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Results for “Polymerase Chain Reaction”

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At least 55 records · Page 3Linked to original sources

PubMed1993

[Identification of enterotoxin-producing Clostridium perfringens by the polymerase chain reaction].

Polymerase chain reaction was applied to identify enterotoxin-producing Clostridium perfringens by amplifying a segment of the C. perfringens enterotoxin gene. All of the four enterotoxin-positive reference strains tested were PCR positive while an enterotoxin-negative strain was PCR positive. All 17 clostridial strains (16 species) other than C. perfringens were PCR negative. With clinical strains isolated from various clinical specimens in Japan, Korea, and Thailand, all three enterotoxin-positive isolates were PCR positive and all 82 enterotoxin-negative isolates were PCR negative. PCR results for amplifying a region containing the initiation codon of the C. perfringens enterotoxin gene also demonstrated complete agreement with enterotoxin producibility. These results suggested that the PCR assay is a rapid and simple test for identifying the enterotoxin-producing C. perfringens without using any cultures and spore treatments.

Base Sequence↗

PubMed2000

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover. CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.

B-Lymphocytes↗

PubMed1994

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction.

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.

Animals↗

PubMed1995

[Detection of human cytomegalovirus in urine by polymerase chain reaction].

Polymerase chain reaction (PCR) amplification was set up with double primer pairs of major immediate-early and late gene in order to detect human cytomegalovirus (HCMV) from urine. This method was applied to the detection of HCMV in clinical samples of urine from renal transplant recipients. The result indicated that the primers did not cross react with other members of the herpes family of virus and human genomic DNA; that the HCMV AD169 tissue culture mixture used and the dilutions to estimate the sensitivity of PCR relative to tissue culture, 2.5 microliters of a 10(-3) dilution of the culture (625fg HCMV DNA) assayed were detected by direct gel analysis; that 25 of 30 samples of urine from the renal transplant recipients (serum HCMV IgM positive) were positive. It is conclude that PCR amplification is a valuable tool for diagnoses of HCMV infection in renal transplant recipients.

Base Sequence↗

PubMed1994

Detection of human cytomegalovirus in urine of infants by polymerase chain reaction.

Polymerase chain reaction (PCR), viral isolation and serological methods were used to diagnose HCMV infection in infants. Specimens of urine and clotted blood were collected from suspected cases of congenital or HCMV infection who attended the Pediatric Clinic, Siriraj Hospital. Prevalence of HCMV infection was found in 3 per cent of infants aged under 14 days and increased to 48 per cent in infants aged over 14 days. PCR was the most sensitive technique, it could detect HCMV infection in 29 per cent of the study infants, whereas, detection rate by isolation was 17 per cent and by specific IgM ELISA was 15 per cent. Sensitivity and specificity of PCR compared with isolation and/or serology were 93 per cent and 96 per cent, respectively. Detection of HCMV in urine by PCR can be used as a sensitive and rapid test for diagnosis of HCMV infection in infants.

Base Sequence↗

PubMed1995

[Detection of Mycoplasma pneumoniae from throat swab by polymerase chain reaction].

Polymerase chain reaction (PCR) with primers directed against the 16S-rRNA gene of Mycoplasma pneumoniae was used to diagnose M. pneumoniae infections, and the results were compared with those of culture and serology methods. Eighty (22%) of 363 throat swab samples from patients with acute respiratory complaints gave positive results by using the PCR method. Sixty-seven (18.5%) of the samples were positive in culture method. Of 35 samples which were unreliable culture results due to contamination with other bacteria, 13 gave positive results in the PCR method. Of the 97 cases obtained throat swabs and paired sera, 28 (28.9%) showed positive results by the PCR assay, and 29 (29.9%) by serology method (particle agglutination test). The positive rate was increased to 36% by using both the PCR and the serology methods. From these results it was concluded that the PCR method is useful for laboratory diagnosis of M. pneumoniae infections.

Base Sequence↗

PubMed1993

Detection of toxigenic Clostridium difficile in stool specimens by the polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of a segment of the toxin A gene was used to detect toxigenic Clostridium difficile directly from stool specimens of patients with antibiotic-associated diarrhea. Although PCR-inhibitory substances were recognized in DNA prepared from stool specimens, the inhibitory substances were eliminated by using an ion-exchange column after phenol-chloroform extraction. Eventually, 39 stool specimens were evaluated by PCR. PCR results for detection of toxigenic C. difficile were in complete agreement with cell culture assay results; all 12 PCR-positive stool specimens were positive by cytotoxin assay, and all 27 PCR-negative specimens were negative by cytotoxin assay. Toxigenic C. difficile was cultured from all PCR-positive specimens. These results suggest that PCR amplification may be an effective method for laboratory diagnosis of C. difficile-associated diarrhea and colitis.

Bacterial Toxins↗

PubMed2001

Detection of babesia species from infected dog blood by polymerase chain reaction.

Polymerase chain reaction (PCR) was first applied to diagnosis of canine babesiosis in Japan. Blood samples from 13 dogs suffering from canine babesiosis were used for examination of specificity and sensitivity of the PCR diagnosis. Of the 13 dogs, three were experimentally infected, and ten were naturally infected with Babesia species in west part of Japan. We designed a nested PCR to amplify the babesial small subunit ribosomal RNA gene and found that only the nested PCR produced a visual band, which were not apparent by the first-round PCR to the positive samples. Specificity of the nested PCR was confirmed by amplification after the second-round PCR. Sensitivity of the nested PCR was examined by diluting the blood samples from infected and uninfected dogs. The nested PCR was found to show positive results on the most diluted blood at 0.0001% parasitemia. These results indicate that the nested PCR is highly sensitive and useful for diagnosis of canine babesiosis.

Animals↗

PubMed1998

Identification of common dermatophytes (Trichophyton, Microsporum, Epidermophyton) using polymerase chain reactions.

Polymerase chain reaction (PCR) fingerprinting detected DNA polymorphisms among frequently isolated species and strains of the genera Trichophyton, Microsporum and Epidermophyton. The patterns generated by this DNA-based method permitted species and strains to be identified. The conventional methods to identify dermatophytes rely on the expression of characteristic morphological features, as well as several physiological properties. Identification is often delayed or problematic because isolates may be slow to form conidia or produce atypical microscopic structures or colony appearances. Using non-specific primers such as (AC)10, (GTG)5, M13 core sequence and AP3, characteristic PCR profiles were generated for 17 species. Intraspecies variables were also observed for four of six varieties of T. mentagrophytes, whereas no detectable DNA variability was found within the three varieties of T. tonsurans. Comparing species-specific PCR fingerprints of clinical isolates with those of type strains, species could be identified by their PCR fingerprints, even if they could not be identified by the accepted phenotypic characteristics.

Arthrodermataceae↗

PubMed1990

Human cytomegalovirus infection during childhood: detection of viral DNA in peripheral blood by means of polymerase chain reaction.

Polymerase chain reaction (PCR) technique was applied to detect cytomegalovirus (CMV) DNA. Two pairs of synthetic oligonucleotide primers were used to amplify DNA from the immediate early 1 and the late antigen genes of CMV, respectively. Either primer sets could detect as few as 0.01 plaque-forming unit of CMV strain AD 169 by Southern blot hybridization. Sixteen CMV clinical isolates were examined and all were found to be positive by the both primer sets. The PCR was used to detect CMV DNA in peripheral blood from six children with elevated anti-CMV antibody titers, who showed abnormal liver-function tests. In three immunocompromised patients, all blood samples were positive for CMV DNA. In three immunocompetent young infants with primary CMV infection, CMV DNA was detected from peripheral blood of one patient during acute phase. Presence of CMV DNA in peripheral blood seemed to be related with the extent of CMV infection, and possibly diagnostic for CMV hepatitis.

Child, Preschool↗

PubMed1994

Detection of Candida albicans DNA in serum by polymerase chain reaction.

Polymerase chain reaction (PCR) was applied in order to improve the diagnosis of disseminated candidosis. A nested PCR technique with 2 primer pairs was used to increase the sensitivity. We were able to detect Candida DNA in serum and tissue samples from experimentally infected mice as well as in serum samples from candidemia patients and patients with deep-seated Candida infection. Our PCR could detect as little as 1 pg Candida albicans DNA. The PCR method was more sensitive than culture in both the mouse experiments and the patients with deep candidosis (5/7 were PCR positive and 0/7 blood culture positive), and of similar sensitivity in candidemia patients (11/17 were 15/17 blood culture positive). The relatively short processing time of PCR, when compared with culturing, its sensitivity, as well as the possibility of using serum samples for analysis, all helped improve the diagnostics in deep-seated candidosis and candidemia.

Adult↗

PubMed2004

Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction.

Polymerase chain reaction (PCR) has been widely used due to its high specificity, sensitivity, and rapid turn-around time. However, inhibitory factors may be co-extracted with the target nucleic acid that will hinder the performance of PCR. In this study, DNA extraction methods for Mycobacterium avium subsp. paratuberculosis were evaluated including rapid lysis, organic extraction, silica-based and magnetic particle-based (MagaZorb) technologies on bacterial cells, and spiked bovine feces. Efficiency of the extraction was determined by PCR end point titration with primers targeting the insertion sequence, IS900. Results of the end point titrations are identical for bacterial cells and spiked feces. Inhibition was observed in PCR with DNA isolated from spiked feces, and a 1/100 dilution was able to alleviate this problem with DNA extracted by MagaZorb. A 1/1000 dilution was required for the other three methods. MagaZorb proved to be more efficient at removing inhibitory factors and required the least labor and completion time. Further evaluation is required for its utilization in other clinical specimens.

Base Sequence↗

PubMed1993

Study on cytomegaloviral infection in acute leukemia patients by polymerase chain reaction.

Polymerase chain reaction (PCR) and blot hybridization by probe labelled with digoxin were used to detect human cytomegalovirus (HCMV) DNA in 31 patients with acute leukemia and 20 controls. The positive rate detected by PCR in acute leukemia was 74.2% and 15.0% in the controls. The difference was statistically significant. The results suggest that acute leukemia patients are the high risk population with high infection rate of HCMV. The positive rate detected by digoxin method in acute leukemia was 58.1%. Six patients showed negative results by digoxin method, but positive by PCR. It was demonstrated that PCR was superior to digoxin method in sensitivity. Anti-HCMV IgM in serum of patients was detected at the same time, the positive rate was 16.1%. Only 6 of the 23 PCR positive patients showed positive anti-HCMV IgM. It suggests that the immune response is weak in acute leukemia patients and HCMV recently infected could not be excluded in the anti-HCMV IgM negative cases.

Adult↗

PubMed1991

Detection of polyadenylated RNA in hepatitis B virus-infected peripheral blood mononuclear cells by polymerase chain reaction.

Polymerase chain reaction (PCR) with a reverse transcriptase step characterized a specific transcription activity in hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) in two patients (1 and 2) with chronic hepatitis positive for antibody to hepatitis B core antigen (HBc). Patient 1 was also coinfected with human hepatitis delta virus. A patient who cleared HBV replication after antiviral treatment with vidarabine served as negative control. HBV-specific RNA poly A sequences were detected by PCR in PBMC of patients 1 and 2 even without detectable HBV DNA (patient 2) as shown by dot blot and PCR assays. RNA sequences were found in both the nucleus and cytoplasm. The demonstration of HBV mRNA sequences within PBMC suggests the transcription of viral DNA, in agreement with the findings of HBV surface antigen in PBMC. The results in patient 1 demonstrated HBV mRNA sequences in leukocytes even without PCR-detectable HBV DNA sequences, likely due to ongoing hepatitis delta virus replication.

Base Sequence↗

PubMed2001

Parasite persistence in treated chagasic patients revealed by xenodiagnosis and polymerase chain reaction.

Polymerase chain reaction (PCR) was compared with xenodiagnosis performed 20 years after trypanocidal chemotherapy to investigate parasite clearance. Eighty-five seropositive individuals for Chagas disease presenting a positive xenodiagnosis were treated with specific drugs; 37 in the acute phase and 48 in the chronic phase. Fifteen chronic asymptomatic patients received a placebo. Treatment in the acute phase led to PCR negative results in 73% of the cases, while xenodiagnosis was negative in 86%. In the chronic phase, PCR was negative in 65% of the patients and 83% led to xenodiagnosis negative results. Regarding the untreated group (placebo), 73% gave negative results by xenodiagnosis, of which 36% were positive by PCR. Individuals that were considered seronegative (n=10), presented unequivocally negative results in the PCR demonstrating the elimination of parasite DNA. Seventeen individuals had their antibodies titers decreased to such a level that the final results were considered as doubtful and 16 of them presented negative PCR. The molecular method represents a clear advantage over conventional techniques to demonstrate persistent infections in Chagas disease patients that underwent chemotherapy.

Acute Disease↗

PubMed1991

Gene-deletion and carrier detections, and prenatal diagnosis of Duchenne muscular dystrophy by analysis of the dystrophin gene amplified by polymerase chain reaction.

Polymerase chain reaction (PCR)-based diagnosis was carried out in 62 patients (57 probands) with Duchenne or Becker muscular dystrophy (DMD or BMD) and 226 members in 57 families. The PCR studies were also performed for carrier detection in 57 mothers and 58 sisters, and prenatal diagnosis of 4 fetuses at risk of DMD. The PCR with 7 sets of primers, which amplify 7 different exon-sequences of the dystrophin gene, detected gene deletion of at least one exon in 49% of the probands. The PCR with the other 4 primer sets, which amplify 3 intragenic loci, and subsequent endonuclease digestion detected in 84% of the mothers a heterozygous pattern in at least one such locus/segment. Using the same primer sets, carrier detection was successful in 5 sisters of familial DMD cases, while recombination between the ERT87 and the 3' end intragenic loci was observed in 11% of family members studied. Prenatal diagnosis was made in all the 4 fetuses; two males were affected, one male fetus non-affected, and the remaining one female fetus a carrier. Thus, the PCR study and the primers used in the present study are useful and convincing for rapid diagnosis of DMD and/or BMD.

Base Sequence↗

PubMedDate not supplied

Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction.

Polymerase chain reaction (PCR) is now widely used in malaria research for analysis of field samples. However, little has been reported regarding loss of sensitivity due to field methodology. Therefore, studies were carried out in relation to blood sampling (anticoagulants, culture medium, filter paper), storage (temperature, time and immediate lysis) and handling (repeated thawing and freezing). The PCR was unaffected by citrate and EDTA but partly inhibited by heparin (inhibition was reversed by heparinase at optimal concentrations). Samples collected on filter paper showed a significant 100-fold lower sensitivity (compared to control samples frozen immediately after collection) when stored at 30 degrees C and 60% humidity; and the paper quality appeared to be critical. Storage of unprocessed whole blood at 4 degrees C, 20 degrees C or 30 degrees C rarely resulted in any loss of sensitivity. Repeated thawing generally resulted in 10-fold loss of sensitivity compared to blood kept frozen until DNA extraction. The presence of antimalarial drug did not apparently affect sensitivity. We conclude that the mode of collection and storage of blood samples may influence the sensitivity of detection of malaria parasites by PCR. This may be critical in studies including individuals with low parasitaemia, mixed infections and comparison of data from different settings.

Animals↗

PubMed1993

Detection of human papillomavirus in squamous intraepithelial lesions by consensus and type-specific polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify human papillomavirus (HPV) in 216 cervical biopsy specimens from women referred to the gynecological out-patient unit for colposcopy because of an abnormal smear. HPV DNA was screened using type-specific primers for HPV6, 11, 16, 18, 31 and 33 (TS-PCR) as well as a consensus primer located in the E1 region of the HPV genome (C-PCR). TS-PCR specificity was validated by Southern blot analysis. Low-grade (SIL 1) and high-grade (SIL 2) squamous intraepithelial lesions were found in 165 biopsies. HPV16 detection was better with PCR than Southern blot, particularly for SIL 1 and SIL 2. The fact that 10% of HPV16 (all SIL 2) were not detected by C-PCR indicates that both PCR techniques should be performed. C-PCR also detects uncharacterized HPV types (8.6% prevalence in our results), mainly in SIL 1 and SIL 2. HPV16, the most frequently isolated type (prevalence 21%), was associated with SIL 2 in 83% of cases. A low HPV prevalence was found in specimens without dysplastic cells. These results suggest that PCR may be an important tool for identifying women at risk for developing dysplasia or cervical cancer.

Adult↗