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Results for “Polymerase Chain Reaction”

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PubMed1994

[Diagnosis of falciparum malaria by polymerase chain reaction].

Polymerase Chain Reaction (PCR) method using a pair of Plasmodium falciparum-specific oligonucleotides as primers was used to detect malaria patient blood samples collected from Yunnan endemic malarious area. The results showed that a DNA fragment of 492 bp was amplified in all 53 microscopically diagnosed P. falciparum samples, but negative in 8 P. vivax samples and 12 normal blood samples. This method seems to have high sensitivity and high specificity.

Animals↗

PubMed2002

A nanoliter rotary device for polymerase chain reaction.

Polymerase chain reaction (PCR) has revolutionized a variety of assays in biotechnology. The ability to implement PCR in disposable and reliable microfluidic chips will facilitate its use in applications such as rapid medical diagnostics, food control testing, and biological weapons detection. We fabricated a microfluidic chip with integrated heaters and plumbing in which various forms of PCR have been successfully demonstrated. The device uses only 12 nL of sample, one of the smallest sample volumes demonstrated to date. Minimizing the sample volume allows low power consumption, reduced reagent costs, and ultimately more rapid thermal cycling.

Polymerase Chain Reaction↗

PubMed1994

Detection of monoclonal B-cell populations in decalcified, plastic-embedded bone marrow biopsies with the polymerase chain reaction.

Polymerase chain reaction (PCR) has been employed successfully for the detection of clonal immunoglobulin gene rearrangements in paraffin-embedded clinical samples. The authors examined whether this technique can also be applied to fixed, decalcified, and plastic-embedded bone marrow biopsies. DNA extracted from 66 glycolmethacrylate-embedded trephine biopsy samples was amplified for the detection of rearranged VDJ regions of the immunoglobulin heavy chain genes using both a single-step and a semi-nested PCR technique. After exclusion of samples with inadequate DNA, clonality was confirmed in 16 (67%) of 24 cases with B cell malignancy, whereas all 11 non-B cell neoplasms, and 6 of 9 cases with normal bone marrow showed evidence of a polyclonal B cell population. Patterns indicating oligo- or monoclonality were observed in three plastic-embedded samples of normal bone marrow, although control PCR of frozen bone marrow samples obtained in parallel showed no evidence of clonality. Repeated PCR of these cases revealed inconsistent bands, probably due to the amplification of rare templates from polyclonal B cells. Decalcified, plastic-embedded bone marrow biopsies are suitable for PCR-based determination of B-cell clonality. To exclude the possibility of false-positive results, monitoring of template DNA quality and independent control amplifications are mandatory.

B-Lymphocytes↗

PubMed1996

Detection of congenital cytomegalovirus infection in Chinese newborn infants using polymerase chain reaction.

Polymerase chain reaction (PCR) amplification was used to detect cytomegalovirus (CMV) in 1000 urine specimens from Chinese newborns for defining the incidence of congenital CMV infection in the Chinese population. The major immediate-early and the late antigen genes of CMV were amplified and detected by gel electrophoresis. There were 18 congenitally infected infants found when tests were performed with one or both primer pairs. Comparing with tissue culture, PCR of both primer sets provided a sensitivity of 94%, a specificity of 100% and a predictive value of positive result of 100%.

China↗

PubMed1997

Specific detection of mousepox virus by polymerase chain reaction.

Polymerase chain reaction was applied to the rapid identification and detection of mousepox virus. This was accomplished by selection of primers targeting the A-type inclusion body protein gene. By investigating 20 strains belonging to five different species of the genus Orthopoxvirus, amplification was achieved only with the seven mousepox virus strains examined. The size of the resulting DNA fragment accounted for 116 base pairs and contained a recognition site for the restriction enzyme HindII, thus confirming its viral origin. Amplification of mousepox virus specific sequences was also possible from infected mouse lung tissue and serum.

Animals↗

PubMed1994

[Clinical and experimental study of diagnosis of viral myocarditis using gene amplification by polymerase chain reaction].

Polymerase chain reaction (PCR) technique was applied for detection of picornavirus genomes in blood and myocardial tissue of patients with viral myocarditis. Additionally, the blood and myocardial tissue of rabbits infected by picornavirus was also screened by PCR. Twelve rabbits infected by piocrnavirus showed pathological change of myocarditis and all of these animals gave a positive PCR result for picornaviral RNA sequences in the blood and myocardial tissue. All 6 control rabbits gave no PCR positive signals. PCR gene amplification actually provides a new diagnostic approach to patients suspected of viral myocarditis.

Adolescent↗

PubMed1992

[Identification of a segment of the gene for the substance B receptor in the human DNA genome using the polymerase chain reaction].

Polymerase chain reaction was applied to human genomic DNA using primers corresponding to the rat substance P receptor cDNA. As a result, a fragment of 94 b.p. was isolated identical to the fragment 771-864 of the above-mentioned cDNA, with the exception of the G796----A substitution (Val----Ile in the amino acid sequence). A comparison of the established sequence with the published structures of tachykinin receptors of NK-1, NK-2 and NK-3 types allows its assignment to the substance P receptor (NK-1 tachykinin receptor) gene detected in the human genome.

Amino Acid Sequence↗

PubMed1994

[Serotype-specific amplification of Rickettsia tsutsugamushi DNA from clinical specimens by nested polymerase chain reaction].

Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose Tsutsugamushi disease and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of Rickettsia tsutsugamushi were detected and identified by nested PCR. The serotypes of patients registered during 1990 to 1992 in Kanagawa Prefecture were identified by nested PCR. Sixty percentage of patients showed Kawasaki types, 20% Karp types, and 20% Kuroki types. This result suggested that the recent Tsutsugamushi disease were mostly caused by Kawasaki types in Kanagawa Prefecture.

DNA Primers↗

PubMed1993

Salmonella identification by the polymerase chain reaction.

Polymerase chain reaction (PCR) primers for genus specific detection of Salmonella have been selected from a Salmonella-specific fragment of 2.3 kilobases (kb). Due to interserovar sequence diversity within this fragment, primer selection was based on DNA sequence alignment of sequences from 20 different Salmonella serovars. The specific PCR product of 429 base pairs (bp) was formed from 144 of 146 salmonella strains tested (116 of 118 serovars). The two false-negative strains belonged to two different serovars of the rarely isolated subspecies IIIa (monophasic S. arizonae). No product was produced in any of 86 non-Salmonella Enterobacteriacea strains tested, covering 41 species from 21 genera.

Base Sequence↗

PubMed1997

Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction.

Polymerase chain reaction (PCR) technique was applied to detect BHV-1 in bovine semen inoculated with BHV-1. The technique was found to be 10(6) times more sensitive than a non-isotopic dot-blot hybridization method in detecting viral genomic DNA. Of the three primer pairs used, the one chosen from glycoprotein gC appeared to be most sensitive as it could detect up to 0.01 TCID50 of BHV-1 in the semen. The technique could be useful in screening breeding bulls or samples of frozen semen prior to use in artificial insemination.

Animals↗

PubMed1995

[Application of Plasmodium falciparum DNA extract from dried blood spot of falciparum malaria patient in polymerase chain reaction].

Polymerase chain reaction (PCR) primered with primers 3,4 and 5,6 was performed using DNA extracted from dried blood spot of a falciparum malaria patient from Mengpeng Township, Yunnan using four different methods, including STE-proteinase K-SDS, methanol-proteinase K-SDS, Chelex-100 and Chelex-100-proteinase K to amplify DNA of apical membrane antigen 1 (AMA-1). A 900 bp DNA band could be seen on the ethidium bromide-stained agarose gel electrophoresis of the PCR products when DNA was extracted using all the above-mentioned methods except STE-proteinase K-SDS method. DNA extracted with Chelax-100 was recommended.

Animals↗

PubMed1991

Specific amplification of Rickettsia tsutsugamushi DNA from clinical specimens by polymerase chain reaction.

Polymerase chain reaction (PCR) was used to detect Rickettsia tsutsugamushi-specific DNA in clinical specimens. The primer pair used for PCR was designed from the nucleotide sequence of the gene encoding the 56-kDa antigen of the Gilliam strain. Theses primers led to a 78-bp fragment by amplifying the genomic DNAs from five serovariants, i.e., the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, and also the DNA from blood clots of patients with scrub typhus, even at the early stage of onset of the disease. This indicates that this method is suitable for the diagnosis of scrub typhus.

Base Sequence↗

PubMed1995

[Detection of pathogenic Entamoeba histolytica DNA in liver abscess aspirates with polymerase chain reaction].

Polymerase chain reaction (PCR) was used to diagnose amebic liver abscess by detecting pathogenic Entamoeba histolytica DNA in liver aspirates from patients with liver abscess. Oligonucleotide primers specific for gene encoding of the 30,000 molecule of pathogenic Entamoeba histolytica were used in the test. Liver aspirates from 23 cases with amebic liver abscess as evidenced by typical clinical findings or very high titres of anti-E. Histolytica antibodies with ELISA were found to be positive with PCR. Fourteen control samples (3 cases of bacterial liver abscess, 1 of liver cancer and 10 of abscess in other sites) were all negative to PCR. The results suggest that PCR is a sensitive and useful method for diagnosing amebic liver abscess.

Animals↗

PubMed1993

Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction.

Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose scrub typhus and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Serotype-specific primers were used in the second PCR amplification. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, were identified by nested PCR. In addition, the serotype identified by PCR with DNA from blood clots was the same as that of the strain isolated from five patients with scrub typhus. These findings indicate that this method is useful for diagnosis and identification of the rickettsial serotype in infected patients.

Base Sequence↗

PubMed2004

Detection of Fasciola gigantica infection in snails by polymerase chain reaction.

Polymerase chain reaction (PCR) was used to detect Fasciola gigantica infection in the snail intermediate host. Fasciola specific primers amplified a 124 bp fragment in PCR when the genomic DNA isolated from F. gigantica infected Lymnaea auricularia snails was used as template. In addition to the 124 bp amplicon, a ladder of DNA fragments representing amplification of the 124 bp repetitive sequences was observed. Genomic DNA of the parasite was used as a positive control, which also gave an amplification of the 124 bp fragment. DNA isolated from non-infected snails was used as a negative control and no amplification of this sequence was observed. This technique is highly specific and sensitive and possesses fairly good prospects of its utility as an epidemiological tool for ascertaining the infectivity status in ubiquitous snail populations.

Animals↗

PubMed1994

[Detection of Mycobacterium tuberculosis in transbronchial biopsy specimens by polymerase chain reaction].

Polymerase chain reaction (PCR) for the rapid detection of Mycobacterium tuberculosis was used to test 30 clinical specimens (transbronchial biopsy specimens) from patients in whom tuberculosis was suspected on chest X-ray. In these patients, 15 cases were histologically diagnosed as tuberculosis. Among them, M. tuberculosis was detected in 8 specimens by PCR. Of 8 PCR positive specimens, 7 were also positive by smear and culture; the other was negative by smear and positive by culture. On the other hand, 15 cases were therapeutically diagnosed as the tuberculosis and these specimens showed negative results in neither PCR nor microbiological tests. We conclude that the PCR method is useful for rapid and direct detection of M. tuberculosis in transbronchial biopsy specimens.

Biopsy↗

PubMed1998

Simple detection of a point mutation in LDL receptor gene causing familial hypercholesterolemia in southern Italy by allele-specific polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of specific alleles allowed the rapid detection of a point mutation (missense Gly528 --> Asp) in exon 11 of the low density lipoprotein receptor gene which was otherwise not detectable by exon amplification and enzymatic digestion as it does not modify the normal restriction pattern. The mutant allele, designated as FH-Palermo-1 from the origin of the first carrier family identified, gave a specific PCR product of 109 bp clearly distinct from the product of 168 bp obtained from other alleles with a nonspecific couple of primers. This method allowed us to distinguish one positive sample mixed with up to 11 parts of normal DNA. Furthermore, the specific amplification product was characterized by a Bsm I restriction site not present in nonspecific products.

Alleles↗

PubMed2006

Evaluation of B-cell clonality in archival skin biopsy samples of cutaneous B-cell lymphoma by immunoglobulin heavy chain gene polymerase chain reaction.

Polymerase chain reaction (PCR)-based detection of clonal T- and B-cells is widely used in the diagnosis of various lymphomas, including those of the skin. A large number of corresponding methods have been published. Recently, for the first time, standardized PCR protocols were developed in common by 14 European centers of lymphoma diagnosis and research (Biomed-2 protocols). Here, we have applied Biomed-2 immunoglobulin heavy chain (IgH)-PCR for clonality detection in primary cutaneous B-cell lymphoma (CBCL) and compared it with previously established methods. The DNA of 43 paraffin-embedded lesional skin biopsies of confirmed CBCL cases [27 follicle center cell lymphoma (FCCL), 11 marginal zone B-cell lymphoma/immunocytoma (MZL/IC) and five large CBCL of the lower leg (CBCL-LL)] were amplified by the Biomed-2 IgH-PCR protocols as well as using four other assays, priming also the three IgH framework regions (FR) 1-3. All PCR products were analysed by fluorescence fragment analysis. Twenty-nine of 43 (67%) CBCL samples (5/5, 100% of CBCL-LL; six of 11, 54.5% of IC/MZL; 18 of 27, 66.7% of FCCL) showed monoclonal B-cell presence complementary in all of the IgH-PCR. The three Biomed-2 PCR indicated together clonality in 24 of 43 samples (56%). Considering each method separately, the Biomed-2 FR3-PCR showed the highest rate of clonality detection (20 of 43, 47%). In conclusion, the Biomed-2 FR3-PCR is recommended for detecting B-cell clonality in archival skin samples of CBCL but should be completed by FR1- and/or FR2-PCR.

Biopsy↗