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PubMed1997

Additional primer sets for an amelogenin gene PCR-based DNA-sex test.

When aligning the human amelogenin X (AmelX)-gene with the human amelogenin Y (AmelY)-gene, 19 regions of absolute homology ranging in size from 22 to 80 bp, 5 deletions located on AmelX from 1 to 6 bp and 5 deletions 1-183 bp long located on AmelY can be observed. The regions of absolute homology are used to design primer sets which span deletions of the X- and/or Y-chromosome. The PCR products generated on the X- and Y-homologues differ in size. New regions of deletions and newly designed primers are described to facilitate the integration of the amelogenin sex test into multiplex PCR reactions.

Amelogenin↗

PubMed1998

On-line coupling of polymerase chain reaction and capillary electrophoresis for automatic DNA typing and HIV-1 diagnosis.

We demonstrate an integrated on-line system with a fused-silica capillary as the microreactor for PCR and capillary gel electrophoresis with laser-induced fluorescence detection for DNA typing and disease diagnosis. Two applications have been investigated: the four short tandem repeat (STR) loci vWA, THO1, TPOX and CSF1PO (CTTv) for DNA typing, and DNA probe for human immunodeficiency virus (HIV-1) diagnosis. The CTTv are important loci in forensic and genetic linkage analysis. The PCR technique is a powerful tool in HIV research because it can detect the presence of the virus before any antibody response in the infected person. Thus it is important for early diagnosis. Multiplexed PCR in a fused-silica capillary, on-line injection, DNA denaturation and calibration based on a standard ladder have been successfully combined. Also, on-line liquid flow management, DNA separation and detection have been completely integrated.

Autoanalysis↗

PubMed2000

Novel method for molecular detection of the two common hereditary hemochromatosis mutations.

We describe a novel molecular screening technique for hereditary hemochromatosis through which HFE genotypes at codon positions 282 and 63 are simultaneously detected. The technique combines multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) and allows automated high-throughput analysis. We used this method to genotype 43 previously characterized anonymous DNA specimens in blinded fashion and found multiplex PCR/DHPLC 100% accurate when compared with PCR/restriction enzyme digestion, yet far more efficient.

Base Sequence↗

PubMed2002

The t(4;22)(q12;q11) in atypical chronic myeloid leukaemia fuses BCR to PDGFRA.

Chronic myeloid leukaemia (CML) is characterized by the presence of the BCR-ABL fusion gene, usually in association with the t(9;22)(q34;q11) translocation. We report here the identification and cloning of a rare variant translocation, t(4;22)(q12;q11), in two patients with a CML-like myeloproliferative disease (MPD). RT-PCR indicated that both patients were negative for BCR-ABL, but FISH analysis suggested that the BCR gene was rearranged. Since other translocations in MPDs frequently involve tyrosine kinases, we designed a multiplex PCR to search for mRNA fusions between BCR and three potential partner genes at 4q12: KIT, KDR and PDGFRA. An unusual inframe BCR-PDGFRA fusion mRNA was identified in both patients, with either BCR exon 7 or exon 12 fused to short BCR intron-derived sequences, which were in turn fused to part of PDGFRA exon 12. Sequencing of the genomic breakpoint junctions showed that the chromosome 22 breakpoints fell in BCR introns whereas the chromosome 4 breakpoints were within PDGFRA exon 12. This is the first report of a fusion gene that involves PDGFRA. Our findings indicate that apparently simple cytogenetic variants of t(9;22) do not always mask a cryptic BCR-ABL fusion, even when found in association with clinical and haematological indications of CML.

Adult↗

PubMed1999

Approximately half of the erythroblasts in maternal blood are of fetal origin.

The enrichment of fetal erythroblasts from the peripheral blood of pregnant women is currently actively pursued for the development of a non-invasive means of prenatal diagnosis. Since erythroblasts in maternal blood are not all of fetal origin, and currently no reliable method exists to distinguish between the maternal and fetal erythroblasts, their use for prenatal diagnosis is not without uncertainty. The purpose of this study was to determine the percentage of fetal erythroblasts in maternal blood at the single cell level and to what extent such cells can reproducibly be used for polymerase chain reaction (PCR)-based prenatal diagnostic analyses. Erythroblasts were enriched from the peripheral blood of rhesus negative pregnant women using magnetic cell sorting (MACS). Single erythroblasts identified morphologically were individually micromanipulated and analysed by a multiplex PCR reaction for the fetal SRY and rhesus D genes. As a control for the PCR reaction the beta-globin gene was used. The PCR results were validated by the results obtained by invasive procedures. In all instances where single erythroblasts were examined, the correct fetal genotype for the two fetal specific loci was detected. Furthermore, our results indicate that approximately 50% of the enriched erythroblasts are of fetal origin.

DNA-Binding Proteins↗

PubMed2006

Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.

Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A-H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes.

Base Sequence↗

PubMed2002

Incidence and molecular analysis of Vibrio cholerae associated with cholera outbreak subsequent to the super cyclone in Orissa, India.

An epidemiological study was carried out to find out the aetiological agent for diarrhoeal disorders in the cyclone and flood affected areas of Orissa, India. Rectal swabs collected from 107 hospitalized diarrhoea patients were bacteriologically analysed to isolate and identify the various enteropathogens. Detection of toxic genes among E. coli and V. cholerae was carried out by polymerase chain reaction (PCR) assay. Of the 107 rectal swabs analysed, 72.3% were positive for V. cholerae O1 Ogawa, 7.2% for V. cholerae O139, 1.2% for E. coli (EAggEC) and 1.2% for Shigella flexneri type 6. Using multiplex PCR assay it was found that all V. cholerae isolates were ctxA positive and El Tor biotype. Strains of V. cholerae O1 were observed to be resistant to nalidixic acid, furazolidone, streptomycin, co-trimoxazole and ampicillin. Except for nalidixic acid, the resistance pattern for O139 was identical to that of O1 strains. Representative strains of V. cholerae were further characterized by randomly amplified polymorphic DNA (RAPD) analysis and ribotyping. Both O1 and O139 V. cholerae strains exhibited the R3 pattern of ribotype and belonged to a similar pattern of RAPD compared with that of Calcutta strains. Early bacteriological and epidemiological investigations have revealed the dominance of V. cholerae O1 among the hospitalized patients in cyclone affected areas of Orissa. Drinking water scarcity and poor sanitation were thought to be responsible for these diarrhoeal outbreaks. Timely reporting and implementation of appropriate control measures could contain a vital epidemic in this area.

Cholera↗

PubMedDate not supplied

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.

DNA↗

PubMed2003

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory.

A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2x10(2) c.f.u. ml(-1). Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.

Cerebrospinal Fluid↗

PubMed2006

Rapid identification of Enterococcus hirae and Enterococcus durans by PCR and detection of a homologue of the E. hirae mur-2 Gene in E. durans.

During an evaluation of PCR for identification of isolates of Enterococcus hirae, a homologue with 82% identity to E. hirae mur-2 was identified in Enterococcus durans and was named mur-2(ed). PCR using primers for two genes (copY and murG) of E. hirae strains showed amplification with E. hirae strains only. PCR (under high-stringency conditions) with primers for the mur-2(ed) gene gave the expected amplification product only with E. durans strains. A combination of murG and mur-2(ed) primers in a multiplex PCR assay differentiated E. hirae from E. durans in all cases. PCR using these primers appears to be a rapid alternative for identification of E. hirae and E. durans isolates.

Bacterial Outer Membrane Proteins↗

PubMed2001

Rapid identification of Enterococcus durans and Enterococcus hirae by PCR with primers targeted to the ddl genes.

Species-specific PCR assays with primers targeted to D-alanine:D-alanine ligase (ddl) encoding genes were developed for the identification of Enterococcus durans and E. hirae. The specificity of the primers was validated in a multiplex PCR on well characterised E. durans (n=30) and E. hirae (n=16) strains, all of which were identified correctly. This PCR procedure offers a reliable and rapid alternative to conventional phenotypic methods for speciation of these enterococci of growing clinical importance.

Bacterial Typing Techniques↗

PubMed2000

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes.

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.

Buffers↗

PubMed2001

The neuroprotective actions of FK506 binding protein ligands: neuronal survival is triggered by de novo RNA synthesis, but is independent of inhibition of JNK and calcineurin.

The immunosuppressant FK506 displays substantial neuroprotective and neuroregenerative effects. It is not fully understood to which extent these effects depend on the inhibition of the calcineurin phosphatase (PP2B). The present study has re-addressed this issue using Lie120, a novel highly specific inhibitor of calcineurin, which does not block the enzymatic activity of FKBPs or cyclophilins, respectively. We have determined the effect of FK506 (10-500 nM), V-10,367 (a FK506 derivative which does not block calcineurin; 1-5 microM) and Lie120 (a novel specific inhibitor of calcineurin, 0.1-5 microM) on the cellular survival and the pro-degenerative JNK activity of PC12 and Neuro2A cells following application of 200 microM H(2)O(2). FK506 and V-10,367, but not Lie120, protected both cell lines against H(2)O(2)-mediated death, whereas an increase in JNK1 activity was blocked by FK506 and Lie120, but not by V-10,367. Co-incubation of FK506 and V-10,367 with the mRNA synthesis inhibitor actinomycin D abolished the protective effect of FK506 and V-10,367. This antagonization was effective when actinomycin D was applied 30 min or 1 h, but not 2 or 4 h, after H(2)O(2) suggesting that FKBP-ligands confer their neuroprotection by rapid de novo synthesis of (functionally) anti-apoptotic proteins. The search for the corresponding effector genes revealed that the expression of FKBP25, FKBP38 and FKBP52 (analysis by reverse transcription-polymerase chain reaction (RT-PCR) did not change following H(2)O(2) or FK506, and this was also true for the expression of apoptosis-related genes caspase 3, bax, bcl-2 and bcl-xL (analysis by Multiplex-PCR). Summarizing, neuronal protection by FKBP-ligands is not mediated either by calcineurin or by JNK1 in this experimental set-up, whereas the FK506 mediated inhibition of JNK1 is realized by the inhibition of calcineurin, an effective activator of JNK1 in neurons.

Animals↗

PubMed1999

Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae. Genitourinary infections in males by the Amplicor PCR assay of urine.

The Amplicor CT/NG polymerase chain reaction (PCR) test on urine specimens from males was prospectively evaluated against established specimens and laboratory methods for diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae genitourinary infections, in patients from a remote region of Western Australia. Seventy-three males who were tested for both C. trachomatis and N. gonorrhoeae by both conventional methodology and Amplicor PCR on urine were enrolled in the study. Established testing comprised enzyme immunoassay/immunofluorescence antigen testing (EIA/IF) for C. trachomatis and microscopy and/or culture for N. gonorrhoeae on urethral swabs. Positive test results were confirmed using a set of criteria that included supplemental PCR testing and clinical history. Overall, 13.7% of patients were resolved as positive for C. trachomatis and 52.1% as positive for N. gonorrhoeae. The sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens for C. trachomatis were 80.0% (8/10) and 95.2% (60/63), compared with 60.0% (6/10) and 100.0% (63/63) for EIA/IF on urethral swabs. For N. gonorrhoeae, the sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens were both 100% (38/38 and 35/35, respectively) compared with 86.8% (33/38) and 100% (35/35) for microscopy and/or culture on urethral swabs. The results of this study indicate that the Amplicor CT/NG multiplex PCR test for C. trachomatis and N. gonorrhoeae performed on urine in males provides a highly sensitive, specific, and robust method for the diagnosis of both C. trachomatis and N. gonorrhoeae, for the early detection of both symptomatic and asymptomatic infected individuals.

Chlamydia Infections↗

PubMed2003

Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway.

Germline intragenic mutations in PTEN are associated with 80% of patients with Cowden syndrome (CS) and 60% of patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS). The underlying genetic causes remain to be determined in a considerable proportion of classic CS and BRRS without a polymerase chain reaction (PCR)-detectable PTEN mutation. We hypothesized that gross gene deletions and mutations in the PTEN promoter might alternatively account for a subset of apparently mutation-negative patients with CS and BRRS. Using real time and multiplex PCR techniques, we identified three germline hemizygous PTEN deletions in 122 apparently mutation-negative patients with classic CS (N=95) or BRRS (N=27). Fine mapping suggested that one deletion encompassed the whole gene and the other two included exon 1 and encompassed exons 1-5 of PTEN, respectively. Two patients with the deletion were diagnosed with BRRS, and one patient with the deletion was diagnosed with BRRS/CS overlap (features of both). Thus 3 (11%) of 27 patients with BRRS or BRRS/CS-overlap had PTEN deletions. Analysis of the PTEN promoter revealed nine cases (7.4%) harboring heterozygous germline mutations. All nine had classic CS, representing almost 10% of all subjects with CS. Eight had breast cancers and/or benign breast tumors but, otherwise, oligo-organ involvement. PTEN protein analysis, from one deletion-positive and five PTEN-promoter-mutation-positive samples, revealed a 50% reduction in protein and multiple bands of immunoreactive protein, respectively. In contrast, control samples showed only the expected band. Further, an elevated level of phosphorylated Akt was detected in the five promoter-mutation-positive samples, compared with controls, indicating an absence of or marked reduction in functional PTEN. These data suggest that patients with BRRS and CS without PCR-detected intragenic PTEN mutations be offered clinical deletion analysis and promoter-mutation analysis, respectively.

Base Sequence↗

PubMed2003

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments.

A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

Bacterial Typing Techniques↗

PubMed2005

The molecular diagnosis of porcine viral diseases: a review.

The worldwide occurrence and re-occurrence of transboundary diseases like foot-and-mouth disease or classical swine fever indicates that there is a high need for the development of powerful, robust and high-capacity new diagnostic methods, which are able to detect the causative agents before they could spread to large populations and cause tremendous losses. This article reports the experiences of a research group on the development of molecular methods for the improved diagnosis of a range of porcine viral diseases, including diseases on List A of the Office International des Epizooties (OIE). Nucleic acid hybridisation and various polymerase chain reaction (PCR) assays have been applied for routine diagnosis of a large range of viral diseases. During the last one-and-a-half decade more than 40 nested PCR assays have been developed to detect a variety of DNA and RNA viruses. False positive and negative results are avoided by the use of special tools, practices and internal controls of amplification (mimics). Recently, real-time PCR methods (TaqMan, molecular beacons, Primer-Probe Energy Transfer system) have been developed for the diagnosis of a wide range of diseases, such as foot-and-mouth disease, swine vesicular disease and vesicular stomatitis. Multiplex PCR packages have been developed for the simultaneous detection of eight important viruses of swine. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the diagnostic procedures have become rapid, robust and automated. In order to standardise the real-time PCR assays, the rules of OIE are considered. By following the five steps of OIE standardisation and validation, the new diagnostic procedures are nationally and internationally standardised and harmonised. The rapid, powerful and internationally standardised molecular diagnosis contributes to the reduction of losses caused by the transboundary viral diseases in swine populations.

Animals↗

PubMed2004

Are false negative direct immnufluorescence assays caused by varicella zoster virus gE mutant strains?

Strains of Varicella zoster virus (VZV) have been described recently in which a single base mutation in the gE epitope abrogates binding of the 3B3 monoclonal antibody, which is widely used for virus detection in diagnostic laboratories. These strains, named VZV-MSP, are associated with a distinct phenotype in both in vitro culture and in SCID-hu mice. We investigated the possibility that negative direct immunofluorescence results, using the 3B3 antibody, where the presence of virus was confirmed by polymerase chain reaction (PCR) or tissue culture are due in some cases to the MSP strain of VZV. A total of 249 vesicle fluid specimens from people with suspected shingles were examined using direct immunofluorescence, tissue culture and a nested multiplex PCR for VZV, herpes simplex virus type 1 (HSV-1) and 2 (HSV-2). VZV was detected in 218 of 249 (87.6%) cases. Forty-five confirmed VZV specimens, but with negative (30) or indeterminate (15) immunofluorescence results, were analysed further. PCR was used to amplify a fragment in ORF 68 that encodes the VZV gE ectodmain recognised by 3B3 antibody. The fragments were sequenced and analysed for the single base change G448A (D150N), which is present in VZV-MSP as compared with the reference Dumas strain. No VZV gE mutant (MSP/MSP-like) was detected. Overall, PCR was found to be the most sensitive method of confirming VZV infection. False negative VZV immunofluorescence results are unlikely to be due to virus variants.

Antibodies, Monoclonal↗