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At least 343 records · Page 19Linked to original sources

PubMed1996

Single-tube heminested PCR coupled with 'touchdown' PCR for the analysis of the walleye dermal sarcoma virus env gene.

The nucleotide sequences of geographically distant isolates of a newly characterized retrovirus of fish, the walleye dermal sarcoma virus (WDSV) were compared. Primers were designed based on the nucleotide sequence of a single molecular clone isolated from a tumor collected from a fish in New York state, USA. Conventional polymerase chain reaction (PCR) did not allow the satisfactory amplification of WDSV isolates from a different geographic region (Quebec, Canada), probably owing to the high genetic variability of retroviruses. To allow the successful amplification of Quebec WDSV isolates, heminested PCR and 'touchdown' PCR were combined in a highly sensitive, short and simple protocol taking place in a single tube, thus minimizing cross contamination. This technique allowed the first unambiguous detection of a fish retrovirus in Canada and the phylogenetic analysis of various geographic isolates of this virus. Other potential applications for this technique include PCR amplification using degenerate primers, and amplification of templates with some expected degree of genetic variability.

Animals↗

PubMed2005

Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species.

The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 degrees C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.

Animals↗

PubMed2006

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

In the present study, we analyzed the kinetics of the PCR-suppression effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an improved template-switching full-length cDNA synthesis method that greatly reduces truncated cDNA. This study provides a general guide for the design of PS-effect related PCR and is useful for representative or long-transcript enriched cDNA library construction, especially when only a small amount of total RNA is available.

Base Sequence↗

PubMed2004

Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid.

The presence of the human polyomaviruses JCV and BKV in immunocompromised patients can lead to lethal diseases and conditions including progressive multifocal leukoencephalopathy (PML), interstitial nephritis, hemorrhagic cystitis, and kidney allograft rejection. Typically, detection of JCV and BKV in clinical samples has employed standard PCR amplification for viral nucleotide sequences, with subsequent confirmation for viral genome specificity of PCR products by southern blot hybridization. Here, we directly tested a validated PCR-southern protocol with a TaqMan real-time PCR protocol (Applied Biosystems) to assay clinical samples of urine and cerebrospinal fluid. We found equal specificity and sensitivity with both methods. However, real-time allowed for absolute viral-genome quantitation without the use of radionucleotides and was performed more rapidly, in as little as 24 h. Such advantages are important to consider in the effort to establish international standardization of controls for the detection of JCV and BKV, which would aid in screening confidence and the reliable assessment of anti-viral therapies.

BK Virus↗

PubMed2005

Use of quantitative real-time PCR (qRT-PCR) to measure cytokine transcription and viral load in murine cytomegalovirus infection.

A quantitative real-time PCR (qRT-PCR) assay was developed to measure cytokine transcription profiles and viral load during sub-clinical and clinical infection with murine cytomegalovirus (MCMV). Primers/fluorogenic probes specific for mouse cytokines and for the immediate early gene 1 (IE1) of MCMV were used to quantitate cytokine responses and viral load in various organs of MCMV infected mice. Increased mRNA levels of TNF-alpha, INF-gamma and IL-10 were detected in the spleens, lungs and livers of clinically infected mice at 5 days post-infection. Transcription of these cytokines was 2-5-fold lower (p=0.07 for each cytokine) in the spleens and 10-100-fold lower in the lungs (p=0.03 for INFgamma, not significant for IL-10 and TNFalpha) and livers (p<0.05 for each cytokine) of sub-clinically infected mice. Clinical MCMV infection induced high levels of IL-6 in the lungs and spleens of infected animals, while no significant transcription of IL-6 was detected in any organ during sub-clinical infection (p<0.05). The timing of peak amounts of INF-gamma, IL-10 and IL-6 observed in the spleens of clinically infected mice correlated with high viral loads in these organs. Cytokine expression rose in the salivary glands later, at day 15, corresponding to the increase in salivary gland viral load. The qRT-PCR demonstrates that infection with MCMV induces an organ-specific cytokine response characterized by the production of TNF-alpha, INF-gamma, IL-6 and IL-10 which correlates with severity of the disease (sub-clinical versus clinical) and with viral load. In summary, qRT-PCR is a sensitive and accurate method to study MCMV infection and host responses to the virus.

Animals↗

PubMed2005

A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities.

The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/FR1-GC) that amplifies almost the entire 18S rRNA gene for the DGGE analysis of fungal communities in soil samples from 36 sites. This direct PCR system failed to amplify the fragment of interest from the total DNA extracted from most of the soils tested. Thus, we developed a new semi-nested PCR system based on the initial amplification of over 1,700 bp of the 18S rRNA gene with a new primer combination, followed by a subsequent amplification with NS1/FR1-GC. By means of the PCR approach developed in this study distinct 18S rRNA gene amplicons could be reproducibly generated for all soil samples. Amplification tests with 101 soil fungal isolates showed that with the new semi-nested system 18S rRNA gene fragments could be obtained from more fungi than with the direct approach. The subsequent DGGE separation of community amplicons resulted in a high resolution and revealed reproducible complex soil fungal communities specific for each site, despite a minor variability between replicates of the same sample. The semi-nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a robust, reliable and sensitive tool for the analysis of soil fungal community structure.

Cluster Analysis↗

PubMed2006

PCR and PCR-RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum.

Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the Lamiaceae family; its active ingredient, the neoclerodane diterpene salvinorin A, is a psychotropic molecule that produces hallucinations. A comparative evaluation of S. divinorum fresh and dried leaves, S. officinalis fresh leaves, and dried powdered leaves claimed to be S. divinorum was done. HPLC-MS data confirmed the presence of salvinorin A in both S. divinorun leaf extracts and the powdered leaves, whereas no salvinorin A was found in S. officinalis. The non-transcribed spacer (NTS) in the 5S-rRNA gene of all leaf samples and the dried powdered leaves was amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 500bp for S. divinorum and 300bp for S. officinalis) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences, great diversities were found in the spacer region of the two species. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR-restriction fragment length polymorphism (PCR-RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in S. divinorum NTS region at 428-433bp. For TaqI, multiple sites (161-164, 170-173, and 217-220bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235-238bp). The results of this work show that the combined use of analytical chemical (HPLC-MS) and molecular (DNA fingerprinting) methods lead to the precise and unequivocal identification of S. divinorum.

Base Sequence↗

PubMedDate not supplied

Trypanosoma brucei: trypanosome strain typing using PCR analysis of mobile genetic elements (MGE-PCR).

We describe the development of a single-primer amplification system, which uses the trypanosomal mobile genetic element RIME as a molecular marker for the differentiation of Trypanosoma brucei stocks. Using a well-characterised set of T. brucei stocks from southeast Uganda, Kenya and Zambia, we have evaluated the application of this technique, termed MGE-PCR (mobile genetic element PCR) for the typing of trypanosome strains. The technique revealed considerable variation between stocks and was sufficiently specific to amplify trypanosomal DNA in the presence of host DNA. The results showed a clear distinction between human-infective and non-human-infective stocks. Comparative studies on these stocks using markers for the human serum resistance associated (SRA) gene, which identifies human-infective stocks, demonstrated complete agreement between MGE-PCR derived groups and human-infectivity status. Furthermore, MGE-PCR detects high levels of variability within the T. b. brucei and T. b. rhodesiense groups and is therefore a powerful discriminatory tool for tracking individual T. brucei genotypes and strains.

Animals↗

PubMed2000

Sensitivity of the nested-polymerase chain reaction (PCR) assay for Brugia malayi and significance of 'free' DNA in PCR-based assays.

The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.

Animals↗

PubMed2002

Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR).

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in Côte d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire (n = 141), Angola (n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world (n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene (ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.

Animals↗

PubMed2002

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

DNA Probes↗

PubMed1999

A simple and reliable protocol for the detection of apple stem grooving virus by RT-PCR and in a multiplex PCR assay.

Primers were identified which amplify specifically a 499 bp fragment in the coat protein coding region of apple stem grooving virus (ASGV) genome. These primers were used in various RT-polymerase chain reaction (PCR) analyses for the detection of ASGV in Chenopodium quinoa, Nicotiana occidentalis, and in species of Malus and Pyrus. Isolates of ASGV in Malus and Pyrus from locations in Canada, China, Israel, Japan, Nepal, Pakistan, South Africa, and the U.S.A. were reliably detected in leaf and bark (budwood) tissue. Storage of the tissues at -80 degrees C for more than 4 months did not affect the reliability of detection by immunocapture (IC) RT-PCR. Triton-X was not necessary for the detection of ASGV by IC/RT-PCR, and it was also possible to combine the antibody incubation and virus sap incubation into a single step without any obvious loss in sensitivity. A Tube Capture (TC) RT-PCR procedure was developed that eliminated the need for antibody binding of the virus. Phosphate buffered saline with 2% PVP was identified as the most effective sample-grinding buffer for the detection of ASGV by IC/RT-PCR and TC/RT-PCR. TC/RT-PCR facilitated the simultaneous detection (multiplex PCR) of ASGV and cherry mottle leaf virus.

Base Sequence↗

PubMed1998

An integrated multiplex-PCR and PCR-RFLP typing system for markers associated with seronegative arthritides.

The system was designed with emphasis on the identification HLA-B alleles and genotypes associated or potentially associated with seronegative arthritides. By using a combination of multiplex SSP and PCR-RFLPs, the assays can be economically performed on a large range of sample sizes in diagnosis and epidemiology. 24 HLA-B alleles and subtypes can be discriminated, including options for PCR-RFLP or sequence specific amplification of the allele groups B27 and B60 (B*4001 and B*4007). In addition, the internal control carries central MHC polymorphisms, which can help to identify HLA extended halplotypes. False negatives, caused by preferential amplification of the internal control under suboptimal PCR conditions, were prevented by employing new, optimized PCR buffer. Four of the HLA-B primers were pooled into a multiplex reaction whose products were subtyped by digestion with seven restriction endonucleases. Specificity and sensitivity were verified in a panel of 68 homozygous cell lines and 200 heterozygous samples. An HLA-B*27-B*40 hybrid allele was observed in 3 out of 95 B*27-positive individuals from Berlin, Germany. Such an allele could be mistyped by some published assays as a B*27/B*40 heterozygote, a genotype reported to confer an increased risk for ankylosing spondylitis.

Alleles↗

PubMed1999

High resolution HLA-DQB1 typing by combination of PCR-RFLP and PCR-SSCP.

A reliable method for high-resolution HLA-DQB1 typing using a combination of PCR- restriction fragment length polymorphism (RFLP) and PCR-single strand conformation polymorphism (SSCP) analysis is described. The second exon of the DQB1 gene was subjected to PCR using generic primers and digested with two restriction enzymes, MspA1I and HaeIII, and the DQB1 alleles were divided into seven groups. According to the RFLP patterns, appropriate group specific primers for DQ5, 6 and DQ2, 3, 4 groups were used to selectively amplify the alleles and the SSCP technique was used to distinguish the individual alleles. A total of 88 quality control samples of various ethnic groups distributed in the International Cell Exchange and HLA DNA Exchange programs and the ASHI/CAP Proficiency Tests were investigated by the PCR-RFLP/SSCP method. The concordance between our typing results and the consensus results of the surveys were 100%, and a total of 14 DQB1 alleles in 49 homozygous and heterozygous combinations were all correctly identified by the method described. This method is accurate, economical and relatively easy to interpret and well suited for routine clinical and research uses.

HLA-DQ Antigens↗

PubMed1998

Detection of tumor-derived DNA in exfoliated cells of urine sediments with a novel quantitative PCR method based on allele-specific PCR.

We have established a novel quantitative method based on the allele-specific PCR, which uses the linearly amplified fragment of the PCR products as the internal control. The improved characteristics of the procedure are the high sensitivity for quantitation of the mutant alleles at ratios of up to 1:10000 and the reduced necessity of the optimization of the PCR conditions for each mutation. Using this modified allele-specific PCR, we could quantify the tumor alleles in the urine sediments of three patients with urothelial cancers that harbored different p53 gene mutations. This method can be applied to other genetic targets that have other types of alterations, such as deletions or insertions.

Alleles↗

PubMed2003

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests.

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.

Animals↗

PubMed2004

A preliminary trial using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) on the same feedstuffs to detect tissues of animal origin.

A preliminary study using multi-target polymerase chain reaction (multiplex PCR) and restriction fragment length polymorphism (PCR-RFLP) was done on the same feedstuffs to detect animal tissues. The results of the two methods differ somewhat: PCR-RFLP did not detect any signal in any sample, but multiplex PCR detected a signal in one sample. These findings could be a basis for further investigations.

Animal Feed↗

PubMed2002

A comparative analysis of FISH, RT-PCR, PCR, and immunohistochemistry for the diagnosis of mantle cell lymphomas.

Mantle cell lymphoma (MCL) diagnosis first relies on morphology and phenotype that may overlap with other B-cell lymphomas. Therefore, the demonstration of t(11;14)(q13;q32), the cytogenetic hallmark of MCL, is considered of diagnostic value. By studying a series of 35 MCL with characteristic morphology and phenotype (CD5+, CD10-, CD20+, CD23-), we have evaluated the applicability and the sensitivity of interphase fluorescence in situ hybridization (FISH) for t(11;14) detection and other techniques: (1) polymerase chain reaction (PCR) for amplification of t(11;14) genomic breakpoint, (2) competitive RT-PCR for the detection of cyclin D1 transcripts overexpression, and (3) immunohistochemistry (IHC) for cyclin D1 protein detection. Tissues from different origins were analyzed: lymph nodes (n = 24), spleen (n = 3), digestive biopsy (n = 3), tonsils (n = 3), and skin (n = 2). Interphase FISH was performed either on touch preparations (n = 11) and frozen (n = 9) or paraffin sections (n = 15). FISH analysis detected t(11;14) in 34/35 cases (97%) and demonstrated a recurrent CCND1 amplification in t(11;14)+ nuclei of the three blastoid MCL variants of our series. Genomic PCR analysis, hampered by the scattering of 11q13 breakpoints, was positive in only 13/35 cases (37%). RT-PCR analysis was applicable on nonepithelial tissues (27/35) and showed cyclin D1 transcript overexpression in all tested cases (27/35). IHC for cyclin D1 protein was performed either on frozen (n = 12) or on paraffin sections (n = 23), and its sensitivity was higher on paraffin sections (91%) than on frozen sections (25%). A cyclin D1 protein immunoreactivity was observed in 24/35 cases (69%). Our study emphasizes on the use of FISH analysis for the direct detection of t(11;14) because its applicability and sensitivity largely exceeded those of other techniques. It may also provide some informations on secondary cytogenetic changes of potential clinical relevance.

Antigens, CD20↗