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.

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 An Extended Multilocus Sequence Typing (MLST) Scheme for Rapid Direct Typing of Leptospira from Clinical Samples

2016 ◽  
Vol 10 (9) ◽  
pp. e0004996 ◽  
Author(s):  
Sabrina Weiss ◽  
Angela Menezes ◽  
Kate Woods ◽  
Anisone Chanthongthip ◽  
Sabine Dittrich ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Clinical Samples ◽  
Mlst Scheme

scholarly journals DjinniChip: Evaluation of a novel molecular rapid diagnostic device for the detection of Chlamydia trachomatis in trachoma-endemic areas

2020 ◽  
Author(s):  
Tamsyn R Derrick ◽  
Natalia Sandetskaya ◽  
Harry Pickering ◽  
Andreas Kölsch ◽  
Athumani Ramadhani ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Low Cost ◽  
Bacterial Species ◽  
Clinical Signs ◽  
Field Conditions ◽  
Clinical Samples ◽  
Chlamydia Trachomatis Infection ◽  
Low Concentrations ◽  
Control Programmes ◽  
The Right

Abstract Background The clinical signs of active trachoma are often present in the absence of ocular Chlamydia trachomatis infection, particularly following mass drug administration. Treatment decisions following impact surveys and in post-control surveillance for communities are currently based on the prevalence of clinical signs, which may result in further unnecessary distribution of mass antibiotic treatment and the increased spread of macrolide resistance alleles in ‘off-target’ bacterial species. We therefore developed a simple, fast, low cost diagnostic assay (DjinniChip) for diagnosis of ocular C. trachomatis for use by trachoma control programmes. Methods The study was conducted in London, Germany and Tanzania. For clinical testing in Tanzania, specimens from a sample of 350 children between the ages of 7 to15 years, which were part of a longitudinal cohort that began in February 2012 were selected. Two ocular swabs were taken from the right eye. The second swab was collected dry, kept cool in the field and archived at -80 o C before sample lysis for DjinniChip detection and parallel nucleic acid purification and detection/quantification by qPCR assay. Results Djinnichip was able to reliably detect >10 copies of C. trachomatis per test and correctly identified 7/10 Quality Control for Molecular Diagnostics C. trachomatis panel samples, failing to detect 3 positive samples with genome equivalent amounts >10 copies. Djinnichip performed well across a range of typical trachoma field conditions and when used by lay personnel using a series of mock samples. In the laboratory in Tanzania, using clinical samples the sensitivity and specificity of DjinniChip for C. trachomatis was 66% (95% CI 51–78) and 94.8 (95% CI 91–97%) with an overall accuracy of 90.1 (95% CI 86.4 – 93). Conclusions DjinniChip performance is extremely promising, particularly its ability to detect low concentrations of C. trachomatis and its usability in field conditions. The DjinniChip requires further development to reduce inhibition and advance toward a closed system. DjinniChip results did not vary between local laboratory results and typical trachoma field settings, illustrating its potential for use in low-resource areas to prevent unnecessary rounds of MDA and to monitor for C. trachomatis recrudescence.

scholarly journals SARS-CoV-2 R.1 lineage variants prevailed in Tokyo in March 2021

2021 ◽  
Author(s):  
Katsutoshi Nagano ◽  
Chihiro Tani-Sassa ◽  
Yumi Iwasaki ◽  
Yuna Takatsuki ◽  
Sonoka Yuasa ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Disease Severity ◽  
Genome Sequencing ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Nasopharyngeal Swab ◽  
Whole Genome ◽  
Crucial Issue ◽  
Chain Reaction ◽  
Polymerase Chain

Background: The spread of SARS-CoV-2 variants, such as B.1.1.7 and B.1.351, has become a crucial issue worldwide. Therefore, we began testing all patients with COVID-19 for the N501Y and E484K mutations associated with SARS-CoV-2. Study design: Nasopharyngeal swab samples from 108 patients who visited our hospital between February and April 2021 were analyzed. The samples were analyzed using reverse transcription-polymerase chain reaction with melting curve analysis to detect the N501Y and E484K mutations. A part of the samples were also subjected to whole genome sequencing. Clinical parameters such as mortality and admission to the intensive care unit were analyzed to examine the association between increased disease severity and the E484K mutation. Results: The ratio of cases showing the 501N+484K mutation rapidly increased from 8% in February to 46% in March. Whole genome sequencing revealed that the viruses with 501N+484K mutation are R.1 lineage variants. Evidence of increased disease severity related to the R.1 variants were not found. Conclusions: We found that the R.1 lineage variants rapidly prevailed in Tokyo in March 2021.

scholarly journals Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs

2020 ◽  
Author(s):  
Katherine E. Bowden ◽  
Sandeep J. Joseph ◽  
John Cartee ◽  
Noa Ziklo ◽  
Damien Danavall ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Intracellular Bacterium ◽  
Clinical Samples ◽  
Sexual Networks ◽  
Whole Genome ◽  
Target Enrichment ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Improve Efficiency ◽  
Sex With Men

AbstractChlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. U.S. cases have been steadily increasing for more than a decade in both the urogenital tract and rectum. C. trachomatis is an obligate intracellular bacterium that is not easily cultured, limiting the capacity for genome studies to understand strain diversity and emergence among various patient populations globally. While Agilent SureSelectXT target-enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival and rectal samples, efficiencies are only 60-80% for ≥95-100% genome coverage. We therefore re-designed and expanded the RNA bait library to augment enrichment of the organism from clinical samples to improve efficiency. We describe the expanded library, the limit of detection for C. trachomatis genome copy input, and the 100% efficiency and high-resolution of generated genomes where genomic recombination among paired vaginal and rectal specimens from four patients was identified. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, among geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.ImportanceChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, and there is limited information on rectal C. trachomatis transmission and its impact on morbidity. To improve efficiency of previous studies involving whole genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we expanded the RNA bait library to augment enrichment of the organism from clinical samples. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis. We show the new system is sensitive for near identical genomes of C. trachomatis from two body sites in four women. Further, we provide a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital and rectal infections, and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

scholarly journals A Multiplex Snapback Primer System for the Enrichment and Detection ofJAK2V617F andMPLW515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Zhiyuan Wu ◽  
Yunqing Zhang ◽  
Xinju Zhang ◽  
Xiao Xu ◽  
Zhihua Kang ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Amplification Efficiency ◽  
Primer Sets ◽  
Low Ionic Strength ◽  
Multiplex System

A multiplex snapback primer system was developed for the simultaneous detection ofJAK2V617F andMPLW515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets forJAK2andMPLmutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.

scholarly journals Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

2008 ◽  
Vol 54 (6) ◽  
pp. 964-972 ◽  
Author(s):  
Clara R L Teo ◽  
Wen Wang ◽  
Hai Yang Law ◽  
Caroline G Lee ◽  
Samuel S Chong
Keyword(s):  
Huntington Disease ◽  
Trinucleotide Repeat ◽  
Neurodegenerative Disorder ◽  
False Positive Rate ◽  
Screening Method ◽  
Melting Curve ◽  
Single Step ◽  
Cag Repeat ◽  
Clinical Samples ◽  
Melting Curve Analysis

Abstract Background: Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG trinucleotide repeat in exon 1 of the HTT (huntingtin) gene and typically has an adult onset. Molecular diagnosis and screening for HD currently involve separate amplification and detection steps. Methods: We evaluated a novel, rapid microplate-based screening method for HD that combines the amplification and detection procedures in a single-step, closed-tube format. We carried out both the PCR for the HTT CAG-repeat region and the subsequent automated melting-curve analysis of the amplicon in the same wells on the plate. To establish cutoff melting temperatures (Tms) for each allelic class, we used a panel of reference DNA samples of known CAG-repeat sizes that represent a range of HTT alleles [normal (≤26 repeats), intermediate (27–35 repeats), reduced penetrance expanded (36–39 repeats), and fully penetrant expanded (≥40 repeats)]. We also measured well-to-well variation in Tm across the thermal block and validated cutoff Tms with DNA samples from 5 different populations. We also conducted a blinded validation analysis of clinical samples from an additional 40 HD-affected and 30 unaffected individuals. Results: We observed a strong correlation between CAG-repeat size and amplicon Tm among the reference DNA samples. Use of the Tm cutoffs we established revealed that 5 samples from unaffected individuals had been misclassified as affected (1.1% false-positive rate). All samples from HD-affected and unaffected individuals were correctly identified in the blinded analysis. Conclusions: This simple and scalable homogeneous assay may serve as a convenient, rapid, and accurate screen to detect the presence of pathologic expanded HD alleles in symptomatic patients.

scholarly journals High-Resolution Melting Curve Analysis of Genomic and Whole-Genome Amplified DNA

2008 ◽  
Vol 54 (12) ◽  
pp. 2055-2058 ◽  
Author(s):  
Michael H Cho ◽  
Dawn Ciulla ◽  
Barbara J Klanderman ◽  
Benjamin A Raby ◽  
Edwin K Silverman
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Dna Melting ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
High Resolution Melting Curve

Abstract Background: High-resolution melting curve analysis is an accurate method for mutation detection in genomic DNA. Few studies have compared the performance of high-resolution DNA melting curve analysis (HRM) in genomic and whole-genome amplified (WGA) DNA. Methods: In 39 paired genomic and WGA samples, 23 amplicons from 9 genes were PCR amplified and analyzed by high-resolution melting curve analysis using the 96-well LightScanner (Idaho Technology). We used genotyping and bidirectional resequencing to verify melting curve results. Results: Melting patterns were concordant between the genomic and WGA samples in 823 of 863 (95%) analyzed sample pairs. Of the discordant patterns, there was an overrepresentation of alternate melting curve patterns in the WGA samples, suggesting the presence of a mutation (false positives). Targeted resequencing in 135 genomic and 136 WGA samples revealed 43 single nucleotide polymorphisms (SNPs). All SNPs detected in genomic samples were also detected in WGA. Additional genotyping and sequencing allowed the classification of 628 genomic and 614 WGA amplicon samples. Heterozygous variants were identified by non–wild-type melting pattern in 98% of genomic and 97% of WGA samples (P = 0.11). Wild types were correctly classified in 99% of genomic and 91% of WGA samples (P &lt; 0.001). Conclusions: In WGA DNA, high-resolution DNA melting curve analysis is a sensitive tool for SNP discovery through detection of heterozygote variants; however, it may misclassify a greater number of wild-type samples.

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