Detection Of Polymerase Chain Reaction Products Research Articles

Polymerase Chain Reaction-Based Technique for the Detection of Wuchereria bancrofti in Human Blood Samples, Hydrocele Fluid, and Mosquito Vectors

Oligonucleotide primers were designed to amplify a 490-basepair DNA fragment in the 5' end of the pWb 12 repeated DNA sequence in Wuchereria bancrofti for specific amplification of W. bancrofti DNA by the polymerase chain reaction (PCR). A single microfilaria in 100 microliter of blood or added to 1 ml of blood, a single third-stage larva in a pool of 20 uninfected mosquitoes, or 0.4 pg of W. bancrofti genomic DNA added to 100 microliter of human blood or serum can be detected by this PCR method. The parasite DNA in human blood and hydrocele samples and in mosquitoes was isolated free of any PCR inhibitors using simple purification techniques. Detection of PCR products was carried out by agarose gel electrophoresis, followed by staining with ethidium bromide and visualization under ultraviolet illumination. The results indicate that the PCR method is species-specific, rapid, and more sensitive than that of DNA probes and routine microscopy.

HIV INFECTION OF HUMAN CARTILAGE

Infection of human cartilage with HIV in vivo has not previously been reported. Specimens of articular cartilage taken at postmortem from ten patients who were HIV-positive were examined. Two had AIDS and eight were believed to have stage-2 disease. The standard polymerase chain reaction (PCR) protocol was modified to allow semiquantitative analysis of the samples. Oligonucleotide primers labelled with 32P gamma-ATP were used to detect a segment of HIV DNA and a control DNA gene segment (HLA genome) to estimate the ratio of infected cells. The 32P-labelled PCR products were separated on acrylamide gels and visualised directly by autoradiography and computer densitometry. Infection of human cartilage in vivo was demonstrated in nine of the ten samples in which the PCR analysis was positive. The other did not react sufficiently to produce detectable radiolabelled PCR product despite repeated DNA digestion and extraction. Cartilage infected with HIV could be a potential source of HIV when used in operations.

Analysis of double-stranded polymerase chain reaction products from the Bacillus cereus group by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The analysis of polymerase chain reaction (PCR) products by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) has been achieved. Specifically, a 105 base-pair nucleotide portion of the ribosomal spacer region was amplified in two members of the B. cereus group (i.e. B. thuringiensis and B. cereus) using PCR. These amplified regions were then analyzed by gel electrophoresis and ESI-FTICR. Based on the predicted sequence of the PCR products for each organism, the mass measurement using ESI-FTICR matched the theoretical mass within experimental error and was consistent with gel electrophoresis results. In contrast, for the typical several hour time-scale of the gel electrophoresis experiment, the mass spectrometric analysis was completed in a matter of minutes. To our knowledge, this constitutes the first report demonstrating the ionization and detection of a double-stranded PCR product by ESI-MS. This preliminary result indicates the potential use of ESI-MS to analyze PCR products on a rapid time-scale, with potential for medical and taxonomic applications.

Mathematical considerations of competitive polymerase chain reaction

Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1–5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals.

Macroporous hydrophobic cloth (polymacron) as a solid phase for nucleic acid probe hybridizations

Polyester cloth (polymacron) was employed as a solid phase onto which polymerase chain reaction (PCR) products from theListeria monocytogenes hlyA gene were dot blotted and sensitively detected by hybridization with nucleic acid probes. This inexpensive solid phase exhibited a high DNA binding capacity and provided ease of handling and washing between each reaction step, resulting in simple hybridization procedures for PCR product detection.

Polymerase chain reaction and non-radioactive gene probe based identification of mosquito larvicidal strains of Bacillus sphaericus and monitoring of B. sphaericus 1593M, released in the environment

In attempts towards an accurate monitoring in the environment, the status of the deliberate release of Bacillus sphaericus, a powerful mosquito larvicidal agent, as well as to evolve a rapid method of screening for potent isolates of B. sphaericus, we have developed a polymerase chain reaction (PCR) method of analysis. Using specific primers spanning the 5′ and 3′ ends of the coding sequences of these two mosquito larvicidal genes, a 1.3 and a 1.1 kb product from gene A and a 2.6 kb product from gene B have been amplified by PCR. The primers and the products amplified from them in PCR are highly specific for B. sphaericus. We used digoxigenin based non-radioactive chemiluminescence method for the detection of PCR products and the sensitivity of the method was high enough to detect the presence of 1 to 5 cells of B. sphaericus. A simple and inexpensive sample processing procedure has also been developed for direct PCR amplification of B. sphaericus DNA from field samples collected from areas where it had been applied. Several highly toxic to non-toxic strains of B. sphaericus were screened with these primers by PCR for the presence of either or both of these toxin genes. The results indicate that there is a good correlation between the presence of both genes with higher toxicity of the strains.

A polymerase chain reaction-enzyme-linked immunosorbent assay method for detecting human papillomavirus in cervical carcinomas and high-grade cervical cancer precursors

To develop and evaluate a novel polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA)-based method for detecting high-oncogenic-risk human papillomaviruses (HPV). An HPV assay based on PCR amplification of a region of the E6 open reading frame and ELISA detection of PCR products that specifically identify high-oncogenic-risk HPV types (eg, types 16, 18, 31, 33, 35, 39, 45, 56, 58, and 65) was developed. Dacron swabs were used to obtain samples from the cervices of 371 women referred for colposcopy. The swabs were analyzed using the PCR-ELISA method. The results of HPV DNA testing were then compared with the results of a repeat Papanicolaou smear and cervical biopsy obtained at the same visit. The sensitivity of the PCR-ELISA HPV test for detecting invasive cervical cancer or high-grade squamous intraepithelial lesions (SIL) was 90%. High-oncogenic-risk HPVs were detected in six of seven women with biopsy-confirmed invasive cervical cancer, 74 of 81 women with biopsy-confirmed high-grade SIL, 58 of 128 women with biopsy-confirmed low-grade SIL, and 46 of 155 women with no evidence of cervical disease by colposcopy and cervical biopsy. When used in conjunction with a repeat Papanicolaou test, 97% of the women with invasive cervical carcinoma and high-grade SIL lesions were identified. The PCR-ELISA-based HPV detection provides the potential for an automated, rapid, and sensitive test for cervical cancer and high-grade cervical lesions.

Improved microplate immunoenzymatic assay of PCR products for rapid detection of Mycoplasma pneumoniae

We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction

Background: The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. Objectives: In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). Study design: The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. Results: In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. Conclusion: This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.

Nonradioactive labeling and high-sensitive detection of PCR products.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.

Colorimetric detection of competitive PCR products for quantification of hepatities C viremia

A method based on competitive polymerase chain reaction (PCR) and colorimetric detection of the amplified products was developed to quantify hepatitis C virus (HCV) genomes. Serum samples were obtained from patients who were treated with interferon alpha (IFN-α). After reverse transcription of the HCV RNA, the cDNA was coamplified with a serially diluted cloned HCV competitor DNA using nested PCR. The competitor DNA consisted of the amplified region of the wild type HCV cDNA with an internal region substituted with the lac operator ( lacO) sequence. The PCR products were quantitated specifically by a colorimetric solid-phase assay. The results suggest that the method is well suited for analysing the kinetics of the anti-HCV effects during IFN-α treatment. The quantification assay is simple, reliable and suitable for quantitating HCV genomes in a large number of clinical samples.

Analysis of polymerase chain reaction product by capillary electrophoresis and its application to the detection of single base substitution in genes

Capillary gel electrophoresis (CGE) was studied for the direct analysis of polymerase chain reaction (PCR) amplified samples. A low cross-linked polyacrylamide gel (3%T, 0.5%C) was used for CGE with treated and untreated silica capillaries. CGE showed high reproducibility and resolution in the separation of DNA fragments ( ca. 100–1000 base pairs) produced by PCR. The CGE system was applied to the detection of an amplification refractory mutation system (ARMS) and PCR—restriction fragment length polymorphism (PCR-RFLP), which are detection methods of single base substitution in genes using PCR. With the CGE system, full automation of PCR product detection is feasible.

Determination of DRB alleles using PCR amplification and allele-specific primers.

HLA class-II allelic diversity is commonly defined using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or the combination of PCR and restriction fragment length polymorphism methods (PCR-RFLP). Nevertheless, the identification of the DRB polymorphism by PCR-SSO is a time-consuming procedure and the PCR-RFLP is cumbersome. A rapid technique which allows a precise and extensive HLA-DRB typing is required, particularly in order to study the role of class-II matching in organ transplantation. A DRB typing method based on the detection and length of PCR products amplified using combination of allele specific primers has been developed. Thirty-four DRB alleles (29 DRB1, 4DRB3, 1DRB4) can be detected using 29 primers distributed into 19 amplification mixtures.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA

A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and differentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogroups and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample preparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combined with proteinase treatment. Regardless of the method used, the PCR assay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold excess of indigenous bacteria. When the samples were enriched overnight in a nonselective medium, the sensitivity was increased to approximately 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR products with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualization of amplified fragments in a microtiter plate format with an optical density reader. DIANA and gel electrophoresis showed complete concordance in their discrimination between positive and negative samples. The combination of immunomagnetic separation, nested PCR, and DIANA makes possible the development of a fully automated analytic process which requires a minimum of laboratory manipulations.

High-performance liquid chromatography to assess the effect of serum storage conditions on the detection of hepatitis C virus by the polymerase chain reaction

The effect of various serum storage conditions on the detection of hepatitis C virus (HCV) by the polymerase chain reaction (PCR) was assessed. 50 μl aliquots of serum from four HCV PCR positive patients were subjected, in triplicate, to: (a) storage at −20°C for 1, 5, 10 days; (b) storage at room temperature (RT) for 1, 2, 7 days; (c) 1, 3, 5, and 10 successive freeze-thaw cycles; (d) incubation at 37°C, and 56°C for 1, 3, 24 h; and (e) storage in guanidium-thiocyanate extraction buffer at RT, and 4°C for 1, 5, 10 days. PCR products were detected by agarose gel electrophoresis (AGE) and quantitatively by high-performance liquid chromatography (HPLC). Only storage in extraction buffer at RT for 5–10 days and incubation at 56°C for 24 h appeared to result in a loss of ⩾50% of detectable HCV PCR product. Up to 10 successive freeze-thaw cycles, storage at −20°C for up to 10 days or at RT for up to 7 days, storage in extraction buffer at RT for 1 day or at 4°C for up to 10 days, and incubation at 37°C for up to 24 h resulted in minimal PCR signal loss. HPLC was a reproducible method of detecting and quantitating HCV PCR products, and was more sensitive than AGE.

Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis

The complexity, expense, and susceptibility to contamination of the polymerase chain reaction (PCR) are all issues which need to be overcome if PCR is to be used outside of research laboratories. We addressed these problems with respect to the diagnosis of tuberculosis. First, we simplified the procedure for extracting Mycobacterium tuberculosis DNA from sputum samples. Two methods of sample preparation were compared: the chaotrope-silica method and a novel, more simple chloroform method. Second, we developed a colorimetric method for product detection. This method was as sensitive and specific as agarose gel electrophoresis for detection of PCR product. By using a one-tube nested protocol, 5 to 50 genome equivalents of M. tuberculosis DNA were detected. The simplified colorimetric PCR was compared with microscopy and culture for detection of M. tuberculosis in clinical specimens of sputum. A total of 171 sputum samples were investigated from 108 patients, 12 of whom were subsequently found to have tuberculosis by culture and/or microscopy. PCR of samples prepared by the chaotrope-silica method had a sensitivity of 75% and a specificity of 100% whereas PCR of samples prepared by the chloroform method had a sensitivity of 92% and a specificity of 99% when compared with the sensitivities and specificities of the combined classical microbiological methods for the diagnosis of tuberculosis. The simplified colorimetric PCR in combination with the chloroform sample preparation method was at least as sensitive as microscopy but had a greater specificity because samples with atypical mycobacteria were not detected by PCR. The sensitivity of the method for detection of smear-negative and extrapulmonary tuberculosis remains to be investigated.

Immunoglobulin heavy chain rearrangements in primary brain lymphomas. A study using PCR to amplify CDR‐III

Primary brain lymphomas (PBLs) have only rarely been analysed for immunoglobulin heavy chain (IgH) rearrangements. In this study, DNA was extracted from paraffin blocks in 23 cases of PBL and examined for IgH rearrangements using the polymerase chain reaction (PCR) to amplify the complementarity-determining region III (CDR-III) of rearranged IgH genes. Fifteen of the cases were phenotyped on paraffin-embedded tissue using a pan-B and pan-T antibody (L26 and UCHL-1, respectively). The remaining eight cases were not phenotyped for lack of tissue. For comparison, we used DNA extracted from paraffin blocks of normal brain, lymph nodes with lymphoid hyperplasia, and non-lymphoid malignancies. PCR products were examined by polyacrylamide gel electrophoresis. Among the ten B-cell PBL; four had a pattern indicative of IgH rearrangement, one had a germline pattern, and five had no detectable PCR products. Among the five T-cell PBLs, one had a germline pattern and four had no detectable products. Among the eight untyped PBLs, two had IgH rearrangement, four had a germline pattern, and two gave no detectable products. DNA from non-lymphoid tissues gave a consistent germline pattern, while DNA from polyclonal lymphoid populations (lymph node) had a pattern of polyclonal IgH rearrangement. In a dilution study, a clonal rearrangement could be detected as long as the clone's DNA constituted at least 10 per cent of the total DNA. PCR to amplify CDR-III can be successfully applied to DNA extracted from paraffin blocks, and it detected a clonal rearrangement in 50 per cent of cases that gave a detectable pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison study of human papillomavirus prevalence by the polymerase chain reaction in low risk women and in a gynaecology referral group at elevated risk for cervical cancer

The reported prevalence of human papillomavirus (HPV) type 16 in the genital tracts of women with various gynaecological conditions is highly variable. In particular, some results with the polymerase chain reaction (PCR) technique have suggested that HPV-16 is a ubiquitous or very common virus. We undertook this study to help clarify the current confusion. PCR with HPV consensus L1 primers and specific E6 primers was used to study 89 women attending two gynaecology referral clinics, as well as 99 women attending a health maintenance organization (HMO) clinic; 70 of these latter women had no current or prior history of genital HPV disease. HPV-16 was detected in less than 5% of cytologically normal women from either group and in 17% (6/36) and 31% (9/29) of women with cervical intraepithelial neoplasia (CIN) from the referral clinic and the HMO, respectively. The other high-risk or intermediate-risk HPVs (types 18, 31, 33 or 35) were less prevalent than HPV 16 in all groups of women. A majority of the HPV types detected by the L1 primers in normal women were uncharacterized HPVs. Overall these uncharacterized HPVs were detected in 37% (46/123) of the normal women and in 48% (31/65) of the women with CIN. Using the most sensitive PCR product detection method employed in the study, HPV DNA was detected in 36% (4/11) of swab specimens obtained from the external abdomen.