Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2630-2640 ◽  
Author(s):  
J. T. Tambong ◽  
R. Xu ◽  
E. S. P. Bromfield
Keyword(s):  
Root Zone ◽  
Phylogenetic Analyses ◽  
Melting Curve ◽  
Pcr Amplification ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Its1 Region ◽  
A Genome ◽  
Pure Cultures ◽  
Intercalating Dye

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNAIle and tRNAAla in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

Differentiation and Characterization by Molecular Techniques of Bacillus cereus Group Isolates from Poto Poto and Dégué, Two Traditional Cereal-Based Fermented Foods of Burkina Faso and Republic of Congo

2007 ◽  
Vol 70 (5) ◽  
pp. 1165-1173 ◽  
Author(s):  
HIKMATE ABRIOUEL ◽  
NABIL BEN OMAR ◽  
ROSARIO LUCAS LÓPEZ ◽  
MAGDALENA MARTÍNEZ CAÑAMERO ◽  
ELENA ORTEGA ◽  
...  
Keyword(s):  
Melting Curve ◽  
Pcr Amplification ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Toxin Gene ◽  
Fermented Foods ◽  
Sequencing Analysis ◽  
16S Rdna Sequencing ◽  
Bacillus Cereus Group ◽  
Rdna Sequencing

Poto poto (a maize sourdough) and dégué (a pearl millet–based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.

scholarly journals Rapid, Sensitive, and Discriminating Identification of Naegleria spp. by Real-Time PCR and Melting-Curve Analysis

2006 ◽  
Vol 72 (9) ◽  
pp. 5857-5863 ◽  
Author(s):  
Bret S. Robinson ◽  
Paul T. Monis ◽  
Phillip J. Dobson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Cooling Systems ◽  
Wide Range ◽  
Intercalating Dye ◽  
Medical Importance ◽  
Single Primer

ABSTRACT The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

scholarly journals New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group

2015 ◽  
Vol 53 (11) ◽  
pp. 3430-3437 ◽  
Author(s):  
Shamira J. Shallom ◽  
Natalia S. Moura ◽  
Kenneth N. Olivier ◽  
Elizabeth P. Sampaio ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Rrna Gene ◽  
Nucleotide Position ◽  
Inducible Resistance ◽  
Clarithromycin Resistance

Members of theMycobacterium abscessusgroup (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducibleerm(41) gene or via acquired mutations in therrl(23S rRNA) gene. Strains harboring a truncation or a T28C substitution inerm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncatederm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 oferm(41) and 23S rRNArrlgene mutations at position 2058 and/or 2059. Truncated and full-sizeerm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showedrrlmutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an activeerm(41), inactiveerm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.

scholarly journals Human Papillomavirus (HPV69/HPV73) Coinfection associated with Simultaneous Squamous Cell Carcinoma of the Anus and Presumed Lung Metastasis

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 349
Author(s):  
Stephanie Shea ◽  
Marina Muñoz ◽  
Stephen C. Ward ◽  
Mary B. Beasley ◽  
Melissa R Gitman ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Sanger Sequencing ◽  
Tissue Sample ◽  
Melting Curve ◽  
Pcr Amplification ◽  
Human Papillomaviruses ◽  
Melting Curve Analysis ◽  
Anal Squamous Cell Carcinoma

Background: Human papillomaviruses (HPVs) have been linked to a variety of human cancers. As the landscape of HPV-related neoplasia continues to expand, uncommon and rare HPV genotypes have also started to emerge. Host-virus interplay is recognized as a key driver in HPV carcinogenesis, with host immune status, virus genetic variants and coinfection highly influencing the dynamics of malignant transformation. Immunosuppression and tissue tropism are also known to influence HPV pathogenesis. Methods: Herein, we present a case of a patient who, in the setting of HIV positivity, developed anal squamous cell carcinoma associated with HPV69 and later developed squamous cell carcinoma in the lungs, clinically presumed to be metastatic disease, associated with HPV73. Consensus PCR screening for HPV was performed by real-time PCR amplification of the L1 gene region, amplification of the E6 regions with High-Resolution Melting Curve Analysis followed by Sanger sequencing confirmation and phylogenetic analysis. Results: Sanger sequencing of the consensus PCR amplification product determined that the anal tissue sample was positive for HPV 69, and the lung tissue sample was positive for HPV 73. Conclusions: This case underscores the importance of recognizing the emerging role of these rare “possibly carcinogenic” HPV types in human carcinogenesis.

scholarly journals High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shlomo Pilo ◽  
Gal Zizelski Valenci ◽  
Mor Rubinstein ◽  
Lea Pichadze ◽  
Yael Scharf ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Multilocus Sequence Typing ◽  
Phylogenetic Trees ◽  
Melting Curve ◽  
Pcr Amplification ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Whole Genome ◽  
Mlst Scheme ◽  
Low Concentrations

Abstract Background Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA. Results In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. Conclusions The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

scholarly journals The rates of mutations associated with macrolide resistance in Mycoplasma genitalium among patients with non-gonococcal sexually transmitted infections in Smolensk and Tula

2019 ◽  
Vol 21 (4) ◽  
pp. 330-339
Author(s):  
L.M. Zubareva ◽  
Inna A. Edelstein ◽  
N.S. Rudneva ◽  
Andrey V. Romanov ◽  
T.A. Vlasova ◽  
...  
Keyword(s):  
Melting Curve ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
Epidemiological Surveillance ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Common Mutation ◽  
Resistance Mutations ◽  
Nucleotide Substitutions ◽  
Two Cities

Objective. In this study, we examined the prevalence of macrolide-resistant M. genitalium in two Russian cities, Smolensk and Tula, between 2013 and 2017. Materials and Methods. DNA’s samples were isolated from urethral and cervicovaginal swabs using primary screening and tested for macrolide resistance-associated mutations by real-time PCR. This technology makes it possible to identify any nucleotide substitutions in the 23S rRNA M. genitalium gene at positions 2058, 2059, 2611 M. genitalium by melting curve analysis after the amplification. Results. According to the study in two cities (Smolensk and Tula) macrolide resistance-associated mutations were found in 3, 65% of isolates (21⁄574). The A2058G transition 23S rRNA MGE was the most common mutation that is associated with macrolide resistance: 5⁄12 (41.6%) – Smolensk, 8⁄9 (88.8%) – Tula. Rare substitutions have been reported at position A2058T 23S rRNA MGE and at position C2611T23S rRNA MGE. All received data is published at the AntiMicrobial Resistance Map (AMRmap) (http://AMRmap.com). Conclusions. According to our study, the frequency of macrolide-resistance mutations in M. genitalium was not more than 4% in two cities of Central Russia during 2013–2017. Despite the relatively low rates of resistance of M. genitalium to macrolides in Smolensk and Tula, our data emphasize the need for epidemiological surveillance of resistance in this pathogen.

scholarly journals A Novel SimpleProbe PCR Assay for Detection of Mutations in the 23S rRNA Gene Associated with Macrolide Resistance in Mycoplasma genitalium in Clinical Samples

2016 ◽  
Vol 54 (10) ◽  
pp. 2563-2567 ◽  
Author(s):  
Marianne Gossé ◽  
Hilde Lysvand ◽  
Brita Pukstad ◽  
Svein Arne Nordbø
Keyword(s):  
Melting Curve ◽  
Mycoplasma Genitalium ◽  
Curve Analysis ◽  
23S Rrna ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Rrna Gene ◽  
Content Type ◽  
23S Rrna Gene ◽  
Resistant Strains

Macrolide-resistant strains ofMycoplasma genitaliumare an increasing problem throughout the world, and the implementation of a rapid and sensitive assay for mutation detection to guide treatment is needed. Macrolide-resistant strains have been shown to contain base substitutions in positions 2058 and 2059 (Escherichia colinumbering) in region V of the 23S rRNA gene. In this study, we present a SimpleProbe PCR followed by melting curve analysis to differentiate between macrolide-resistant mutants and wild types. The assay was performed on 159Mycoplasma genitalium-positive samples, and the results were compared with DNA sequencing. We also looked at the prevalence of macrolide-resistant strains in a Norwegian population. Of 139 samples characterized successfully by sequencing, 54 (39%) were wild types and 85 (61%) were mutants, consisting of 59 (42%) A2059G, 24 (17%) A2058G, 1 (1%) A2058T, and 1 (1%) A2059C mutation. The melting curve analysis correctly differentiated between wild-type and mutant strains in all cases, but it could not identify the different mutant types. The SimpleProbe PCR proved to be a simple, rapid, and reliable method for the detection of macrolide-resistant isolates ofMycoplasma genitaliumin a clinical setting.

scholarly journals Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15) mRNA in Various Human Cell Types

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Sang Mee Hwang ◽  
Mi Jung Kim ◽  
Ho Eun Chang ◽  
Yun Ji Hong ◽  
Taek Soo Kim ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Human Platelet ◽  
Human Fibroblasts ◽  
Leukemia Cell ◽  
Embryonic Stem ◽  
Melting Curve ◽  
Leukemia Cell Line ◽  
Melting Curve Analysis ◽  
Platelet Antigen ◽  
Human Platelet Antigen

CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

scholarly journals Genetic Diversity of Enteric Viruses in Children under Five Years Old in Gabon

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 545
Author(s):  
Gédéon Prince Manouana ◽  
Paul Alvyn Nguema-Moure ◽  
Mirabeau Mbong Ngwese ◽  
C.-Thomas Bock ◽  
Peter G. Kremsner ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Preventive Measures ◽  
Phylogenetic Analyses ◽  
Enteric Viruses ◽  
Study Cohort ◽  
Viral Agent ◽  
A Genome ◽  
Stool Samples ◽  
Pcr Techniques ◽  
High Diversity

Enteric viruses are the leading cause of diarrhea in children globally. Identifying viral agents and understanding their genetic diversity could help to develop effective preventive measures. This study aimed to determine the detection rate and genetic diversity of four enteric viruses in Gabonese children aged below five years. Stool samples from children <5 years with (n = 177) and without (n = 67) diarrhea were collected from April 2018 to November 2019. Norovirus, astrovirus, sapovirus, and aichivirus A were identified using PCR techniques followed by sequencing and phylogenetic analyses. At least one viral agent was identified in 23.2% and 14.9% of the symptomatic and asymptomatic participants, respectively. Norovirus (14.7%) and astrovirus (7.3%) were the most prevalent in children with diarrhea, whereas in the healthy group norovirus (9%) followed by the first reported aichivirus A in Gabon (6%) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. Phylogenetic analysis of the 3CD region of the aichivirus A genome revealed the presence of two genotypes (A and C) in the study cohort. Astrovirus and sapovirus showed a high diversity, with five different astrovirus genotypes and four sapovirus genotypes, respectively. Our findings give new insights into the circulation and genetic diversity of enteric viruses in Gabonese children.

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