scholarly journals A 7-member SNP Assay on the iPlex MassARRAY Platform Provides a Rapid and Affordable Alternative to Typing Major AfricanStaphylococcus aureusTypes

2018 ◽  
Author(s):  
Justin Nyasinga ◽  
Cecilia Kyany’a ◽  
Raphael Okoth ◽  
Valerie Oundo ◽  
Daniel Matano ◽  
...  
Keyword(s):  
Regional Distribution ◽  
Accurate Method ◽  
Turnaround Time ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Detection Rates ◽  
Dominant Type ◽  
Clonal Distribution ◽  
Typing Methods ◽  
Mrsa Strains

AbstractBackgroundData on the clonal distribution ofStaphylococcus aureusin Africa is scanty, partly due to high costs and long turnaround times imposed by conventional genotyping methods such asspaand multilocus sequence typing (MLST) warranting the need for alternative typing approaches. This study applied and evaluated the accuracy, cost and time of using iPlex massARRAY genotyping method on Kenyan staphylococcal isolates.MethodsFifty four clinicalS. aureusisolates from three counties were characterized using iPlex massARRAY,spaand MLST typing methods. Ten Single Nucleotide Polymorphisms (SNPs) from theS. aureusMLST database were assessed by iPlex massARRAY.ResultsThe iPlex massARRAY assay grouped the isolates into 14 SNP genotypes with 9/10 SNPs interrogated showing high detection rates (average 89%). spaand MLST typing revealed 22spatypes and 21 STs that displayed unique regional distribution.spatype t355 (ST152) was the dominant type and t2029 and t037 (ST 241) were observed among MRSA strains. MassARRAY showed 83% and 82% accuracy againstspaand MLST typing respectively in isolate classification. Moreover, massARRAY identified all MRSA strains and a novelspatype. MassARRAY had reduced turnaround time (<12 hrs) compared tospa(3 days) and MLST (20 days) typing. The iPlex massARRAY cost approximately 18 USD compared tospa(30 USD) and MLST (126 USD) typing based on consumable costs/isolate.ConclusionUpon validation with a larger collection of isolates, iPlex massARRAY could provide a faster, more affordable and fairly accurate method of resolving AfricanS.aureusisolates especially in large surveillance studies.

scholarly journals Reverse complement-PCR, an innovative and effective method for multiplexing forensically relevant single nucleotide polymorphism marker systems

BioTechniques ◽  
2021 ◽  
Author(s):  
Magdalena M Bus ◽  
Erik AC de Jong ◽  
Jonathan L King ◽  
Walter van der Vliet ◽  
Joop Theelen ◽  
...  
Keyword(s):  
Dna Typing ◽  
Substantial Improvement ◽  
Single Step ◽  
Degraded Dna ◽  
Nucleotide Polymorphisms ◽  
Discrimination Power ◽  
Single Nucleotide ◽  
Reverse Complement ◽  
Pcr Multiplex ◽  
Typing Methods

DNA analyses from challenging samples such as touch evidence, hairs and skeletal remains push the limits of the current forensic DNA typing technologies. Reverse complement PCR (RC-PCR) is a novel, single-step PCR target enrichment method adapted to amplify degraded DNA. The sample preparation process involves a limited number of steps, decreasing the labor required for library preparation and reducing the possibility of contamination due to less sample manipulation. These features of the RC-PCR make the technology a unique application to successfully target single nucleotide polymorphisms (SNPs) in fragmented and low copy number DNA and yield results from samples in which no or limited data are obtained with standard DNA typing methods. The developed RC-PCR short amplicon 85 SNP-plex panel is a substantial improvement over the previously reported 27-plex RC-PCR multiplex that will provide higher discrimination power for challenging DNA sample analyses.

scholarly journals Genetic Study of IL6, GDF5 and PAPPA2 in Association with Developmental Dysplasia of the Hip

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 986
Author(s):  
Stefan Harsanyi ◽  
Radoslav Zamborsky ◽  
Lubica Krajciova ◽  
Milan Kokavec ◽  
Lubos Danisovic
Keyword(s):  
Autosomal Dominant ◽  
Developmental Dysplasia ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Dominant Type ◽  
Skeletal Disorders ◽  
Autosomal Dominant Type ◽  
Dysplasia Of The Hip ◽  
Il6 Gene ◽  
Gdf5 Gene

Background: Developmental dysplasia of the hip (DDH) is one of the most prevalent skeletal disorders. DDH is considered a pathologic condition with polygenic background, but environmental and mechanic factors significantly contribute to its multifactorial etiology. Inheritance consistent with autosomal dominant type has also been observed. Single-nucleotide polymorphisms (SNPs) in various genes mostly related to formation of connective tissue are studied for a possible association with DDH. Methods: We genotyped three SNPs, rs1800796 located in the promoter region of the IL6 gene, rs143383 located in the 5′ untranslated region (UTR) of the GDF5 gene and rs726252 located in the fifth intron of the PAPPA2 gene. The study consisted of 45 subjects with DDH and 85 controls from all regions of Slovakia. Results: Association between DDH occurrence and studied genotypes affected by aforementioned polymorphisms was confirmed in the case of rs143383 in the GDF5 gene (p = 0.047), where the T allele was over-expressed in the study group. Meanwhile, in the matter of IL6 and PAPPA2, we found no association with DDH (p = 0.363 and p = 0.478, respectively). Conclusions: These results suggest that there is an association between DDH and GDF5 polymorphisms and that the T allele is more frequently presents in patients suffering from DDH.

scholarly journals Rapid high-resolution melting genotyping scheme for Escherichia coli based on MLST derived single nucleotide polymorphisms

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matej Bezdicek ◽  
Marketa Nykrynova ◽  
Karel Sedlar ◽  
Stanislava Kralova ◽  
Jana Hanslikova ◽  
...  
Keyword(s):  
In Silico Analysis ◽  
Discriminatory Power ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
E Coli ◽  
Mlst Scheme ◽  
Large Sets ◽  
Processing Cost ◽  
Typing Methods ◽  
Efficient Processing

AbstractRoutinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726–0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726–0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones’.

scholarly journals Single-nucleotide polymorphism in the SCCmec-orfX junction distinguishes between livestock-associated MRSA CC398 and human epidemic MRSA strains

2009 ◽  
Vol 14 (49) ◽  
Author(s):  
U Reischl ◽  
J Frick ◽  
S Hoermansdorfer ◽  
H Melzl ◽  
M Bollwein ◽  
...  
Keyword(s):  
Direct Detection ◽  
Integration Site ◽  
Melting Curve ◽  
Animal Origin ◽  
Strain Identification ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Mrsa Strains ◽  
The Right

A number of real-time PCR assays for direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic Tm value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.

scholarly journals Sequence-Based Epidemiology of an OXA-48 Plasmid during a Hospital Outbreak

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Laura Hidalgo ◽  
Mark de Been ◽  
Malbert R. C. Rogers ◽  
Anita C. Schürch ◽  
Jelle Scharringa ◽  
...  
Keyword(s):  
The Netherlands ◽  
Illumina Miseq ◽  
Conjugative Plasmid ◽  
Nucleotide Polymorphisms ◽  
Dutch Hospital ◽  
Single Nucleotide ◽  
Related Sequence ◽  
Hospital Outbreak ◽  
Typing Methods ◽  
Miseq Platform

ABSTRACT A large OXA-48 outbreak in The Netherlands involved the spread of OXA-48-producing Enterobacteriaceae among at least 118 patients, suggesting horizontal transfer of this resistance gene through one or more plasmids. Elucidating transmission dynamics of resistance plasmids is hampered by the low resolution of classic typing methods. This study aimed to investigate the molecular epidemiology of plasmids carrying the OXA-48 carbapenemase using a next-generation sequencing approach. A total of 68 OXA-48-producing Enterobacteriaceae isolates collected from the hospital outbreak, as well as 22 non-outbreak-related OXA-48-producing Enterobacteriaceae isolates from The Netherlands, Libya, and Turkey were selected. Plasmids were sequenced using the Illumina MiSeq platform, and read sets were assembled and analyzed. In all plasmids, blaOXA-48 was embedded in transposon Tn1999.2 and located on a ca. 62-kb IncL/M conjugative plasmid in 14 different species. There were a maximum of 2 single nucleotide polymorphisms (SNPs) between the core sequence alignment of all plasmids. Closely related sequence variants of this plasmid were detected in nonoutbreak isolates from The Netherlands and other countries. Thirty-one of 89 OXA-48-producing isolates also harbored blaCTX-M-15, which was not located on the blaOXA-48-carrying plasmid. Sequencing of four plasmids harboring blaCTX-M-15 revealed extensive plasmid heterogeneity. A ca. 62-kb plasmid was responsible for the OXA-48 outbreak in a Dutch hospital. Our findings provide strong evidence for both within-host interspecies and between-host dissemination of plasmid-based OXA-48 during a nosocomial outbreak. These findings exemplify the complex epidemiology of carbapenemase-producing Enterobacteriaceae (CPE).

Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing

2006 ◽  
Vol 55 (8) ◽  
pp. 1061-1070 ◽  
Author(s):  
Erin P. Price ◽  
Venugopal Thiruvenkataswamy ◽  
Lance Mickan ◽  
Leanne Unicomb ◽  
Rosa E. Rios ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Variable Region ◽  
Turnaround Time ◽  
Resolving Power ◽  
Campylobacter Coli ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Bacterial Populations ◽  
Snp Typing ◽  
Short Variable Region

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogen Campylobacter jejuni. SNPs were identified in silico using the program ‘Minimum SNPs’, which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-D SNPs identified in this study were derived from the combined C. jejuni/Campylobacter coli multilocus sequence typing (MLST) database. Seven SNPs were found that provided a D of 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-D SNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69 C. jejuni isolates were subjected to MLST and flagellin A short variable region (flaA SVR) sequencing and combined with a population of 84 C. jejuni and C. coli isolates previously characterized by these methods. Within this collection of 153 isolates, 19 flaA SVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST and flaA SVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided a D of 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of the flaA SVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaA SVR. This investigation has shown that a seven-member C. jejuni SNP typing assay, used in combination with sequencing of the flaA SVR, efficiently discriminates C. jejuni isolates.

scholarly journals Polymorphic Amplified Typing Sequences and Pulsed-Field Gel Electrophoresis Yield Comparable Results in the Strain Typing of a Diverse Set of BovineEscherichia coliO157:H7 Isolates

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Indira T. Kudva ◽  
Margaret A. Davis ◽  
Robert W. Griffin ◽  
Jeonifer Garren ◽  
Megan Murray ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Gel Electrophoresis ◽  
Turnaround Time ◽  
Pulsed Field Gel Electrophoresis ◽  
Strain Typing ◽  
Nucleotide Polymorphisms ◽  
O157 Strain ◽  
Single Nucleotide ◽  
Pulsed Field ◽  
Quick Turnaround Time

Polymorphic amplified typing sequences (PATS), a PCR-basedEscherichia coliO157:H7 (O157) strain typing system, targets insertions-deletions and single nucleotide polymorphisms atXbaI andAvrII restriction enzyme sites, respectively, and the virulence genes (stx1,stx2,eae,hlyA) in the O157 genome. In this study, the ability of PATS to discriminate O157 isolates associated with cattle was evaluated. An in-depth comparison of 25 bovine O157 isolates, from different geographic locations across Northwest United States, showed that about 85% of these isolates shared the same dendogram clade by PATS and pulsed-field gel electrophoresis (PFGE), irrespective of the restriction enzyme sites targeted. The Pearson’s correlation coefficient,r, calculated at about 0.4, 0.3, and 0.4 forXbaI-based,AvrII-based and combined-enzymes PATS and PFGE similarities, respectively, indicating that these profiles shared a good but not high correlation, an expected inference given that the two techniques discriminate differently. Isolates that grouped differently were better matched to their locations using PATS. Overall, PATS discriminated the bovine O157 isolates without interpretive biases or sophisticated analytical software, and effectively complemented while not duplicating PFGE. With its quick turnaround time, PATS has excellent potential as a convenient tool for early epidemiological or food safety investigations, enabling rapid notification/implementation of quarantine measures.

Nuclear receptors in disease: the oestrogen receptors

2004 ◽  
Vol 40 ◽  
pp. 157-167 ◽  
Author(s):  
Maria Nilsson ◽  
Karin Dahlman-Wright ◽  
Jan-Åke Gustafsson
Keyword(s):  
Nuclear Receptors ◽  
Target Genes ◽  
Receptor Subtype ◽  
Target Tissue ◽  
Oestrogen Receptors ◽  
Nucleotide Polymorphisms ◽  
Cell Type ◽  
Single Nucleotide ◽  
Beneficial Effects ◽  
The Family

For several decades, it has been known that oestrogens are essential for human health. The discovery that there are two oestrogen receptors (ERs), ERalpha and ERbeta, has facilitated our understanding of how the hormone exerts its physiological effects. The ERs belong to the family of ligand-activated nuclear receptors, which act by modulating the expression of target genes. Studies of ER-knockout (ERKO) mice have been instrumental in defining the relevance of a given receptor subtype in a certain tissue. Phenotypes displayed by ERKO mice suggest diseases in which dysfunctional ERs might be involved in aetiology and pathology. Association between single-nucleotide polymorphisms (SNPs) in ER genes and disease have been demonstrated in several cases. Selective ER modulators (SERMs), which are selective with regard to their effects in a certain cell type, already exist. Since oestrogen has effects in many tissues, the goal with a SERM is to provide beneficial effects in one target tissue while avoiding side effects in others. Refined SERMs will, in the future, provide improved therapeutic strategies for existing and novel indications.

Development of a simple method for the determination of single nucleotide polymorphisms

2010 ◽  
Vol 34 (8) ◽  
pp. S75-S75
Author(s):  
Weifeng Zhu ◽  
Zhuoqi Liu ◽  
Daya Luo ◽  
Xinyao Wu ◽  
Fusheng Wan
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Simple Method ◽  
Single Nucleotide
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