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At least 235 records · Page 13Linked to original sources

PubMed2006

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

Automation↗

PubMed2003

One-tube semi-nested PCR-ELISA for the detection of human cytomegalovirus DNA sequences; comparison with hybridization-based and semi-nested-based PCR-ELISA procedures.

Amplification of DNA targets by polymerase chain reaction (PCR) followed by their colorimetric detection in an enzyme-linked immunosorbent assay (ELISA) is increasingly used in both immunological research and clinical practice. Several methods for the labeling and detection of amplified DNA sequences have been previously described. In this study, we compared the conventional hybridization-based PCR-ELISA with a modified semi-nested and one-tube semi-nested PCR-ELISA for the detection of human cytomegalovirus (HCMV) DNA. Amplified DNA sequences were labeled with biotin and 2,4-dinitrophenyl (DNP), and detected by DNP-specific monoclonal antibody conjugated to alkaline phosphatase. Using a cloned HCMV DNA as a template, we found that the one-tube semi-nested PCR-ELISA gave a strong positive response when 20 copies of the template were used, whereas both the hybridization-based and the semi-nested-based PCR-ELISA required at least 200 template copies. The claim of higher sensitivity and robustness of the one-tube semi-nested assay was also supported by the analysis of plasma samples from patients treated for HCMV infection. Since the modified one-tube semi-nested PCR-ELISA is quick, sensitive and easy-to-perform, it can be used with advantage in routine identification of DNA targets in clinical and other specimens.

Cytomegalovirus↗

PubMed2004

Allele-frequency determination of BsmI and FokI polymorphisms of the VDR gene by quantitative real-time PCR (QRT-PCR) in pooled genomic DNA samples.

The vdr gene is a candidate for osteoporosis susceptibility, with conflicting results in association studies. We have designed and optimized an individual allele-specific and DNA pooling PCR-based methodology to quantitate BsmI and FokI polymorphisms of the vdr gene and studied single-nucleotide polymorphisms (SNPs) from pooled DNA samples. The allele frequency in DNA pooling experiments has been analyzed by kinetic PCR: quantitative real-time PCR (QRT-PCR). A Spanish cohort of 225 healthy postmenopausal women was studied. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA) and quantitative ultrasound calcaneous densitometry. Allele-specific PCR amplification of BsmI and FokI genes showed full concordance with the PCR-RFLP approach. The prevalence of the three BsmI VDR genotypes was 19.1, 44.9 and 36.0% for BB, Bb and bb, respectively. In the case of the FokI locus, the prevalence of genotypes was 40.4, 48.0 and 11.6% for FF, Ff and ff, respectively. No positive correlation was found between polymorphism and BMD. The DNA pooling procedure was validated. No differences were found in allele frequencies and T-score data obtained using the high throughput DNA pooling approach, as compared to known individual frequencies. In our hands, this is a very useful approach to study quantitative (thus polygenic) traits like osteoporosis susceptibility.

Absorptiometry, Photon↗

PubMed2006

A one-step, gel-based RT-PCR assay with comparable performance to real-time RT-PCR for detection of classical swine fever virus.

Classical swine fever, a notifiable disease to the Office International des Epizooties (OIE), is a highly contagious viral disease affecting both domestic pigs and wild boars. Rapid, sensitive, and specific detection of the causing agent classical swine fever virus (CSFV) is therefore essential for diagnosis and control of the disease. Most protocols for gel-based PCR consist of two steps, reverse transcription followed by PCR. Such a protocol is time consuming, laborious and more prone to contamination. Two highly sensitive and fast one-step RT-PCR assays were developed for gel-based and real-time detection of CSFV, and their performances were compared to that of a published real-time assay. The results showed that the gel-based assay had comparable performance to the real-time RT-PCR assays for detection of the virus. A detection limit of 50 copies was achieved by both assays. It is concluded that the one-step gel-based RT-PCR assay provides the simplest and most sensitive method for detection of CSFV in cell culture material or clinical samples, that can be applied in laboratories without facilities for real time PCR assays. The one-step format minimizes the risk for cross contamination and the hands-on time. The real-time assay is suitable for high-throughput screening of the virus in large populations.

Animals↗

PubMed2002

Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus.

Chicken anemia virus (CAV) is a ubiquitous pathogen of poultry. A CAV specific TaqMan-based PCR and RT-PCR assay for real-time quantitation of viral load and relative quantitation of virus-specific transcript levels was developed. Detection of viral DNA copy number from infected MDCC-CU147 cells was determined by extrapolation from a CAV plasmid-based standard curve. Viral load increased proportionally with increasing cell number harvested, increasing from 4x10(2) copies in 250 cells with 38% virus positive cells in an indirect immunofluorescence assay to 8x10(5) copies in 250,000 cells with 64% infected cells. The estimated average viral copy number per infected cell ranged from 5 to 14. Strain-specific primers were developed to distinguish between the Cux-1 and CIA-1 strains of CAV. These primers exhibited a 3 to 4 log differential in amplification comparing homologous versus heterologous virus-primer combinations. The sensitivity of the real-time assay was found to be comparable to a nested PCR assay using DNA samples from a SPF poultry flock exposed to the SH-1 strain of CAV. The real-time PCR detected from 1.7 to 4.2 target molecules in three out of four samples that were positive by nested PCR using 50% of the DNA used in the nested PCR. Relative viral transcript levels for Cux-1 and CIA-1 infected cell cultures increased proportionally with increasing cell numbers harvested for RNA extraction. This assay will be important for both diagnosis and in understanding the complex pathogenesis of CAV infection.

Animals↗

PubMed1998

A multiplex PCR assay for the simultaneous detection of human herpesvirus 6 and human herpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products.

A multiplex polymerase chain reaction (PCR) method was developed for the simultaneous detection of human herpesviruses 6 and 7 (HHV-6; HHV-7) in clinical samples, using primers which amplify a section of the HHV-6 U67 gene and the HHV-7 homologue of the HHV-6 U42 gene. Comparison of the multiplex assay with the respective single PCR assays, using cloned HHV-6 and HHV-7 sequences as targets for amplification, showed equivalent sensitivity and specificity for the assays. To demonstrate the use of multiplex PCR for the analysis of clinical samples, serum and saliva from infants were analysed using this technique. The results showed that a clear distinction can be made between the amplicons of HHV-6 and HHV-7, without loss of sensitivity or specificity. There was complete concordance between the respective single PCR assays, and the multiplex PCR. HHV-6 amplicons derived from the multiplex PCR analysis were typed by differential AvaII restriction endonuclease digestion, in which HHV-6 variant A amplicons are cleaved but those of variant B remain undigested. These results were compared to HHV-6 variant typing by an established method, the results of which showed complete concordance between assays. It is proposed that this multiplex assay, where HHV-6 positive samples may be typed directly from the reaction products, is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of HHV-6 and HHV-7.

Child↗

PubMed2003

Subtyping of foodborne and environmental isolates of Escherichia coli by multiplex-PCR, rep-PCR, PFGE, ribotyping and AFLP.

A total of 54 isolates were characterized by multiplex-PCR for toxin genes and genotyped using several DNA fingerprinting methods: using repetitive extragenic palindromes (REP) and Box primers (rep-PCR), amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE) and ribotyping. The known-pathogenic strains tested were from food and clinical samples (34 strains) and included serovars O157:H7, O111:H8, O111:H11, O91:H21 and O55:H7. Two type cultures, Escherichia coli K12 (ATCC 29425) and DUP-101 (ATCC 51739), were included as known non-pathogenic strains and an additional 17 previously unclassified isolates from animal fecal samples. Comparisons of genomic DNA fingerprint patterns using unweighted pair group method with arithmetic averages (UPGMA) cluster analysis of Jaccard similarity indices indicated that all methods tested showed a greater similarity between the E. coli O157:H7 strains than to other isolates. On the basis of these studies, we propose that AFLP, REP-PCR, Box-PCR and ribotyping techniques can all be used for discriminating O157:H7 isolates and are preferred for large-scale screening because of the speed and ease of the methods. The PFGE method is the best to discriminate between subtypes of O157:H7 associated with specific outbreak investigations; however, it is more time consuming and unnecessary if subtyping is not required. There are differences between the dendrograms generated from each method and the relationship between the other strains analyzed. However, the fingerprint profiles of the O157:H7 isolates were virtually identical using REP-PCR and Box-PCR enabling easy distinction of the group. Thus, these typing methods have the potential to aid investigators in identifying the source of an outbreak to prevent or control further spread of E. coli O157:H7.

Bacterial Toxins↗

PubMed2002

Detection of Bacillus cereus in foods by colony hybridization using PCR-generated probe and characterization of isolates for toxins by PCR.

Isolates of Bacillus cereus from traditional Indian foods were detected by colony hybridization using the PCR-generated phospholipase (PL-1) probe. In all, 29 isolates picked up by the probe were confirmed as B. cereus by conventional cultural and biochemical characteristics. All the isolates reacted positively in PCR with phospholipase (PL-1) primers. Among the native isolates, 11 of them showed the discontinuous pattern of haemolysin BL activity in gel diffusion assay. Though 14 isolates reacted positively in PCR with primers (Ha-1) specific to the B gene of haemolysin BL, only four of them showed both the presence of gene and haemolysin BL activity. More than 50% of the isolates indicated their potential enterotoxigenicity by reacting positively with primers specific for the BceT gene encoding for diarrhoeal enterotoxin. PCR with primers for different inverse (IS) repeat elements revealed that isolates carrying transposon IS 231-P 231-1 did not carry IS 240-P 240. Some of the isolates were devoid ofthese IS elements. The study demonstrated the potential of using of a PCR-generated labelled PL-1 probe for the direct detection of B. cereus in food samples and PCR for characterizing the toxigenic isolates.

Bacillus cereus↗

PubMed1999

PCR assay for detection of the E. coli O157:H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E. coli O157:H7 in ground beef.

A PCR assay targeting the 3' end of the eae-gene of E. coli O157:H7 has been developed. It was shown to be specific for the E. coli O157:H7 eae-gene. Sensitivity of the PCR assay was 1 pg DNA or 10(3) cfu per PCR reaction. Subsequently, a study was conducted to examine the effect of the food matrix and the sample preparation method on PCR detection of non-viable cells using heat-killed E. coli O157:H7 in ground beef as a model system. Inoculated ground beef samples were subjected to either selective enrichment or immediately prepared for PCR analysis. Four sample preparation methods were applied: centrifugation, buoyant density centrifugation (BDC), immunomagnetic separation (IMS), and chelex extraction. Production of false-positive results due to amplification of the DNA of dead cells occurred if the number of heat-killed cells exceeded 10(8) cfu/g. For inocula <10(8) cfu/g, PCR results for heat- killed E. coli O157:H7 cells depended upon the sample preparation method. It was shown that the inclusion of two washings steps of the bacterial pellet before DNA extraction was the critical factor for elimination of false-positive results due to heat-killed cells in the centrifugation and BDC procedure. IMS did not produce false-positive results due to heat-killed cells (two washing steps were included in the IMS procedure). With the chelex-extraction method, false-positive results due to heat-killed cells were obtained. An effect of the ground beef food matrix on the presence of amplifiable DNA was noted.

Adhesins, Bacterial↗

PubMed2005

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Animals↗

PubMed2001

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders.

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

Base Sequence↗

PubMed2001

Polymerase chain reaction (PCR) for human herpesvirus 8 and heteroduplex PCR for clonality assessment in angiolymphoid hyperplasia with eosinophilia and Kimura's disease.

BACKGROUND: Recently, human herpesvirus 8 (HHV-8) has been isolated from almost all cases of Kaposi's sarcoma. It has not been found in most cutaneous hemangioproliferative disorders other than Kaposi's sarcoma. Benign vascular lesions including Kimura's disease were not found to contain the HHV-8 DNA sequence. However, there has been contradictory data concerning the presence of HHV-8 in angiolymphoid hyperplasia with eosinophilia (ALHE). Clonality studies in ALHE and Kimura's disease were rare. METHODS: We performed polymerase chain reaction (PCR)-based analysis to determine whether HHV-8 is present and heteroduplex analysis of rearranged T-cell receptor (TCR) gene for clonality assessment in paraffin-embedded skin biopsy samples of 7 ALHE and 2 Kimura's disease, taken from immunocompetent patients. RESULTS: HHV-8 could not be identified in all the cases of ALHE and Kimura's disease. Although 2 cases (2/7) of ALHE and 2 cases (2/2) of Kimura's disease showed positive result for PCR analysis of TCR, all the cases were negative for heteroduplex-PCR. CONCLUSIONS: We suggest that HHV-8 may not involve in a pathogenetic role in ALHE and Kimura's disease and the failure to demonstrate clonality may be consistent with the reactive nature of these diseases and lack of malignant transformation. In addition, heteroduplex-PCR can be applied to confirm doubtful cases of lymphoma in that heteroduplex-PCR is more specific than PCR as seen in our study.

Adolescent↗

PubMed2003

Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

Animals↗

PubMed2003

Comparison of real-time PCR and conventional hemi-nested PCR for the detection of Bordetella pertussis in nasopharyngeal samples.

We directly compared a conventional hemi-nested PCR assay with a real-time (LightCycler) PCR assay for the detection of Bordetella pertussis in nasopharyngeal samples. Of the 152 samples tested, 49 (32%) were positive by first-round conventional PCR, 56 (37%) were positive by the hemi-nested PCR assay, and 39 (26%) were positive by the real-time assay. All samples testing positive with the real-time assay were also positive by the hemi-nested assay (both first- and second-round PCR), and all culture-positive samples tested positive by both PCR assays. These results suggest that the hemi-nested assay is more sensitive than the real-time assay for detecting B. pertussis.

Adolescent↗

PubMed2004

Genetic analysis of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) and arbitrarily primed PCR: gel analysis compared with microchip gel electrophoresis.

OBJECTIVES: To assess the applicability of a newly emerging microchip gel electrophoresis for rapid strain differentiation among clinical isolates of Pseudomonas aeruginosa, and to compare this technique with the traditional gel method for DNA separation. METHODS: One hundred clinical strains of P. aeruginosa obtained from a hospital in northwestern Ohio were tested for reactivity to 3 serotype-specific monoclonal antibodies by enzyme-linked immunosorbent assay. Twelve strains (4 from each serogroup) were selected for DNA analysis by polymerase chain reaction (PCR)-based, single primer DNA fingerprinting methods with 3 different primers: 1 enterobacterial repetitive intergenic consensus PCR and 2 arbitrarily primed PCRs. The PCR products were analyzed by agarose slab gel and microchip gel electrophoresis. RESULTS: Of the 100 clinical isolates tested, 39% (4%, 14%, and 21%) were found to be serotypes 0:3, 0:6, and 0:11, respectively. Twelve strains were chosen for DNA analysis by PCR. The PCR products were analyzed by agarose slab gel electrophoresis and on microchips to determine interspecies diversity. Both methods demonstrated that different serotypes exhibited different electrophoretic patterns. Two strains (clinical strains 6 and 7, serotype 0:6) showed identical patterns, indicating a high degree of relatedness. CONCLUSION: In all cases, there was concordance between the electrophoretic patterns detected by the two methods. The capability of conducting both PCR and microchip gel electrophoresis offers an opportunity for an automated and rapid method for genetic analysis and differentiation among strains of P. aeruginosa and other microorganisms.

Bacterial Typing Techniques↗

PubMed2002

DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium leprae to assess the efficacy of multi-drug therapy for leprosy.

DNA-PCR and reverse transcription (RT)-PCR for the 18-kDa protein of Mycobacterium leprae were used to examine the efficacy of multi-drug therapy (MDT) in leprosy. MDT was administered for 0-24 months. Fourteen (63.6%) of 22 patients showed positive PCR results after treatment for 12 months and the positive results decreased to 30% after 24 months of MDT. These results did not correlate with the bacterial index (BI) or the IgM antibody titre for the phenolic glycolipid (PGL)-1. One-dimensional densitometric analysis of agarose gels from PCR from the longitudinal study showed a gradual reduction of the 360-bp band after 12-24 months of MDT. RT-PCR for mRNA of the 18-kDa protein successfully tracked bacterial RNA changes in the biopsies and confirmed a decrease in the RNA of M. leprae in patients after MDT for 12 months. Thus, DNA- and RT-PCR for the 18-kDa protein of M. leprae are effective in assessing the efficacy of MDT for leprosy.

Animals↗

PubMed1992

HLA-DQB1 allele typing by a new PCR-RFLP method: correlation with a PCR-SSO method.

We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to follow, requiring only one DQB1 generic amplification and 5 endonucleases to assign 14 out of 15 HLA-DQB1 alleles. In addition, we determined that by using one generic amplification and two enzymes (Sau96 I and Hae III) it was possible to type the generic specificities: DQw2, DQw4, DQw5, DQw6, and DQw7, DQw8-9, providing a practical alternative for serological HLA-DQ generic typing. We also performed a side-by-side correlation with a PCR-SSO typing method and found an almost 100% concordance between the methods. The limitations of these methods were: 1) the PCR-RFLP method did not allow the differentiation between the HLA-DQB1*0602 and *0603 alleles; 2) the PCR-SSO method gave crosshybridization signals in the detection of *0302 or *0303 alleles. Our results suggested that both methods, PCR-RFLP and PCR-SSO, are useful alternatives for HLA-DQB1 typing.

Alleles↗

PubMed1998

The PCR-SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR-SSP primers and primer mixes.

An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class II means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class II sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class II typing set. The SSP Manager is available on request from the authors.

Antisense Elements (Genetics)↗