis leakage through mineral trioxide aggregate (MTA) apical obturations of pulpless teeth with open apices. Prepared root canals of 34 extracted teeth were given a standard apical foramen opening and received orthograde apical obturation with MTA; three groups had 1-, 2-, or 3-mm thickness. Sterilized specimens were inoculated with E. faecalis and incubated in sterile medium. DNA extracted from the specimens was amplified by polymerase chain reaction, which yielded a specific segment of E. faecalis 16S rDNA. On day 10 of incubation, no specimens were contaminated. On day 50, almost 17% of specimens were contaminated, with no statistically significant difference between groups (Chi-square = 0.48; df = 2; p = 0.787). Therefore, MTA provides an adequate seal even in cases of orthograde apical obturation of pulpless teeth with open apices.

Aluminum Compounds↗

PubMed1992

Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction.

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.

Alanine Transaminase↗