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At least 181 records · Page 10Linked to original sources
PubMed2004

An allele-specific genotyping method for rat lyst (lysosomal trafficking regulator) gene.

Two spontaneous mutant beige rats, with phenotypes resembling human Chediak- Higashi syndrome (CHS), were found independently in two inbred strains. Both beige mutations were identified to be recessive alleles in the Lyst locus on rat chromosome 17 and the alleles were denoted Lyst(bg) and Lyst(bg-Kyo). As it is almost impossible to discriminate these mutations phenotypically, we developed an allele-specific genotyping method for the Lyst gene. The nested PCR amplification was followed by restriction fragment length polymorphism (RFLP) analysis. By this method, we could discriminate the mutant Lyst(bg), Lyst(bg-Kyo) alleles, and the normal Lyst allele, easily and accurately.

Alleles↗
PubMed1999

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

Bacterial Outer Membrane Proteins↗
PubMed2001

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.

Amino Acid Sequence↗
PubMed2000

Coordinated expression of Hoxb genes and signaling molecules during development of the chick respiratory tract.

To elucidate the molecular mechanism for regulating the region-specific morphogenesis of the chicken respiratory tract, we analyzed the spatiotemporal expression patterns of the Hoxb genes, Bmp-2, Bmp-4, Wnt-5a, and Wnt-11 in the developing respiratory tract. We found region-specific expression of these genes in the mesenchymal layer of the respiratory tract. Before bronchial branching proceeds, Hoxb genes show nested expression patterns around the ventral-distal tip of the lung bud. As morphogenesis proceeds, these expression domains correspond to the morphological subdivisions of the chick respiratory tract. Hoxb-5 and Hoxb-6 expression domains demarcate the trachea, bronchial tree, and air sacs. Particularly the expression domains of Hoxb-6 to -9 correspond to the morphological subdivisions of the air sacs along the proximodistal axis. Bmp-4 and Bmp-2 are expressed throughout the entire pulmonary mesenchyme and its dorsal half, respectively. Wnt-5a and Wnt-11 are expressed in the tracheal mesenchyme. Interestingly, the expression domain of Bmp-2 is complementary to the Hoxb-6 domain. The respiratory mesenchyme influences the process of epithelial branching during morphogenesis. By tissue recombination experiments, we found that the dorsal and the ventral pulmonary mesenchyme, demarcated by Hoxb-6 expression, have different inductive capacities toward the tracheal epithelium. These observations suggest the possibility that Hoxb genes are involved in the system specifying regional differences in morphogenesis and cytodifferentiation of respiratory tract. In addition, it is possible that BMPs and WNTs mediate region-specific epithelial-mesenchymal interaction in this system.

Animals↗
PubMed2001

The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia.

The T76 mutation in the pfcrt gene has been linked to chloroquine (CQ) resistance in Plasmodium falciparum. PCR-based analysis of pfcrt alleles was performed on pre-treatment samples from 107 individuals who had P. falciparum infections and lived in Papua, Indonesia. The results of a 28-day, in-vivo test revealed clinical resistance to CQ in 79 (74%) of the samples. The crude sensitivity of the pfcrt T76 assay for detecting the CQ-resistant infections in the samples was 96% and the crude specificity 52%. Discordance between pfcrt genotype and in-vivo phenotype was analysed either by genotyping of the merozoite surface protein-2 (to distinguish re-infection from recrudescence) or by amplification of the P. falciparum-specific small-subunit ribosomal RNA (ssrRNA) gene, using nested PCR (to detect any sub-patent but resistant parasites in infections misclassified as sensitive by the in-vivo test). When adjusting for the results of these analyses, the sensitivity and specificity of the pfcrt T76 assay for detecting the CQ-resistant infections became 93% and 82%, respectively. Overall, the present results indicate that the pfcrt T76 assay may be used to forecast therapeutic failure caused by CQ resistance. Validation requires exploration of the phenotype classifications based on the results of in-vivo tests, using genetic analyses that distinguish re-infection from recrudescence and detect microscopically subpatent parasitaemias.

Adult↗
PubMed2006

[Detection of promoter methylation of p16 gene in hematological malignant cell lines by nested methylation specific polymerase chain reaction].

This study was aimed to investigate the efficiency of modified methylation-specific polymerase chain reaction i.e. nested methylation-specific polymerase chain reaction, used to detect the promoter methylation of p16 gene in six hematological malignant cell lines, and to explore the application in selection of hematological malignant cell lines with promoter hypermethylation, and make them to be an idel cell models for studying the relationship between gene methylation and expression. DNAs were denatured by NaOH and then were subjected to bisulfite modification and a nested-MSP was used to amplify the promoter region, nested MSP product of p16 gene promoter was analyzed and sequenced. The results showed that the hypermethylation of p16 gene was detected in CA46 and U266, however, Molt4, K562, HL-60 and Jurkat cell lines were unmethylated. In conclusion, p16 gene methylation in hematological malignant cell lines can be perfectly detected by nested-MSP method, which is simple, sensitive and specific for screening all kinds of hematological malignant cell lines with p16 gene methylated.

Base Sequence↗
PubMed2001

Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene.

The aim of this study was to determine if fluorescent PCR could be used instead of nested PCR, for the detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in clinical specimens, to improve the sensitivity without increasing the risk for cross-contamination. We investigated and compared the sensitivity of single PCR, fluorescent PCR and nested PCR for the detection of IS900, an insertion sequence specific for M. paratuberculosis. A previously described extraction method for clinical specimens, based on xylene, was evaluated regarding its suitability for routine diagnostic work. The sensitivity of each PCR system was assessed by analysing a serial dilution of M. paratuberculosis DNA. To improve the reliability of the PCR and to facilitate the interpretation of the PCR results, a positive internal control molecule ("mimic") was developed and used for single and fluorescent PCR. In nested PCR, an existing mimic was used. The efficiency of recovering DNA of M. paratuberculosis from clinical specimens by the extraction method and detection of the organism by PCR was studied by analysing spiked ileum mucosa specimens. The final evaluation was performed on seventeen ileum mucosa specimens, previously found positive for M. paratuberculosis by bacterial culture. Twelve of the samples were positive by fluorescent PCR and nested PCR, and 10 samples were positive by single PCR. The use of mimics showed inhibition in specimens harbouring few M. paratuberculosis organisms, illustrating the effect of inhibitory substances in combination with small amounts of M. paratuberculosis DNA. We conclude that the extraction method was not adequate to recover small amounts of M. paratuberculosis and that inhibitory substances were still present in the processed specimens, but that the method is useful for identifying positive samples. Fluorescent PCR was a suitable alternative to both single PCR and nested PCR for the detection of M. paratuberculosis.

Animals↗
PubMed1998

Molecular monitoring of tumor cell contamination in leukapheresis products from stage IV neuroblastoma patients before and after positive CD34 selection.

BACKGROUND: Autologous peripheral blood stem cell (PBSCs) are frequently used to reconstitute hematopoiesis following administration of megatherapy in children with advanced stage IV neuroblastoma. Some centers prefer the use of autografts enriched for CD34+ progenitor cells because the positive selection procedure is believed to reduce indirectly tumor cell contamination. PROCEDURES: In this study, we monitored the efficiency of tumor cell purging following CD34 selection in PBSCs from seven patients with advanced neuroblastoma by using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, Amplification of tissues-specific mRNA transcript of tyrosine hydroxylase gene with nested primers enabled the detection of residual neuroblastoma cells with a sensitivity of one malignant-cell per 10(6) normals. RESULTS: Using this method, contaminating tumor cells were detected in seven of nine leukapheresis products of the patients. After positive immunoselection of CD34+ cells on Ceprate column, only one of nine enriched stem cell fraction still contained tumor cells detectable by RT-PCR. In six cases, PCR positive PBSCs became PCR negative after selection. CONCLUSIONS: We conclude that tumor cell contamination may be frequently detected in PBSC harvests of stage IV neuroblastoma patients by sensitive molecular analysis. The load of contaminating malignant cells might be reduced following CD34 selection.

Antigens, CD34↗
PubMed1998

Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion.

With the spread of resistance to chloroquine, the combination of sulphadoxine and pyrimethamine is growing in importance for the treatment of infection with the malaria parasite Plasmodium falciparum. Mutations in the dhfr gene of P. falciparum have been associated with resistance to pyrimethamine. Recently, several polymorphisms have been identified in the P. falciparum dhps gene which may correlate with sulphadoxine-resistance. Simple and rapid tests have been developed to detect these polymorphisms, using PCR followed by restriction digestion. These tests can accurately identify all the polymorphisms described to date at codons 16, 51, 59, 108, and 164 in the dhfr gene and those at codons 436, 437, 540, 581, and 613 in the dhps gene. A nested system has been developed which allows the accurate detection of these polymorphisms in samples of fingerprick blood collected on glass fiber membranes and filter papers, some with very low parasitaemias.

Animals↗
PubMed2005

Prevalence and genetic diversity of Entamoeba histolytica in an institution for the mentally retarded in the Philippines.

A total of 113 mentally retarded patients residing in a mental institution in Metropolitan Manila, Philippines, were screened for the presence of Entamoeba histolytica based on microscopy and polymerase chain reaction (PCR). Anti-E. histolytica antibodies were also screened in 97 serum samples collected using immunofluorescence antibody (IFA) test. Parasitological examination showed E. histolytica/Entamoeba dispar in 43 cases (38.05%), while PCR detected 74 cases (65.48%) positive for E. histolytica and 6 cases (5.30%) positive for E. dispar. Interestingly, these 6 samples were coinfected with E. histolytica. IFA test revealed that 80.41% (78/97) of the respondents possessed significant antibody titers for intestinal infection of E. histolytica. Of this number, there were 5 patients negative in IFA test but positive in PCR. The genetic diversity of E. histolytica isolates was also investigated by analyzing polymorphism in the serine-rich gene by nested PCR on DNA directly extracted from stool specimens. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded six distinct DNA banding patterns among the 74 stool isolates. An apparent clustering of E. histolytica strains was observed in patients living in different residential cottages of the institution. These results indicate the high prevalence of E. histolytica in an institution for the mentally retarded in the Philippines.

Animals↗
PubMed2005

Cryopreserved human B cells as an alternative source for single cell mRNA analysis.

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.

Antigens, Surface↗
PubMed2005

Detection of novel oral species and phylotypes in symptomatic endodontic infections including abscesses.

This study was undertaken to investigate the occurrence of several uncultivated phylotypes and newly named bacterial species in symptomatic endodontic infections. Samples taken from cases clinically diagnosed as acute periradicular abscesses or acute periradicular periodontitis were surveyed for the presence of 12 taxa by means of a 16S rRNA-gene-based nested or hemi-nested PCR assay. The most prevalent of the target taxa were Dialister invisus, Olsenella uli, Granulicatella adiacens, and Synergistes clones BA121 and E3_33. Findings revealed that novel phylotypes and newly named species can take part in the microbiota associated with symptomatic endodontic infections and a pathogenetic role is suspected.

Actinobacteria↗
PubMed2006

Culture-independent detection of Eikenella corrodens and Veillonella parvula in primary endodontic infections.

Eikenella corrodens and Veillonella parvula are normal cultivable inhabitants of the oral cavity but their presence in endodontic infections has not been as common as it could be anticipated. This might have been because of shortcomings of culture techniques when it comes to bacterial isolation or identification. The present study intended to survey samples from primary endodontic infections for the presence of E. corrodens and V. parvula using a culture-independent 16S rRNA gene-based nested PCR protocol. Genomic DNA was isolated directly from samples taken from different forms of periradicular lesions, and the presence of E. corrodens and V. parvula was determined by nested PCR. Specificity for each primer pair was confirmed by sequence analysis of PCR products from positive clinical samples. V. parvula and E. corrodens were, respectively, detected in 33% and 14% of the root canals associated with chronic apical periodontitis. Both V. parvula and E. corrodens were found in 10% of the cases diagnosed as acute apical periodontitis. V. parvula and E. corrodens were detected in 21% and 26% of the samples from acute apical abscesses, respectively. In general, species-specific nPCR allowed the detection of V. parvula in 24% and E. corrodens in 18% of the samples taken from primary endodontic infections. Findings confirmed that V. parvula and E. corrodens can take part in the microbiota of primary endodontic infections, but in prevalence values somewhat higher when compared to most of the previous culture studies that had reported recovery of these species.

Acute Disease↗
PubMed1999

Bacteriological investigation of an outbreak of Clostridium perfringens food poisoning caused by Japanese food without animal protein.

An outbreak of Clostridium perfringens food poisoning occurred in a senior citizen's home in Japan. Japanese food, spinach boiled with fried bean curd, was considered to be the causative food as a result of the detection of the C. perfringens enterotoxin gene by nested PCR. The number of enterotoxin-positive C. perfringens was enumerated as 4.3 x 10(5)/g in the causative food by the MPN method combined with nested PCR. By cultivation, enterotoxin-positive C. perfringens was isolated from all the fecal specimens of patients tested and the causative food. The isolates from patients were serotypable, heat-resistant and the majority produced enterotoxin, however most isolates from the causative food were nonserotypable, enterotoxin-negative and heat-sensitive.

Aged↗
PubMed1997

Characterization and cellular localization of PSG in rat testis.

In order to establish the rat testis as a model system for studying the human pregnancy-specific beta1-glycoprotein (PSG), expression and cellular distribution of PSG in rat testis were examined. Three partial PSG cDNAs, namely, rnCGM6, rnGCM7, and rnCGM8 were obtained when rat testis cDNA libraries were screened with a human placental PSG cDNA probe. Unlike the human PSGs, the rat PSGs show less nucleotide and amino acid sequence homology among family members. The rat PSGs also have multiple truncated leader sequences followed by immunoglobulin variable-like N domains while human PSGs have a single N domain. Examination of the testis, intestine, kidney, liver, lung, and muscle of male rats by reverse transcription-polymerase chain reaction (RT-PCR) with nested gene-specific primers showed that rnCGM6 was present only in the testis, while rnCGM8 was present in the testis, intestine and lung. On the other hand rnCMG7 was found in all tissues examined. Furthermore, rnCGM7 transcript was present in all somatic cells examined whereas rnCGM6 was predominantly in myoid cells and rnCMG8 in Leydig cells. These results suggest that there is cell-specificity in the expression of PSGs in the rat testis and that the rat testis is a good model for studying the biological activities of the PSGs.

Amino Acid Sequence↗
PubMed1998

Molecular genetic evidence for parallel evolution in a marine gastropod, Littorina subrotundata.

Littorina subrotundata from wave-exposed rocky shores differ consistently in shell and radula morphology from those found in wave-protected salt-marshes. To determine if the two morphological forms of this gastropod represent separate species, clades, or ecotypes, DNA sequencing and single-stranded conformational polymorphism (SSCP) analysis were used to assay variation in a 480 bp fragment of the mitochondrial cytochrome-b gene. Several nested analyses of molecular variance (AMOVA) were then performed to test if grouping populations by geographical region or habitat type better explained the distribution of cytochrome-b haplotypes among some northeastern Pacific populations. The analysis by geographic region resulted in a significant variance component that explained 53% of the variance, whereas the analysis by habitat type was not significant. These results, along with previous studies showing that the differences in shell morphology among different forms have a heritable component, suggest that the two forms are ecotypes and not separate species or clades as had been previously proposed. These results also imply that each ecotype has evolved independently in each geographic area and that the morphological similarity of individuals from the same habitat type is most likely the result of parallel evolution.

Analysis of Variance↗
PubMed1999

Studies of genetic relationships between bovine, caprine, cervine, and rangiferine alphaherpesviruses and improved molecular methods for virus detection and identification.

The glycoprotein B (gB) and D (gD) genes from five ruminant alphaherpesviruses, bovine herpesvirus 1 (BHV-1), bovine herpesvirus 5 (BHV-5), caprine herpesvirus 1 (CapHV-1), cervine herpesvirus 1, and rangiferine herpesvirus 1, were partially sequenced. The nucleotide sequence alignments revealed a highly conserved gB gene, with homologies ranging between 87.2 and 99.6%, and a more variable gD gene, with homologies ranging between 71.3 and 98.9%. The phylogenetic analysis of the gB and gD nucleotide and deduced amino acid sequences revealed that BHV-5 is the most closely related virus to the BHV-1 subtype 1 and BHV-1 subtype 2 cluster and that CapHV-1 is the most distantly related virus. The phylogenetic data showed a close relationship of all the studied viruses with suid herpesvirus 1. On the basis of sequence data for the gB gene, a nested PCR combined with restriction enzyme analysis (REA) of the PCR products was developed for the simultaneous detection and identification of the viruses that were studied. Nested primers from highly conserved sequence stretches were selected in order to amplify a region of 294 bp in all five viruses, and a subsequent REA of the PCR products allowed specific identification. A mimic molecule that served as an internal standard of the amplification efficiency was constructed. The practical diagnostic applicability of the assay was evaluated with clinical samples consisting of semen and organ specimens from experimentally infected animals.

Alphaherpesvirinae↗