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Conventional PCR (conventional + pcr)
Selected AbstractsDistinctiveness of the cagA Genotype in Children and Adults with Peptic Symptoms in South ChinaHELICOBACTER, Issue 4 2009Juan Li Abstract Background:,Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3, region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China. Materials and Methods:, One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3, region of CagA by conventional PCR and DNA sequencing. Results:,H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults (p < .05). Thirty-eight of 39 (97.4%) cagA -positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs , ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%) , were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%). Conclusion:,cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China. [source] Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real-time and conventional PCR assaysJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2010H.G. Shen Abstract Aim:, To develop spa multiplex real-time and conventional PCR assays to detect and differentiate between spaA, spaB and spaC genes within Erysipelothrix spp. Methods and Results:, For evaluation of the assays, 28 Erysipelothrix spp. reference strains, 25 tissues from pigs inoculated with reference strains of serotypes 1, 2, 5, 10 or 18, and 15 diagnostic samples were used. SpaA was found to be present in Erysipelothrix rhusiopathiae serotypes 1a, 1b, 2, 5, 9, 12, 15, 16, 17, 23 and N; spaB was detected in E. rhusiopathiae serotypes 4, 6, 8, 11, 19 and 21 and spaC was detected in E. sp. strain 2 serotype 18. Spa-related genes were not detected in E. tonsillarum strains (serotypes 3, 7, 10, 14, 20, 22, 24, 25, 26) or E. sp. strain 1 (serotype 13). With the spa multiplex real-time PCR assay, it was also possible to further differentiate spaB into spaB1 (serotypes 4, 6, 8, 19 and 21) and spaB2 (serotype 11). Overall, spaA was detected in seven experimental tissue samples and six diagnostic tissue samples, and spaC in two experimental tissue samples. The detection limits were determined to be five colony-forming units (CFU) per reaction for the spa multiplex real-time PCR assay and 4000 CFU per reaction for the conventional PCR assay. Conclusions:, Both spa PCR assays were specific and reproducible in the identification of spa types in Erysipelothrix spp. Significance and Impact of the Study:, The described spa PCR assays may be useful tools for investigating spa prevalence among strains isolated from field tissues and to determine the role of the Spa proteins in vaccine protection and pathogenesis. [source] Validation of a real-time PCR for Haemophilus parasuisJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2010C. Turni Abstract Aims:, To validate a real-time PCR test for the diagnosis of Glässer's disease, a major pig disease caused by Haemophilus parasuis. Methods and Results:, The specificity of a real-time PCR amplifying the inf,B gene was validated with 68 H. parasuis isolates and 36 strains of closely related species. As well, 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were tested with the real-time PCR, and the results were compared with culture and a conventional PCR. The real-time PCR produced significantly more positive results than the conventional PCR (165 vs 86). Conclusions:, The sensitivity of the real-time PCR combined with high specificity makes it a very valuable tool for the diagnosis of Glässer's disease. Significance and Impact of Study:, This new method will improve the ability of laboratories to diagnose Glässer's disease, especially in laboratories where the culture method for H. parasuis is not optimal. [source] Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysisJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2006J.E. Burton Abstract Aims:, To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. Methods and Results:, Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. Conclusions:, Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. Significance and Impact of the Study:, Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates. [source] Chronic shedding of Campylobacter species in beef cattleJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004G.D. Inglis Abstract Aims:, To determine the prevalence of chronic shedding of Campylobacter species by beef cattle, a longitudinal study of shedding patterns was conducted in a cohort of 60 beef steers over a 4-month period. Methods and Results:, Steers were maintained in a simulated feedlot setting but individually in pens to minimize transmission among animals. At each collection time, campylobacters in faeces were detected using conventional PCR. In addition, quantities of Campylobacter jejuni and C. lanienae in faeces were measured using real-time quantitative (RTQ) PCR. All of the steers tested shed Campylobacter species during the course of the study, and overall, 90% of the 299 samples tested were positive for Campylobacter DNA. The majority of the animals (86%) shed campylobacters at ,4 sample times. The most prevalent taxon detected in bovine faeces was C. lanienae (56% of samples) followed by C. jejuni (13%), C. hyointestinalis (8%), and C. fetus (2%). No C. coli was detected, and 13% of the faecal samples contained two or more of the above species. Seven (12%) and 34 (57%) animals shed C. jejuni and C. lanienae at ,3 sample times, respectively. For both C. lanienae and C. jejuni, a substantial number of cells were detected in faeces using RTQ-PCR; 27% of the samples positive for C. jejuni contained populations >104 cells g,1 (maximum of 5 × 105 cells g,1), and 44% of samples positive for C. lanienae possessed populations >106 cells g,1 (maximum of 4 × 108 cells g,1). A significant correlation was observed between shedding of C. lanienae and the severity of liver abscesses. In 27% of the samples, an amplicon was obtained for genus-specific but not for the species-specific primers. Sequencing of the partial 16S rRNA gene suggested the presence of at least two undescribed Campylobacter species but this has yet to be confirmed. Conclusions:, A high percentage of feedlot cattle shed large quantities of Campylobacter species in their faeces over a protracted period of time (ca 112 days). Significance and Impact of the Study:, This is the first study of longitudinal shedding patterns of campylobacters in beef cattle using PCR-detection methods. In addition, this is the first use of RTQ-PCR to directly quantify C. jejuni or C. lanienae in faeces. The results of the study show that a large number of cattle (>85%) chronically shed campylobacters in feedlots. [source] Prevalence of human papillomaviruses in urine samples of male patients infected with HIV-1 in Sao Paulo, Brazil,JOURNAL OF MEDICAL VIROLOGY, Issue 12 2009Fernando A.M. Costa Abstract Human papillomavirus is a DNA virus that includes 118 genotypes. HPV16 is responsible for 80% of cervical cancer in women. Men are important reservoirs and major transmitters of HPV to their partners. The aim of this study was to detect HPV DNA and to determine the prevalence of HPV types 6, 11, 16, and 18 in urine samples of men infected with HIV-1. This study included 223 patients infected with HIV-1 from the Center of Reference on HIV/AIDS (CRT-SP) and an outpatient clinic of HIV. Urine samples were collected and after DNA extraction real-time PCR was performed for detection of HPV DNA. Positive samples were then tested by conventional PCR using type-specific primers for the four HPV types. A total of 223 men infected with HIV-1 were tested, 81% of whom were on HAART. Four (5.8%) were positive for HPV6, 18 (26.1%) were positive for HPV11, 22 (31.9%) were positive for HPV16 and five (7.2%) were positive for HPV18 by conventional PCR. Twenty (29%) patients had other HPV types and five patients (1.5%) had multiple types. The mean T CD4+cells count was 517 and 441,cells/mm3 (P,=,0.30), in HPV negative and positive men, respectively. The HIV viral load was higher in the HPV negative group than for in the men with HPV (P,=,0.0002). A 30.9% prevalence of HPV was found in asymptomatic urine samples of men infected with HIV-1. This study suggests that urine may be a useful specimen for HPV screening. J. Med. Virol. 81:2007,2011, 2009. © 2009 Wiley-Liss, Inc. [source] Detection of human bocavirus in respiratory, fecal, and blood samples by real-time PCR,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2009Sarah J. Tozer Abstract Human bocavirus (HBoV) has been detected worldwide in respiratory samples. Two real-time PCR assays, targeting the non-structural protein (NP-1) and viral protein (VP-1) genes, were designed and validated to detect HBoV in patients with respiratory disease, gastroenteritis, or systemic illness. Sensitivity of the NP-1 and VP-1 assays were equal to the conventional PCR assay previously described by Allander et al. [2005: Proc Natl Acad Sci USA 102: 12891,12896] being 100%, and giving specificity of 94% and 93%, respectively. There was no cross-reaction identified with unrelated respiratory agents, or to human DNA. The limits of detection were 10 copies of genomic DNA equivalents per reaction for both assays. The assays were used to screen three different sample populations, combined nose, and throat swabs (n,=,96) from children with acute respiratory disease, fecal samples (n,=,375) from adults, and children with gastroenteritis and whole blood (n,=,229) collected from 31 immunocompromised children taken over an 18-month period. In total 17 (18%) respiratory samples and 18 (4.8%) fecal samples were identified as having HBoV present. Of the pediatric whole blood specimens investigated, HBoV was detected in six (2.6%) samples from four patients. In summary, two real-time PCR assays targeting different genes were designed and validated for use as screening methods for the detection of HBoV. HBoV was found in three different specimen types: parent-collected combined nose,throat swabs, fecal samples collected from symptomatic individuals and whole blood from immunocompromised children. J. Med. Virol. 81:488,493, 2009. © 2009 Wiley-Liss, Inc. [source] Comparative analysis of putative periodontopathic bacteria by multiplex polymerase chain reactionJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2008M. Morikawa Background and Objective:, The polymerase chain reaction (PCR) has been applied for the rapid and specific detection of periodontopathic bacteria in subgingival plaque and is potentially of clinical benefit in the diagnosis and treatment of periodontitis subjects. However, several technical points need to be modified before the conventional PCR detection system can be used by clinicians. Material and Methods:, To develop a PCR-based technique more applicable for clinical use than conventional PCR, we established a multiplex PCR for five putative periodontopathic (Treponema denticola, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Tannerella forsythia) and two nonperiodontopathic (Streptococcus sanguinis and Streptococcus salivarius) species of bacteria using whole-plaque suspension as templates, and detected bacteria in subgingival plaque taken from 85 subjects at the supportive periodontal therapy stage after active periodontal treatments. Results:, Among putative periodontopathic bacteria, the detection frequency of T. denticola and P. gingivalis was elevated in parallel with higher probing pocket depth and clinical attachment loss, and had 4.2,14.1 times increasing odds of the clinical parameters tested. Detection of any of the five species of putative periodontopathic bacteria markedly increased the odds ratio of a higher probing pocket depth, clinical attachment loss and bleeding on probing. Conclusion:, The multiplex PCR system developed in this study enabled the detection of all the bacteria under investigation in one reaction tube in a less time- and labor-intensive manner than conventional PCR. These results support the potential clinical use of multiplex PCR for detecting periodontopathic bacteria and for evaluating therapeutic strategies and predicting the prognosis for each subject. [source] Detection of Phytophthora nicotianae in Soil with Real-time Quantitative PCRJOURNAL OF PHYTOPATHOLOGY, Issue 1 2010Junli Huang Abstract Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora, the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/,l in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/,l and in TaqMan PCR 1.2 fg/,l, and real-time PCR was 104,105 times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/,l. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method. [source] Presence of HCV-RNA after ultracentrifugation of serum samples during the follow-up of chronic hepatitis C patients with a sustained virological response may predict reactivation of hepatitis C virus infectionALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 5 2009I. CASTILLO Summary Background, Concentration of viral particles by ultracentrifugation of serum prior to PCR allows detection of hepatitis C virus (HCV) RNA in patients with undetectable viral RNA by conventional PCR assays. Aim, To analyse if HCV-RNA is detected after serum ultracentrifugation in chronic hepatitis C patients with a sustained virological response to antiviral therapy (defined as serum HCV-RNA negativity by conventional assays 6 months after the end of therapy). Methods, HCV-RNA was tested using real-time PCR in ultracentrifuged sera collected during the post-treatment follow-up (mean: 42 ± 27 months) in 57 sustained virological responders (SVR). Results, After serum ultracentrifugation, HCV-RNA was detected on at least one occasion during the follow-up in 29/57 (51%) SVR. Thirteen (23%) of these 57 SVR suffered a reactivation 18 ± 8 months after the end of therapy (reappearance of serum HCV-RNA detectable by conventional assays). Among reactivated patients, 11/13 (85%) had HCV-RNA in ultracentrifuged serum samples (detectable 10 ± 5 months before reactivation), while HCV-RNA was positive after ultracentrifugation in 18/44 (41%) long-term SVR (P = 0.01). Persistence of detectable HCV-RNA after serum ultracentrifugation was associated with reactivation (P = 0.001). Conclusions, Serum ultracentrifugation prior to PCR allows detection of HCV-RNA in SVR and its persistence may predict late reactivation. [source] Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: A Review of Detection Methods and Geographic Distribution,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005PARKE A. RUBLEE Abstract. Molecular methods, including conventional PCR, real-time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets. [source] A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation,APMIS, Issue 5 2008BENJAMIN EDVINSSON Active infection with Toxoplasma gondii in immunocompromised transplant recipients can lead to toxoplasmosis, which may have a rapid disease course and in some cases be fatal. It is of paramount importance to diagnose toxoplasmosis at an early stage, and to initiate specific treatment to improve the outcome. Polymerase chain reaction (PCR) is today the primary diagnostic tool to diagnose toxoplasmosis in immunocompromised patients. Timely diagnosis may, however, be difficult if toxoplasmosis is at first asymptomatic. To investigate the magnitude of toxoplasmosis after bone marrow transplantation (BMT), we conducted a screening study by PCR where 21 autologous and 12 allogeneic BMT recipients were included. Peripheral blood samples were taken one week prior to BMT; thereafter, blood samples were drawn weekly for the first 6 months, and monthly up to one year after BMT. The samples were analyzed by conventional PCR and real-time PCR. T. gondii DNA was detected in peripheral blood from one patient 5 days post allogeneic BMT. There were no clinical signs of toxoplasmosis. Medical records were reviewed and showed a previously undiagnosed eye infection in another allogeneic BMT recipient. These two patients were seropositive for T. gondii. We concluded that monitoring for T. gondii DNA in peripheral blood samples using PCR might be a valuable method for identifying toxoplasma-seropositive stem cell transplant recipients. [source] Identification of wild type and variants of oestrogen receptors in polymorphonuclear and mononuclear leucocytesCLINICAL ENDOCRINOLOGY, Issue 1 2006Denis Stygar Summary Objective, ,Leucocytes play an important role in the pathogenesis of autoimmune and cardiovascular diseases. Clinical and epidemiological observations indicate that the sex steroid hormones, particularly oestrogens, may regulate leucocyte functions. The assumption that oestrogens have a direct effect on leucocytes has to be supported by identification of functional oestrogen receptors (ER) in leucocytes. This study aimed at investigating the presence of ER subtypes in different types of leucocytes isolated from peripheral blood of female and male donors. Design and patients, ,A total of nine men (age range 18,43 years) and nine women (age range 19,42 years) all healthy blood donors, were recruited for the study. The donors did not receive any medication or hormonal contraceptives for the last three months. Ten millilitres of peripheral blood was collected from each donor. Polymorphonuclear leucocytes (PMN) and peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation. Measurements, ,ER, and ER, mRNA expression was measured by real-time reverse transcriptase,polymerase chain reaction (RT-PCR), and ER proteins were analysed by Western blot in the PBMC and PMN leucocyte populations. In addition, expression profiles of ER variant isoforms were characterized by conventional PCR using the splice-targeted primer approach. Results, ,Although we detected wild-type ER, and ER, mRNAs in PBMC but not in PMN cells, the ER, and ER, proteins were found in both cell types using Western blot. We observed that both ER, and ER, proteins differ in size between PMN and PBMC, suggesting that the two leucocyte populations contain diverse variant isoforms of ER, and ER,. RT-PCR analysis of exon-deleted ER splice variants revealed that PBMC express several exon-deleted variants of ER, and ER,, along with wild-type receptor, whereas the PMN cells only express exon-deleted variant isoforms and no wild-type ER, or ER,. Conclusions, ,Our study demonstrates the presence of ER, and ER, in PBMC and PMN cells from female and male donors. The ER, and ER, genes have complex transcriptional profiles, with many receptor variant isoforms being expressed. Considering the diversity of ER isoforms in leucocyte subtypes, we conclude that the expected effect of oestrogen would be highly cell type-specific. Further studies are needed to test the functional activity of ER isoforms and their relation to disease. [source] Visual detection of IS6110 of Mycobacterium tuberculosis in sputum samples using a test based on colloidal gold and latex beadsCLINICAL MICROBIOLOGY AND INFECTION, Issue 11 2006P. Upadhyay Abstract The IS6110 sequence was detected visually in sputum samples of tuberculosis patients using a bi-probe system. One of the probes was an oligonucleotide conjugated to colloidal gold particles, complementary to one end of the target strand. The other probe was an oligonucleotide conjugated to latex beads complementary to the other end of the target strand. In a reaction mix, these two probes bind to the target strand, and the latex beads are then separated by filtration. Bound latex beads have gold colloid particles at the other end of the target strand. These gold colloid particles were made visible to the naked eye by silver autometallography on the ,invisible' colloidal gold particles. The lower detection limit was 50 ng of genomic DNA of Mycobacterium tuberculosis. This new test, together with conventional PCR, was performed on DNA extracted from sputum samples of suspected tuberculosis patients. The new test was simple to perform, the results were visible to the naked eye, and the test was highly specific, as even single point mutations in the target strand sequence could be differentiated. The test could be useful in field-level laboratories because it requires no sophisticated equipment. [source] Comparison of real-time PCR and conventional hemi-nested PCR for the detection of Bordetella pertussis in nasopharyngeal samplesCLINICAL MICROBIOLOGY AND INFECTION, Issue 7 2003T. P. Anderson 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. [source] | |||||||||||||