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Results for “Multiplex PCR”

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PubMed2006

Hepatitis B virus genotypes and precore/core-promoter mutations in Tunisian patients with chronic hepatitis B virus infection.

OBJECTIVE: The aim of this study was to determine the frequency of genotype and precore/core-promoter mutations in chronic hepatitis B virus (HBV) infected individuals in Tunisia. METHODS: We studied 164 Tunisian patients (38 HBeAg-positive and 126 HBeAg-negative) with chronic HBV infection. Genotypes and precore/core-promoter mutations were studied using Inno-LiPA and Multiplex-PCR and PCR-RFLP methodology. RESULTS: Alanine aminotransferase (ALT) levels were higher in HBeAg-positive compared with HBeAg-negative patients (p<0.05). Patients with HBeAg-positive chronic hepatitis B were younger than HBeAg-negative chronic hepatitis B patients. The 164 genotypes were distributed as follows: 1 genotype A (0.6%), 1 genotype B (0.6%), 3 genotype C (1.82%), 139 genotype D (84.75%), and 20 mixed genotypes (12.2%). In the precore region (41.5%) of the patients had exclusively PC mutant and (50.9%) had a mixture of wild-type and variant sequences. PC variant was more commonly found in HBeAg-negative patients than in HBeAg-positive patients (94.5% vs. 87.8%), respectively. The mutations in the core promoter were more common in HBeAg-negative patients (65.4%) than in HbeAg-positive patients (18.2%). These results indicate that genotype D is predominant in Tunisia. Precore mutation occurred invariably among HBeAg-positive and HBeAg-negative patients, whereas core-promoter mutations were more frequently found in HBeAg-negative patients. CONCLUSION: Analysis of these mutants may prove useful for clinical evaluation and choice of therapy.

Adolescent↗

PubMed2003

Use of ribosomal DNA sequence data to characterize and detect a neogregarine pathogen of Solenopsis invicta (Hymenoptera: Formicidae).

A neogregarine parasite of the red imported fire ant, Solenopsis invicta, was discovered recently in Florida and tentatively placed in the Mattesia genus based on morphological characterization. S. invicta infected with this Mattesia species exhibited a characteristic yellowing of the cuticle which was designated Mattesia "yellow-head disease" (YHD). The 18S rRNA gene sequence from Mattesia YHD was elucidated and compared with the neogregarine pathogens, Mattesia geminata and Ophriocystis elektroscirrha. The sequence data support the previous conclusion that Mattesia YHD is a new species that infects S. invicta. Furthermore, high sequence identity between Mattesia YHD, M. geminata (95.7%), and O. elektroscirrha (86.2%) correctly place the YHD organism in the Mattesia genus and Neogregarinorida order. Oligonucleotide primer pairs were designed to unique areas of the 18S rRNA genes of Mattesia YHD and S. invicta. Multiplex PCR resulted in sensitive and specific detection of Mattesia YHD infection of S. invicta.

Animals↗

PubMed2006

Macrophage-activating lipopeptide-2 induces cyclooxygenase-2 and prostaglandin E(2) via toll-like receptor 2 in human placental trophoblast cells.

We have examined whether toll-like receptor (TLR)2-mediated stimulation by macrophage-activating lipopeptide-2 (MALP-2), originally purified from Mycoplasma fermentans, induces cyclooxygenase (COX)-2 and prostaglandin (PG)E(2) in human placental trophoblast cells. The signaling mechanism by which MALP-2 exerts its effect has also been examined. Human placental trophoblast cells isolated from term placenta were used. TLR expression in trophoblast cells was confirmed by multiplex PCR and immunocytochemistry, and examined whether MALP-2 induces COX-2 and PGE(2) by Northern blotting, RT-PCR, Western blotting and ELISA, respectively. The activation of NF-kappaB and MAP kinases (ERK1/2 and p38) was examined by Western blotting. The effects of inhibitors of NF-kappaB, MEK1/2 and p38 on MALP-2-induced PGE(2) production were also evaluated. TLR2, TLR6 and TLR4 were expressed in human placental trophoblast cells. MALP-2 significantly induced COX-2 expression and enhanced PGE(2) production in a dose-dependent manner. MALP-2 induced the activation of NF-kappaB, ERK1/2 and p38 MAPK. Inhibitors of NF-kappaB, MEK1/2 and p38 blocked MALP-2-inducible PGE(2) production. TLR2-mediated stimulation by MALP-2 induces COX-2 and PGE(2) in human placental trophoblast cells via NF-kappaB and MAP kinases pathways.

Cells, Cultured↗

PubMed2005

Usage of dried blood spots for molecular diagnosis and monitoring HIV-1 infection.

The usage of dried blood spots as specimens for diagnosis and monitoring of HIV-1 infection in Thailand was evaluated. EDTA blood samples, which were collected from 100 HIV seronegative and 109 HIV seropositive individuals, were tested on dried blood spots; Whatman, Schleicher and Schuell (S&S) No. 903 and S&S IsoCode filter paper. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by an "in-house" multiplex PCR and a commercial Amplicor HIV-1 PCR test (Roche, version 1.0). HIV-1 RNA qualitative (QL) and quantitative (QT) detection was determined by Nucleic Acid Sequence Based Amplification (NASBA). The average DNA per blood spot recovered from Whatman and S&S IsoCode was not statistically different (p = 0.512) with a range of 218.9+/-46.84 and 225.63+/-88.33 microg, respectively. The concordance of HIV-1 proviral DNA detection by PCR from dried blood spots Whatman and S&S IsoCode was 94% versus 89.4% for sensitivity and 100% versus 100% for specificity. The sensitivity and specificity of HIV-1 RNA QL detection in dried blood spots was 89.7 and 97.5%, respectively. The HIV-1 RNA QT from dried blood spots showed a good correlation in paired dried blood spots and plasma with Pearson correlation, r = 0.817 (R2 = 0.667, P < 0.05). The data showed that dried blood spots could be used for the diagnosis and monitoring of HIV-1 infection.

Blood↗

PubMed2004

STR typing of ductal adenocarcinomas of the pancreas and healthy control tissue in 18 individuals.

Recently, different work groups have demonstrated that short tandem repeat (STR) typing of various tumor tissues may lead to erroneous results due to tumor microsatellite instability (MSI). This may have considerable implications for genetic profiling of tumor tissue, e.g. in paternity testing or sample individualization. To elucidate whether this is true for ductal adenocarcinomas (DAC) of the pancreas, we genetically investigated tumor and corresponding healthy tissue from 18 patients using a commercially available multiplex PCR kit commonly used in forensic laboratories. STR typing of the samples revealed no differences between tumor and healthy tissue in 17 out of 18 samples. One sample, however, showed an allele expansion at locus D21S11. In heterozygous cases, peak heights varied strongly at different loci, mocking a loss of heterozygozity. This investigation shows that even though tetranucleotide MSI in pancreatic DAC is a rare event, the interpretation of genetic profiles obtained from cancerous samples can be difficult and lead to misinterpretations.

Aged↗

PubMed2005

Identification, characterization and forensic application of novel Y-STRs.

Y-chromosomal polymorphic STRs are a powerful tool for forensic and evolutionary studies. Within the last decade, a series of Y-STR systems have been developed and demonstrated to be suitable for a variety of forensic applications including sexual assault cases and paternity testing. This review describes our recent studies on novel male-specific Y-STRs, involving identification, development of a multiplex-PCR system, population study and forensic application.

Chromosomes, Human, Y↗

PubMed2006

Genetic polymorphisms of 15 STR loci of Chinese Dongxiang and Salar ethnic minority living in Qinghai Province of China.

Fifteen autosomal STRs loci were analyzed from two samples of 178 healthy unrelated autochthonous individuals of Chinese Dongxiang and Salar ethnic minority groups using a multiplex PCR system. Allele frequencies distribution and statistical parameters for all STR loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, were determined by the AmpFISTR Identifiler Kit. The observed genotype frequencies and expected of genotype frequencies were evaluated by chi(2)-test and the Fisher exact tests. chi(2)-test showed that the agreement with Hardy-Weinberg equilibrium (p>0.05) was for all studied STR loci of two populations. The data in the present study can be used greatly for routine forensic application in the region, and enrich Chinese ethnical genetic informational resources.

Alleles↗

PubMed2006

Cardioprotective effect of des-Aspartate-angiotensin-I (DAA-I) on cytokine gene expression profile in ligation model of myocardial infarction.

We investigate the influence of des-Aspartate-angiotensin-I (DAA-I) on the cytokine expression profile in a rodent model of myocardial infarction. Myocardial infarction model was created in female Wistar rats by coronary artery ligation. Animals were randomized to receive intravenously either a daily dose of 1.2 mug DAA-I/kg body weight (group 1; n = 60) or saline (group 2; n = 60) for 14 days after infarction. Heart function was assessed by echocardiography. Animals were euthanized at 1, 3, 7, 14 and 31 days. Morphometric analysis using tetrazolium chloride staining revealed that infarct size was reduced by 32.2% (p < 0.05) in group 1 after 14 days of DAA-I treatment. Left ventricular ejection fraction in group 1 improved significantly (73.4%) as compared to group 2 (47.7%; p < 0.001). Immunostaining for immune cells at the infarct site showed that CD8+ lymphocytes infiltration at the infarct site declined in group 1 (15 +/- 5 cells) as compared to group 2 (50 +/- 6 cells; p < 0.001). Infiltration of monocytes and macrophages remained high at day 14 in group 2 (126 +/- 40 cells) as compared to group 1 (49 +/- 11 cells; p = 0.006). Multiplex PCR was done for differential gene expression of various pro-inflammatory cytokines. IL-6, TNF-alpha, TGF-beta and GM-CSF expression were significantly down-regulated in the infarct, peri-infarct and contra-lateral zones of the left ventricle in group 1 as compared to group 2. IL-6, TGF-beta and GM-CSF expression started to decline from day 1 of DAA-I treatment while TNF-alpha expression only reduced after 7 days of DAA-I treatment. We conclude that DAA-I prevented infarct expansion through suppression of inflammatory cytokines and immune cell infiltration in the infarct region.

Angiotensin I↗

PubMed2004

Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization.

A microarray-based method for characterization of six Clostridium perfringens toxin genes: iA (iota toxin), cpa (alpha toxin), cpe (enterotoxin E), etxD (epsilon toxin), cpb1 (beta toxin 1),and cpb2 (beta toxin 2) was developed and evaluated using 17 C. perfringens isolates. Three individual oligonucleotide probes (oligoprobes), complementary to the unique sequences of each toxin gene, were designed and immobilized on a surface of aldehyde-coated glass slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all six genes. Single-stranded DNA (ssDNA) samples for microarray analysis were prepared by following a primer extension of amplicons in the presence of one primer. Fluorescent moieties (Cy3) were incorporated into the ssDNA by chemical modification of guanine bases. The presence of toxin genes in C. perfringens was established by hybridization of the fluorescently labeled ssDNA representing different samples to the microarray gene-specific oligoprobes. Results of the study showed sensitivity and specificity of genotyping C. perfringens using multiple microarray-based assays.

ADP Ribose Transferases↗

PubMed2005

Molecular identification of Yersinia enterocolitica isolated from pasteurized whole milk using DNA microarray chip hybridization.

A DNA microarray chip of four virulence genes and 16S ribosomal DNA gene conserved region among all Gram negative species, including Yersinia, as a positive control was developed and evaluated using 22 Yersinia enterocolitica isolates. Eight different oligonucleotide probes (oligoprobes) with an average size of 22 bp, complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all five genes, and single stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer of all genes. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed specificity of genotyping Y. enterocolitica using multiple microarray-based assays. Final validation of the chip's ability to identify Y. enterocolitica genes from adulterated pasteurized whole milk was confirmed and successful. The limit of chip detection of virulence genes in pasteurized whole milk was found to be 1000 CFU per hybridization.

Animals↗

PubMed2006

DNA microarray for detection of antibiotic resistance determinants in Bacillus anthracis and closely related Bacillus cereus.

We developed a multiplex PCR for amplification of ten genes involved in resistance to ciprofloxacin, doxycycline, rifampin, and vancomycin in Bacillus anthracis and closely related Bacillus cereus. Enzymatic labelling of PCR products followed by hybridization to oligonucleotide probes on a DNA microarray enabled simultaneous detection of resistance genes tetK, tetL, tetM, tetO, vanA, and vanB and resistance-mediating point mutations in genes gyrA, gyrB, parC, and rpoB. The presented assay allows detection of clinically relevant antibiotic resistance determinants within 4h and can be used as a time-saving tool supporting conventional culture-based diagnostics.

Bacillus anthracis↗

PubMed2006

DNA microarray for the detection of therapeutically relevant antibiotic resistance determinants in clinical isolates of Staphylococcus aureus.

An oligonucleotide microarray was constructed for the rapid and sensitive molecular detection of antibiotic resistance determinants in Staphylococcus aureus. The array is equipped with oligonucleotide capture probes for the detection of 10 clinically and therapeutically relevant antibiotic resistance genes and -mutations (mecA, aacA-aphD, tetK, tetM, vat(A), vat(B), vat(C), erm(A), erm(C), grlA-mutation) as well as several control probes. A microarray concept was established including multiplexed PCR amplification, DNA labeling, hybridization and data processing. This concept was applied to clinical Staphylococcus aureus isolates and results were concordant with those from standard genotypic and phenotypic resistance testing. Our microarray concept offers rapid and accurate identification of antibiotic resistance profiles. It is easily expandable and thus can be adapted to changing clinical and epidemiological requirements in clinical diagnosis as well as in epidemiological studies.

DNA Probes↗

PubMed2006

An outbreak of adenovirus serotype 41 infection in infants and children with acute gastroenteritis in Maizuru City, Japan.

A total of 337 fecal specimens were collected from infants and children with acute gastroenteritis in Maizuru City, Japan from July 2004 to June 2005 and tested for the presence of rotavirus, norovirus, sapovirus, astrovirus, and adenovirus by RT-multiplex PCR. Among diarrheal viruses detected, norovirus was the most prevalent (13.6%, 46 of 337), followed by adenovirus (8%, 27 of 337), group A rotavirus (5%, 17 of 337), astrovirus (1.8%, 6 of 337), and sapovirus (1.8%, 6 of 337), respectively. Adenovirus was subjected to molecular genetic analysis by sequencing. Adenovirus detected in this study was classified into five serotypes, namely Ad1, Ad2, Ad3, Ad5, and Ad41. Of these, Ad41 was the most predominant serotype that accounted for 85.2% (23 of 27). It was noteworthy to point out that Ad41 infection was apparently confined only to the period of 4 months (October 2004 through January 2005). This pattern of infection implied the outbreak of Ad41 in these subjects, which was the first outbreak of acute gastroenteritis attributed to adenovirus in Maizuru City, Japan. Another interesting feature of the study was the existence of two Ad41 subtypes co-circulating in this outbreak. This report confirmed the presence of adenovirus as one of an important cause of acute gastroenteritis among Japanese infants and children.

Acute Disease↗

PubMed2005

Microarray analysis of Bacillus cereus group virulence factors.

Bacillus cereus, B. thuringiensis and B. anthracis are closely related medically and economically important bacterial species that belong to the B. cereus group. Members of the B. cereus group carry genes encoding several important virulence factors, including enterotoxins, phospholipases and exotoxins. Since it is difficult to differentiate among B. cereus group members, and because Bacillus virulence factors are very important for pathogenesis, we explored the use of microarray-based detection of virulence factor genes as a tool for strain identification and for determining virulence. Our method requires an initial multiplex PCR amplification step, followed by identification of the PCR amplicons by hybridization to an oligonucleotide microarray containing genes for all three types of Bacillus virulence factors including B. anthracis virulence factors. The DNA chip described here contains 21 identical arrays used for analysis of seven samples in triplicates. Using the arrays, we found that virulence factors are present in several combinations in the strains analyzed. This work also demonstrates the potential of oligonucleotide microarrays for medical, food safety and biodefense analysis of microbial pathogens.

Bacillus cereus↗

PubMed2006

Rapid laboratory detection of meningococcal disease outbreaks caused by serogroup C Neisseria meningitidis.

Molecular subtyping is of significant importance to the recognition of outbreaks of meningococcal disease caused by serogroup C Neisseria meningitidis. We describe the application of multilocus variable number tandem repeat analysis (MLVA) for the molecular subtyping of N. meningitidis and compare its performance to that of pulsed-field gel electrophoresis (PFGE). For MLVA, a multiplex PCR assay targeting five variable number tandem repeat regions was developed and evaluated using a panel of sporadic and outbreak-associated serogroup C N. meningitidis isolates. MLVA was highly reproducible and provided results within 6 h. Overall, the discriminatory power of MLVA was equivalent to that of PFGE. The utilization of MLVA for subtyping N. meningitidis isolates provides a rapid and safer alternative to PFGE for identifying outbreaks of meningococcal disease. As such, it may provide public health officials with timely information that may minimize the spread of outbreak-related cases through prophylaxis.

Cluster Analysis↗

PubMed2006

Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes.

Streptococcus pneumoniae is a major worldwide human pathogen. This investigation has developed a reliable and accurate DNA microarray method for inter-species differentiation of S. pneumoniae and intra-species differentiation of the 23 groups of S. pneumoniae including serotypes represented in the 23-valent pneumococcal vaccine and the other 20 closely related serotypes. In addition to 16S rDNA probes, serotype- or serogroup-specific probes targeting the capsular polysaccharide synthesis (cps) genes, wzy or capA were generated. We adopted a two-step multiplex PCR to improve the sensitivity of detection to a level of 10(5) cfu/ml in pure culture or 50 ng DNA. A total of 169 isolates (from China, Australia, Canada and New Zealand) including 147 belonging to 23-valent vaccine and closely related serotypes of S. pneumoniae, 11 belonging to other serotypes and 11 of different species commonly isolated from respiratory tract were tested to verify the method. The DNA microarray method developed provides a sensitive means to rapidly identify the members of the most common S. pneumoniae serotypes in patients and to monitor their distribution in different patient groups and geographic locations. Such information is needed for disease surveillance and to monitor vaccine efficacy.

Bacterial Proteins↗

PubMed2005

Lymphocyte Hprt mutant frequency and sperm toxicity in C57BL/6 mice treated chronically with Azathioprine.

Many patients undergoing chronic therapy with the purine analogue Azathioprine (Aza) have highly elevated HPRT lymphocyte mutant frequencies (MFs), and it is likely that these increases are due to selection of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells might result in an increased frequency of the Lesch-Nyhan syndrome. In this study, a mouse model for Aza mutant selection was developed and Aza toxicity was evaluated in the germ cells of treated mice. Groups of 20 male C57BL/6 mice were treated by gavage three times/week with 0, 5, 10, 25, 50, or 100mg/kg Aza, and three to eight mice from each group were sacrificed at various times for up to 23 weeks. Mice treated with 25-100mg/kg Aza were all dead by 14 weeks of treatment. Hprt lymphocyte MF assays indicated that the treated mice had reduced numbers of spleen lymphocytes. Most treated mice had Hprt MFs similar to those of control mice (2.1+/-1.6 x 10(-6)), however, highly elevated MFs were detected in one out of three mice given 5mg/kg for 10 weeks, one out of three mice given 10mg/kg for 10 weeks, and one out of eight mice given 10mg/kg for 23 weeks (e.g., 233 x 10(-6) after 10 weeks of 5mg/kg). Sequence analysis of Hprt cDNA indicated that all mutant clones from one of these mice had a T-->A transversion in the initiation codon. Multiplex-PCR on mutant clones from the other two mice indicated that all the clones from one had a deletion of Hprt exons 2 and 3, while most of the mutants from the other had lost all of the Hprt exons. Measurements of testicular weight, and of sperm count, viability, morphology, and motility found that Aza produced low levels of toxicity in sperm, with the most consistent effect being a reduction in the testicular weight. The data suggest that mice chronically treated with 5 and 10mg/kg Aza (doses similar to those used in humans) have elevated Hprt MFs due to clonal amplification of selected Hprt mutants. The results also suggest that mice treated with these doses of Aza retain reasonable fertility, and will be useful for breeding experiments to examine the possibility of increasing the germ-line transmission of Hprt mutations.

Animals↗

PubMed2005

Mitochondrial DNA diagnosis for taeniasis and cysticercosis.

Molecular diagnosis for taeniasis and cysticercosis in humans on the basis of mitochondrial DNA analysis was reviewed. Development and application of three different methods, including restriction fragment length polymorphism analysis, base excision sequence scanning thymine-base analysis and multiplex PCR, were described. Moreover, molecular diagnosis of cysticerci found in specimens submitted for histopathology and the molecular detection of taeniasis using copro-DNA were discussed.

Animals↗