Gene Polymerase Chain Reaction Products Research Articles

Microbial Profile During Pericoronitis and Microbiota Shift After Treatment.

The microflora of the distal pocket is considered as the major cause of pericoronitis. How the oral microflora changes during pericoronitis and whether different types of impacted third molar harbor the same microflora are still unknown. Saliva, subgingival plaque, and gingival plaque of mandibular third molars (M3Ms) were collected from twelve patients with acute pericoronitis. They were given local irrigation or local irrigation + antibiotics according to symptoms. Samples were harvested at the first visit with pericoronitis, 1 week after treatment, and 6 weeks after treatment. 16S rRNA gene polymerase chain reaction products were generated and sequenced after DNA isolation. Comparison of three sampling sites showed that, the subgingival plaque of M3Ms had most remarkable changes in symptomatic period, including a significant increase in microbial richness, and a convergent trend in microbial composition. After treatment, the subgingival microbiome was altered and largely returned to the state in asymptomatic period. In summary, the distal subgingival microbiota of M3M was most likely to be associated with the pathogenesis of pericoronitis. The post-treatment microbiota shift of M3M proved the effectiveness of treatment. The inclination type of impacted M3Ms and treatment method would also make a difference to the pericoronal microbiota.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis of Three Lipooligosaccharide-Associated Genes of Campylobacter jejuni and Campylobacter coli Isolated From Animal Samples

Background: Several genetic mechanisms are used by Campylobacter spp. to achieve pathogenesis. One of the involved virulence factors is lipooligosacharide-associated genes, which are related to ganglioside mimicry by Campylobacter species. Objectives: The current study was conducted to determine the genetic diversity of 3 LOS-associated genes among Campylobacter jejuni and C. coli isolated from animal fecal samples. Methods: One hundred broiler, cattle, and sheep fecal C. jejuni and C. coli isolates, which had been collected previously from Shiraz slaughterhouses and had been formerly identified by polymerase chain reaction (PCR) reactions, were used in the present study. Campylobacter species were subjected to detect wlaN, cgtB, and waaC genes. The PCR products of three LOS-genes were subjected for restriction fragment length polymorphism (RFLP) using HindIII and AluI restriction enzymes in separate reactions. The most prevalent RFLP patterns in the combination of 2 enzymes were subjected for sequencing and sequence analysis software. Results and Conclusions: Patterns of RFLP for PCR products of all 69 wlaN and 29 cgtB genes were similar yet among 86 waaC gene PCR products, 6 different RFLP patterns were obtained. In conclusion, PCR-RFLP analysis demonstrated considerable variation in gene content and overall sequence heterogeneity in the animal-associated C. jejuni and C. coli LOS biosynthesis genes.

Insights into the recent emergence and expansion of eastern equine encephalitis virus in a new focus in the Northern New England USA.

BackgroundEastern equine encephalomyelitis virus (EEEV) causes a highly pathogenic zoonosis that circulates in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine birds in freshwater hardwood swamps in the northeastern U.S. Epidemic/epizootic transmission to humans/equines typically occurs towards the end of the transmission season and is generally assumed to be mediated by locally abundant and contiguous mammalophagic “bridge vector” mosquitoes.MethodsEngorged mosquitoes were collected using CDC light, resting box, and gravid traps during epidemic transmission of EEEV in 2012 in Addison and Rutland counties, Vermont. Mosquitoes were identified to species and blood meal analysis performed by sequencing mitochondrial cytochrome b gene polymerase chain reaction products. Infection status with EEEV in mosquitoes was determined using cell culture and RT-PCR assays, and all viral isolates were sequenced and compared to other EEEV strains by phylogenetic analysis.ResultsThe host choices of 574 engorged mosquitoes were as follows: Cs. melanura (n = 331, 94.3 % avian-derived, 5.7 % mammalian-derived); Anopheles quadrimaculatus (n = 164, 3.0 % avian, 97.0 % mammalian); An. punctipennis (n = 56, 7.2 % avian, 92.8 % mammalian), Aedes vexans (n = 9, 22.2 % avian, 77.8 % mammalian); Culex pipiens s.l. n = 6, 100 % avian); Coquillettidia perturbans (n = 4, 25.0 % avian, 75.0 % mammalian); and Cs. morsitans (n = 4, 100 % avian). A seasonal shift in blood feeding by Cs. melanura from Green Heron towards other avian species was observed. EEEV was successfully isolated from blood-fed Cs. melanura and analyzed by phylogenetic analysis. Vermont strains from 2012 clustered with viral strains previously isolated in Virginia yet were genetically distinct from an earlier EEEV isolate from Vermont during 2011.ConclusionsCuliseta melanura acquired blood meals primarily from birds and focused feeding activity on several competent species capable of supporting EEEV transmission. Culiseta melanura also occasionally obtained blood meals from mammalian hosts including humans. This mosquito species serves as the primary vector of EEEV among wild bird species, but also is capable of occasionally contributing to epidemic/epizootic transmission of EEEV to humans/equines. Other mosquito species including Cq. perturbans that feed more opportunistically on both avian and mammalian hosts may be important in epidemic/epizootic transmission under certain conditions. Phylogenetic analyses suggest that EEEV was independently introduced into Vermont on at least two separate occasions.

Fast high-throughput screening of H1N1 virus by parallel detection with multichannel microchip electrophoresis.

Influenza is one of the acute respiratory diseases of human caused by the influenza A (H1N1) virus and accounted for major public health concerns worldwide. The polymerase chain reaction (PCR) methods are the most popular tools for clinical diagnosis of influenza A virus. Microchip electrophoresis is a widely used method for DNA molecules separation. Herein, we describe the fast and high-throughput separation of hemagglutinin (HA) and nucleocapsid protein (NP) gene PCR products (116 bp and 195 bp, respectively) by parallel detection with multichannel microchip electrophoresis and programmed step electric field strength (PSEFS).

Diversity and distribution of ammonia-oxidizing Archaea in the seasonally frozen soils in Northeastern China

The genetic diversity and distribution of ammonia-oxidizing Archaea (AOA) in nine seasonally frozen soils sampled around the city of Harbin, China, is analyzed based on archaeal amoA gene. Soil samples are divided into four groups by its properties: fertilized/unfertilized mesic (well-balanced supply of moisture) soils and fertilized/unfertilized hydric (abundant of moisture) soils. Clone libraries based on AOA amoA gene polymerase chain reaction products are constructed, and the phylogenetic analysis at 5% cutoff level shows that AOA members mainly belong to the soil/sediment lineage which includes four clusters, and very few archaeal amoA gene sequences fall into the marine lineage. The four groups of soils have different archaeal amoA gene assemblage, and the available nitrogen and organic carbon are significantly correlated with diversity indexes. The result shows that long-term artificial amendment such as fertilization and agriculture cultivation has an important impact on AOA community shift in terrestrial environment. Moisture may drive the shape of different AOA communities by changing the aerobic environment into anaerobic. Soil composition is another noticeable factor effect AOA community, which can help the shape of a special AOA community with only two species.

Clonal antigen receptor gene PCR products outside the expected size range

Polymerase chain reaction (PCR) of the antigen receptor genes has clinical utility in establishing clonality in lymphoproliferations, which is an important correlate of lymphoid neoplasia. The most frequently used procedures for this purpose were developed by the BIOMED-2 consortium. One of the criteria for establishing monoclonality using PCR of the antigen receptor genes is the finding of an abundant amplicon within a size range determined by the positions of the PCR primers and the known variability in size inherent in the recombination events that assemble a functional antigen receptor gene. However, several cases have been reported in which an amplicon outside this size range has been shown to be a valid indicator of clonality after DNA sequence analysis. In this paper, we will report and discuss several additional cases in which an amplicon outside the accepted size range was consistent with a monoclonal lymphoproliferation. We conclude that oversized and undersized amplicons may indeed represent evidence for a monoclonal lymphoproliferation, but that this interpretation should preferably be confirmed by sequence analysis to avoid a false-positive result.

DETECTION OF HUMAN BOCAVIRUS IN CHINESE CHILDREN WITH RESPIRATORY TRACT INFECTION

INTRODUCTION: Human bocavirus (HBoV), a parvovirus discovered in 2005, was identified as a respiratory pathogen in a proportion of respiratory tract diseases with an unknown causative agent. OBJECTIVE: Our goal was to investigate the role of HBoV in acute lower respiratory tract infection in Chinese children. METHODS: Two hundred forty-five nasopharyngeal aspirates collected from January to December 2006 from hospitalized children with acute lower respiratory tract infection were tested for the presence of HBoV DNA by using polymerase chain reaction (PCR) that targeted the NP-1 gene. Bulk PCR products were subjected to nucleotide sequence analysis. Medical charts were reviewed for clinical features of HBoV infection. RESULTS: HBoV DNA was detected in 11 (4.5%) of the 245 nasopharyngeal aspirates. HBoV infection occurred year-round and peaked in winter. The age range of the children was from 48 days to 18 months. Coinfections of HBoV and respiratory syncytial virus were found in 2 (18.2%) of 11 samples. Nucleotide sequence of the NP-1 gene PCR products showed considerably high identity (99%). Clinical symptoms included cough and wheezing. CONCLUSIONS: HBoV seems to be one of the respiratory pathogens for acute respiratory tract infection in the Chongqing area, particularly in young children. Understanding of the clinical relevance of HBoV infection will require additional studies.

Pre-S Deletion and Complex Mutations of Hepatitis B Virus Related to Advanced Liver Disease in HBeAg-Negative Patients

This longitudinal study investigated the interactions and roles of hepatitis B virus (HBV) genotypes, pre-S deletions, and core promoter and precore mutations on the progression of liver disease in hepatitis B e antigen (HBeAg)-negative patients. A total of 141 HBeAg-negative patients without liver cirrhosis or hepatocellular carcinoma at study entry were recruited for this study, including 45 inactive HBV carriers and 96 patients with HBeAg-negative chronic hepatitis B. The HBV genotypes and the sequences of pre-S, core promoter, and precore regions were determined. Compared with patients without developing liver cirrhosis, patients with the development of liver cirrhosis had higher rates of genotype C; pre-S deletions; C or G1753, T1762/A1764, T1766, and/or A1768 mutants; and G1799 variant. Cox regression analysis showed that older age, higher total bilirubin and HBV DNA levels, pre-S deletions, and T1766 and/or A1768 mutants were significantly associated with the development of liver cirrhosis. HBV with a complex mutation pattern (pre-S deletion, T1762/A1764, and T1766 and/or A1768 mutants) rather than a single mutation was associated with the development of liver cirrhosis, and the patterns of mutation combinations differed between HBV genotype B and C. Moreover, pre-S deletion was a significant risk factor for hepatocellular carcinoma. This study indicated that pre-S deletion and combined mutations of HBV are useful molecular markers for predicting the clinical outcomes of HBeAg-negative patients.

Molecular characterization of the bacteria adherent to human colorectal mucosa

To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.

Identification of Phytoplasmas Associated with Grapevine Yellows in Serbia

AbstractThe molecular identification and characterization of phytoplasmas from infected grapevines in four locations in Serbia are reported. Phytoplasmas were detected and identified by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rDNA. Grapevine yellows were associated with three molecularly distinguishable phytoplasmas: Flavescence dorée phytoplasmas (elm yellows group: 16SrV‐C subgroup) were present only in the Župa Aleksandrovac region; Bois noir phytoplasmas (stolbur group: 16SrXII‐A subgroup) were detected in the other surveyed regions; a mixed infection of European stone fruit yellows (apple proliferation group: 16SrX‐B subgroup) and Bois noir phytoplasmas was identified in one sample. A finer molecular characterization by RFLP analysis of rpS3 and SecY genes of Flavescence dorée phytoplasmas from Župa Aleksandrovac confirmed that the Serbian genotype is indistinguishable from a strain from the Veneto region, Italy. Characterization of the tuf gene of Bois noir phytoplasmas showed lack of amplification of samples from Erdevik. HpaII profiles of tuf gene PCR products of samples from Pali and Radmilovac were identical, and were indistinguishable from one of the two profiles produced by samples from Italian grapevines used as reference strains.

Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene.

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship.

A new platelet polymorphism Duva+, localized within the RGD binding domain of glycoprotein IIIa, is associated with neonatal thrombocytopenia

We report here the identification and characterization of a new platelet alloantigen, Duv(a+), implicated in a case of neonatal thrombocytopenia. Immunochemical studies demonstrated that the epitope was localized on glycoprotein (GP) IIIa. Sequencing of the exons 2 to 15 of GP IIIa gene polymerase chain reaction products from both parents revealed a single base substitution 517C>T (complementary DNA) present in a heterozygous state in DNA from the father leading to amino acid substitution Thr140Ile (ACC>ATC) within the Arg-Gly-Asp binding domain of GP IIIa. Flow cytometry and immunoprecipitation studies of IIb-C517 or T517 IIIa transfected Cos cells allowed us to demonstrate this mutation was responsible for expression of the Duv(a+) epitope. By polymerase chain reaction-single-strand conformational-polymorphism analysis, the mutated allele could not be detected in a population of 100 healthy unrelated donors, indicating a low frequency of occurrence. The Thr140/Ile dimorphism, localized 3 amino acids upstream from the Arg143 involved in the expression of HPA-4a, did not interfere with the binding of an anti-HPA-4a antibody in flow cytometry. Results of functional analysis of wild-type or mutated transfected CHO cells-(1) aggregation in the presence of Ca(++) and soluble fibrinogen after complex activation by dithiothreitol, (2) adhesion on coated fibrinogen, (3) binding of monoclonal antibody PAC-1 or LIBS antibody D3, and (4) outside-in signaling-all suggest that the Thr140Ile polymorphism localized in the Arg-Gly-Asp binding domain of GP IIIa does not affect significantly, if at all, the integrin function. We have shown that the anti-Duv(a+) antibody may inhibit platelet GP IIb-IIIa function.

Molecular analysis of DHFR and DHPS genes in P. falciparum clinical isolates from the Haut–Ogooué region in Gabon

The main objective of this work was to determine the prevalence of mutations in genes coding for the dihydropteroate synthase ( DHPS) and the dihydrofolate reductase ( DHFR) enzymes which are implicated in resistance of P. falciparum to antifolate (pyrimethamine-sulfadoxine (P/S)). In this study, 117 human blood samples were collected at Franceville located in the region of Haut-Ogooué (South-eastern Gabon). In this area, a relatively low level of sensitivity of Plasmodium falciparum to P/S has been reported with 18.2% of RII and 12.1% of RI resistance. A nested polymerase chain reaction was used to amplify a fragment of the DHFR gene containing codon 108, where a point mutation causing a Serine (wild type) to Asparagine or to a Threonine (resistant types) change occurs in pyrimethamine resistant parasites. Eleven DHFR fragments were sequenced and mutations occurring at codons 51, 59 and 108 were analysed. The DHPS gene was amplified by polymerase chain reaction (PCR) and sequenced directly or after cloning. Variant amino acid residues 436, 437, 540, 581, 613 associated with sulfadoxine resistance were analysed. The analysis of codon 108 of the DHFR gene was undertaken for 81 isolates. More than one DHFR P. falciparum genotype was present in 64% of the samples. We showed that 47% of 141 DHFR gene PCR products had Serine (wild phenotype), and 52% had Asparagine. We found one isolate with the Thr-108 confirmed by sequencing of the PCR product. Triple, double and single DHFR mutant at positions 51, 59 and 108 were found. Only codons 436 and 437 of the 38 analysed sequences of the DHPS gene revealed point mutations. These results have been compared with those reported from different sites in Africa, Asia or South-America.

Detection of concurrent/recurrent non-hodgkin's lymphoma in effusions by PCR

A subset of patients with non-Hodgkin's lymphoma (NHL), present with or subsequently develop lymphocytic effusions. Differential diagnosis between reactive lymphocytosis and recurrent low-grade NHL is difficult by cytology alone. We studied the use of polymerase chain reaction (PCR)-based techniques to detect concurrent/recurrent NHL. Both primary tumors and atypical lymphocytic effusions of 12 low-grade B-NHL patients and 4 T-NHL patients were studied. Six pleural effusions (reacitve/carcinomatous), in patients with no history of NHL, were included. Samples were amplified by PCR, using Fr3, Fr2, LJH, and VLJH primers specific for the immunoglobulin heavy chain (IgH) gene and Vγ-8, Vγ9, Vγ10, Vγ11 and Jγ1/Jγ2 consensus primers specific for the T-cell receptor gamma (TCR-γ) gene. IgH gene PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). TCR-γ gene PCR products were analyzed using a novel nonradioactive single-stranded conformational polymorphism (SSCP) procedure. IgH gene rearrangement analysis demonstrated monoclonality in 11 12 primary low-grade B-NHLs. Idential monoclonal bands were found in both primary tumor and effusion in 9 patients. TCR-γ gene rearrangement analysis demonstrated monoclonality in 4 of 4 primary T-NHLs. Identical monoclonal banded patterns were found in both primary tumor and effusion in 3 patients. Our results strongly support the diagnosis of concurrent/recurrent NHL in 13 of 16 (81%) cases of atypical lymphocytic effusions. IgH/PAGE and TCR-γ/SSCP analyses are useful tools in the diagnoses of lymphocytic effusions in patients with NHL.