PCR Sequencing

[Construction PG0839 gene-defective mutant of Porphyromonas gingivalis].

Hua Xi Kou Qiang Yi Xue Za Zhi. 2012 Apr;30(2):192-6, 200

Authors: Liu J, Pan Y, Li C, Lin L, Zhong M

Abstract
OBJECTIVE: In order to determine the function of PG0839 gene from Porphyromonas gingivalis (P. gingivalis) W83 strains, we intended to create a mutant in the PG0839 gene by homologous recombination.
METHODS: 1 584bp PG0839 gene fragment was amplified, digested by BamH I and EcoR I, purified and ligated to pUC19. The recombinant plasmid was designated as pPG0839-1. The erm cassette (2 101 bp) was inserted into the EcoR V restriction site of the PG0839 gene. The resultant recombinant plasmid, pPG0839-2, was used as a donor in the electroporation of P. gingivalis W83. After electroporated and selected on erythromycin brain heart infusion plates, a single colony was collected and designated as PG0839 gene-defective mutant.
RESULTS: A mutant in PG0839 gene was created by insertional inactivation, and inactivation of PG0839 gene was confirmed by restriction endonuclease digestive, sequencing, polymerase chain reaction (PCR) and reverse transcription PCR.
CONCLUSION: A PG0839 gene-defective mutant was created successfully.

PMID: 22594241 [PubMed - in process]

A Novel Nonsense Mutation in HSD17B3 Gene in a Tunisian Patient with Sexual Ambiguity.

J Sex Med. 2012 May 17;

Authors: Ben Rhouma B, Belguith N, Mnif MF, Kamoun T, Charfi N, Kamoun M, Abdelhedi F, Hachicha M, Kamoun H, Abid M, Fakhfakh F

Abstract
Introduction.  17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) isoenzyme is present almost exclusively in the testes and converts delta 4 androstenedione to testosterone. Mutations in the HSD17B3 gene cause HSD17B3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD). Aim.  This study aimed to present the clinical and biochemical features of a Tunisian patient who presented a sexual ambiguity orienting to HSD17B3 deficiency and to search for a mutation in the HSD17B3 gene by DNA sequencing. Methods.  Polymerase chain reaction (PCR) amplification and subsequent sequencing of all the coding exons of HSD17B3 gene were performed on genomic DNA from the patient, her family, and 50 controls. Results.  Genetic mutation analysis of the HSD17B3 gene revealed the presence of a novel homozygous nonsense mutation in the exon 9 (c.618 C > A) leading to the substitution p.C206X. The mutation p.C206X in the coding exons supports the hypothesis of HSD17B3 deficiency in our patient. Conclusion.  The patient described in this study represented a new case of a rare form of 46,XY DSD, associated to a novel gene mutation of HSD17B3 gene. The screening of this mutation is useful for confirming the diagnosis of HSD17B3 deficiency and for prenatal diagnosis. Ben Rhouma B, Belguith N, Mnif MF, Kamoun T, Charfi N, Kamoun M, Abdelhedi F, Hachicha M, Kamoun H, Abid M, and Fakhfakh F. A novel nonsense mutation in HSD17B3 gene in a Tunisian patient with sexual ambiguity. J Sex Med **;**:**-**.

PMID: 22594312 [PubMed - as supplied by publisher]

Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products.

BMC Microbiol. 2012 May 17;12(1):75

Authors: Adimpong DB, Nielsen DS, Sørensen KI, Derkx PM, Jespersen L

Abstract
ABSTRACT: BACKGROUND: Indigenous fermented food products play an essential role in the diet of millions of Africans. Lactic acid bacteria (LAB) are among the predominant microbial species in African indigenous fermented food products and are used for different applications in the food and biotechnology industries. Numerous studies have described antimicrobial susceptibility profiles of LAB from different parts of the world. However, there is limited information on antimicrobial resistance profiles of LAB from Africa. The aim of this study was to characterize 33 LAB previously isolated from three different African indigenous fermented food products using (GTG)5-based rep-PCR, sequencing of the 16 S rRNA gene and speciesspecific PCR techniques for differentiation of closely related species and further evaluate their antibiotic resistance profiles by the broth microdilution method and their haemolytic activity on sheep blood agar plates as indicators of safety traits among these bacteria. RESULTS: Using molecular biology based methods and selected phenotypic tests such as catalase reaction, CO2 production from glucose, colonies and cells morphology, the isolates were identified as Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus ghanensis, Lactobacillus plantarum, Lactobacillus salivarius, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella confusa. The bacteria were susceptible to ampicillin, chloramphenicol, clindamycin and erythromycin but resistant to vancomycin, kanamycin and streptomycin. Variable sensitivity profiles to tetracycline and gentamycin was observed among the isolates with Lb. plantarum, Lb. salivarius, W. confusa (except strain SK9-5) and Lb. fermentum strains being susceptible to tetracycline whereas Pediococcus strains and Lb. ghanensis strains were resistant. For gentamicin, Leuc. pseudomesenteroides, Lb. ghanensis and Ped. acidilactici strains were resistant to 64 mg/L whereas some W. confusa and Lb. plantarum strains had a MIC value of 16 mg/L and 32 mg/L respectively. No beta-haemolytic activity was observed, however, alpha-haemolytic activity was observed in 27% (9) of the strains comprising Lb. salivarius (6), W. confusa (2) and Lb. delbrueckii (1) isolates. CONCLUSIONS: The resistance to kanamycin and vancomycin is probably an intrinsic feature since similar observations were reported in the literature for LAB. Low prevalence of pathogenicity indicator traits were observed among the isolates especially with the presence of poor haemolytic activities and they could therefore be considered as interesting candidates for selection of starter cultures or probiotics for different applications.

PMID: 22594449 [PubMed - as supplied by publisher]

A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays.

BMC Med Genet. 2012 May 17;13(1):34

Authors: Wei LK, Griffiths LR, Hua GS

Abstract
ABSTRACT: BACKGROUND: Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor than can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS 786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C were calculated using the Hardy Weinberg equation. METHODS: The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. RESULTS: The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS 786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). CONCLUSIONS: Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants simultaneously with 100 % concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS 786 T > C variants.

PMID: 22594584 [PubMed - as supplied by publisher]

Molecular detection and analysis of spotted fever group Rickettsia in patients with fever and rash at a tertiary care centre in Tamil Nadu, India.

Pathog Glob Health. 2012 Mar;106(1):40-5

Authors: Prakash JA, Sohan Lal T, Rosemol V, Verghese VP, Pulimood SA, Reller M, Dumler JS

Abstract
BACKGROUND: Detection of specific targets by PCR is used to confirm a diagnosis of spotted fever, but serological tests are still widely used. In this prospective study, nested PCR was performed on skin biopsy specimens to confirm the diagnosis of spotted fever.
METHODS: In 58 clinically suspected cases of spotted fever, nested PCR, to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA and ompB, from skin biopsy of the rash was performed. Sequencing was carried on amplicons representing the four targets to confirm specificity of amplification. This was followed by phylogenetic analysis using MEGA version 4.0 software.
RESULTS: The gltA, 17 kDa, ompA, and ompB genes were detected from skin biopsy specimens in 38, 23, 27, and 22 individuals. Sequence analysis revealed that the gltA, 17 kDa, ompA, and ompB sequences belonged to spotted fever group (SFG) rickettsia. Of the six partial ompA gene sequences, only one was dissimilar to the previously reported 'Candidatus Rickettsia kellyi'.
CONCLUSION: Further evidence indicates that SFG rickettsiae resembling 'Candidatus Rickettsia kellyi' cause fever and rash in southern India. More detailed phylogenetic analysis following isolation of rickettsia in culture is required for providing irrefutable proof for the occurrence of novel spotted fever rickettsiae in this region.

PMID: 22595273 [PubMed - in process]

Identification of a novel Cys146X mutation of SOD1 in familial amyotrophic lateral sclerosis by whole-exome sequencing.

Genet Med. 2012 May 17;

Authors: Wu J, Shen E, Shi D, Sun Z, Cai T

Abstract
Purpose:Familial amyotrophic lateral sclerosis has been linked to mutations in 15 genes, and it is believed these genes account for less than 20-30% of Chinese patients with familial amyotrophic lateral sclerosis. Of the 163 different superoxide dismutase 1 gene mutations in amyotrophic lateral sclerosis 1, only 6.1% of them were from individuals of Chinese origin. Therefore, to quickly learn the causative gene for patients with familial amyotrophic lateral sclerosis in a Chinese pedigree, we opted to apply whole-exome sequencing as a diagnostic tool.Methods:To avoid time-consuming screening of known familial amyotrophic lateral sclerosis candidate genes by PCR-Sanger sequencing, we conducted whole-exome sequencing toward selected individuals of a four-generation familial amyotrophic lateral sclerosis family.Results:Patients in the family showed autosomal dominant features, as well as a mean onset age of 35.3 years, and a mean duration of 2.1 years. By deep sequencing analysis, we identified a novel p.Cys146X SOD1 mutation in all examined patients. Genotype-phenotype and SOD1 structural model analysis revealed the effects of the Cys57-Cys146 disulfide bond formation and the C-terminal dimer contact region on the disease phenotypes.Conclusion:The Cys146X mutation causes familial amyotrophic lateral sclerosis with severe phenotypes. Whole-exome sequencing becomes an attractive diagnostic tool for identifying causative genes, particularly for neurological disorders with unusual phenotypes, pleiotropic malformations, multiple known candidate genes, and complicated inheritance patterns.Genet Med advance online publication 17 May 2012.

PMID: 22595939 [PubMed - as supplied by publisher]

Epidemiological characteristics and genetic structure of blaNDM-1 in non-baumannii Acinetobacter spp. in China.

J Antimicrob Chemother. 2012 May 17;

Authors: Fu Y, Du X, Ji J, Chen Y, Jiang Y, Yu Y

Abstract
OBJECTIVES: The goal of this study was to investigate the epidemiological characteristics and the surrounding genetic structure of bla(NDM-1) in non-baumannii Acinetobacter spp. in China. METHODS: Non-baumannii Acinetobacter spp. were collected from 28 provinces in China and were screened for the presence of bla(NDM-1) using PCR. The following four methods were used to classify the Acinetobacter isolates: the Vitek 2 system, 16S-23S rRNA gene intergenic spacer sequencing, amplified rDNA restriction analysis and partial rpoB sequence analysis. An S1-PFGE assay and Southern blot hybridization were performed to determine the plasmid location of bla(NDM-1). The transferability of bla(NDM-1)-harbouring plasmids was confirmed by conjugation experiments and electrotransformation. The surrounding genetic structure of the bla(NDM-1) gene was analysed using a restriction endonuclease-based cloning approach and primer walking. RESULTS: Among 726 non-baumannii Acinetobacter spp., nine isolates collected from six different provinces and assigned to seven different Acinetobacter spp. contained the bla(NDM-1) gene. None of these isolates was directly infectious to the patients or demonstrated an epidemiological importation from abroad. These bla(NDM-1) genes were located on plasmids that could be transferred to Escherichia coli J53 by conjugation and Acinetobacter baylyi ADP1 by electrotransformation. Seven of the nine strains shared a common genetic structure in which bla(NDM-1) was flanked by two copies of ISAba125. CONCLUSIONS: The clinical challenge posed by bla(NDM-1) is currently minimal in China; however, more attention should be devoted to monitoring the dissemination of this gene due to its potential transferability via the ISAba125-associated transposon.

PMID: 22604448 [PubMed - as supplied by publisher]

Anchored hybrid enrichment for massively high-throughput phylogenomics.

Syst Biol. 2012 May 17;

Authors: Lemmon AR, Emme S, Lemmon EM

Abstract
The field of phylogenetics is on the cusp of a major revolution, enabled by new methods of data collection that leverage both genomic resources and recent advances in DNA sequencing. Previous phylogenetic work has required labor-intensive marker development coupled with single-locus PCR and DNA sequencing on a clade-by-clade and marker-by-marker basis. Here, we present a new, cost-efficient, and rapid approach to obtaining data from hundreds of genes for potentially hundreds of individuals for deep and shallow phylogenetic studies. Specifically, we designed probes for target enrichment of >500 loci in highly-conserved anchor regions of vertebrate genomes (flanked by less conserved regions) from five model species and tested enrichment efficiency in non-model species up to 254 million years divergent from the nearest model. We found that hybrid enrichment using conserved probes (anchored enrichment) can recover a large number of unlinked loci that are useful at a diversity of phylogenetic timescales. This new approach has the potential to not only expedite resolution of deep-scale portions of the Tree of Life but also to greatly accelerate resolution of the large number of shallow clades that remain unresolved. The combination of low cost (∼1% of the cost of traditional Sanger sequencing and ∼3.5% of the cost of high-throughput amplicon sequencing for projects on the scale of 500 loci × 100 individuals) and rapid data collection (∼2 weeks of laboratory time) are expected to make this approach tractable even for researchers working on systems with limited or non-existent genomic resources.

PMID: 22605266 [PubMed - as supplied by publisher]

Mutations in TULP1, NR2E3, and MFRP genes in Indian families with autosomal recessive retinitis pigmentosa.

Mol Vis. 2012;18:1165-1174

Authors: Kannabiran C, Singh H, Sahini N, Jalali S, Mohan G

Abstract
PURPOSE: To identify genes underlying autosomal recessive retinitis pigmentosa (ARRP) by homozygosity mapping. METHODS: Families with ARRP were recruited after complete ophthalmic evaluation of all members and diagnosis of RP by predefined criteria. Genomic DNA from affected members of 26 families was genotyped on Illumina single nucleotide polymorphism (SNP) 6.0 K arrays with standard procedures. Genotypes were evaluated for homozygous regions that were common and concordant between affected members of each family. The genes mapping to homozygous intervals within these families were screened for pathogenic changes with PCR amplification and sequencing of coding regions. Cosegegration of sequence changes with disease was determined within each pedigree, and each variation was tested for presence in 100 unrelated normal controls. RESULTS: A genome-wide scan for homozygosity showed homozygous regions harboring the tubby like protein 1 gene (TULP1; chromosome 6) in one family, the nuclear receptor subfamily 2, group E, member 3 gene (NR2E3; chromosome 15) in three families, and the membrane frizzled-related protein gene (MFRP; chromosome 11) in one family. Screening of the three genes in the respective families revealed homozygous disease-causing mutations in three families. These included a missense mutation in TULP1, a deletion-cum-insertion in NR2E3, and a single base deletion in MFRP. Patients from all three families had a rod-cone type of dystrophy with night blindness initially. The NR2E3 and MFRP genes were associated with fundus features atypical of RP. CONCLUSIONS: This study shows involvement of the TULP1, NR2E3, and MFRP genes in ARRP in Indian cases. Genome-wide screening with SNP arrays followed by a prioritized candidate gene evaluation is useful in identifying genes in these patients.

PMID: 22605927 [PubMed - as supplied by publisher]

Relationship between Oral Anaerobic Bacteria and Otitis Media with Effusion.

Int J Med Sci. 2012;9(3):256-61

Authors: Topcuoglu N, Keskin F, Ciftci S, Paltura C, Kulekci M, Ustek D, Kulekci G

Abstract
Objective: In this study hypothesing the translocation of oral bacteria from oropharynx into the middle ear cavity may be involved in the pathogenesis of otitis media with effusion (OME), we aimed to investigate the presence and similarity of Fusobacterium nucleatum and Treponema denticola in saliva, nasopharyngeal secretion and the middle ear effusion samples from the children with OME.Methods: Totally 20 children with OME undergoing myringotomy and ventilation tube placement were attended. Stimulated saliva samples were collected after otorhinolaryngological and oral examinations were done. The middle ear effusion and nasopharyngeal secretions were collected during the operations. The presence of F. nucleatum and T. denticola were detected using 16SrRNA-based PCR. The clonal similarities of the bacteria were detected in the samples which the same bacteria had been detected in each samples of the same child. After DNA sequencing, clonal similarity was determined by 16SrRNA gene clone library analysis. The sequences from each clone were compared with similar sequences of reference organisms by FASTA search.Results: T. denticola was detected only in four (20%) saliva and in one (5%) nasopharyngeal sample. F. nucleatum was detected in 11 (55%) saliva, eight (40%) nasopharyngeal and six (30%) middle ear effusion samples. Sequences from F.nucleatum clones derived from three different anatomic sites within patients were similar in 33% of OME patients, indicating their genetic relatedness.Conclusions: Bacteria involved in this process most likely originate from the oropharynx since they show a close genetic relatedness with their oropharyngeal counterparts.

PMID: 22606045 [PubMed - in process]