Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers

Okolie, Charles E.; Wooldridge, Karl G.; Turner, David P. J.; Cockayne, Alan; James, Richard

doi:10.1016/j.mcp.2015.03.002

Cited by 26 publications

(19 citation statements)

References 26 publications

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“…Duplex ddPCR assays were developed for (1) Adenoviridae (27) and S. pneumoniae (28), (2) H. influenzae (29) and M. catarrhalis (30, 31), and (3) S. aureus and coagulase-negative Staphylococcus (32, 33) based on published assays. Each assay contained 10 μL 2× ddPCR Supermix for probes (Bio-rad, Hemel Hempstead, UK), primers and probes at custom concentrations (Table 1) and 8 μL of template DNA.…”

Section: Methodsmentioning

confidence: 99%

Active trachoma cases in the Solomon Islands have varied polymicrobial community structures but do not associate with individual non-chlamydial pathogens of the eye

Butcher

Sokana²,

Jack³

et al. 2017

Preprint

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Active trachoma cases in the SolomonAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/134213 doi: bioRxiv preprint first posted online May. 11, 2017; Running header: Microbial correlates of active trachoma in the Solomon Islands 2 Abstract 31 32

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Section: Methodsmentioning

confidence: 99%

Active trachoma cases in the Solomon Islands have varied polymicrobial community structures but do not associate with individual non-chlamydial pathogens of the eye

Butcher

Sokana²,

Jack³

et al. 2017

Preprint

View full text Add to dashboard Cite

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“…Duplex ddPCR assays were developed for ( 1 ) Adenoviridae ( 26 ) and S. pneumoniae ( 2 , 27 ), H. influenzae ( 28 ) and M. catarrhalis ( 29 , 30 ), and S. aureus and coagulase-negative Staphylococcus ( 31 , 32 ) based on the published assays. Each assay contained 10 µL 2× ddPCR Supermix for probes (Bio-rad, Hemel Hempstead, UK), primers and probes at custom concentrations (Table 1 ), and 8 µL of template DNA.…”

Section: Methodsmentioning

confidence: 99%

Active Trachoma Cases in the Solomon Islands Have Varied Polymicrobial Community Structures but Do Not Associate with Individual Non-Chlamydial Pathogens of the Eye

Butcher

Sokana²,

Jack³

et al. 2018

Front. Med.

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BackgroundSeveral non-chlamydial microbial pathogens are associated with clinical signs of active trachoma in trachoma-endemic communities with a low prevalence of ocular Chlamydia trachomatis (Ct) infection. In the Solomon Islands, the prevalence of Ct among children is low despite the prevalence of active trachoma being moderate. Therefore, we set out to investigate whether active trachoma was associated with a common non-chlamydial infection or with a dominant polymicrobial community dysbiosis in the Solomon Islands.MethodsWe studied DNA from conjunctival swabs collected from 257 Solomon Islanders with active trachoma and matched controls. Droplet digital PCR was used to test for pathogens suspected to be able to induce follicular conjunctivitis. Polymicrobial community diversity and composition were studied by sequencing of hypervariable regions of the 16S ribosomal ribonucleic acid gene in a subset of 54 cases and 53 controls.ResultsAlthough Ct was associated with active trachoma, the number of infections was low (cases, 3.9%; controls, 0.4%). Estimated prevalence (cases and controls, respectively) of each non-chlamydial infection was as follows: Staphylococcus aureus: 1.9 and 1.9%, Adenoviridae: 1.2 and 1.2%, coagulase-negative Staphylococcus: 5.8 and 4.3%, Haemophilus influenzae: 7.4 and 11.7%, Moraxella catarrhalis: 2.3 and 4.7%, and Streptococcus pneumoniae: 7.0 and 6.2%. There was no statistically significant association between the clinical signs of trachoma and the presence or load of any of the non-Ct infections that were assayed. Interindividual variations in the conjunctival microbiome were characterized by differences in the levels of Corynebacterium, Propionibacterium, Helicobacter, and Paracoccus, but diversity and relative abundance of these specific genera did not differ significantly between cases and controls.DiscussionIt is unlikely that the prevalent trachoma-like follicular conjunctivitis in this region of the Solomon Islands has a dominant bacterial etiology. Before implementing community-wide azithromycin distribution for trachoma, policy makers should consider that clinical signs of trachoma can be observed in the absence of any detectable azithromycin-susceptible organism.

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“…Various Staphylococcus species are responsible for causing many diseases in humans (1). These bacteria are divided to two coagulase-positive and coagulase-negative and one of the most important groups of nococomial infections that are resistant to a wide range of beta-lactam antibiotics (2).…”

Section: Introductionmentioning

confidence: 99%

Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

Heydari

Alikhani

Tahmasebi

et al. 2019

Arch Clin Infect Dis

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Background: Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. Hence, detection of Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is essential in various infections. Objectives: The aim of this study was to design a melting curve analysis (MCA) assay based on Multiplex Real-Time PCR for rapid detection of staphylococci and antibiotic resistance. Methods: The current study used standard strains of positive and negative coagulase staphylococci. As the first step, serial dilutions were prepared with ratios of 10 8 to 10 1 cfu/mL based on standard bacterial concentration with 0.5 McFarland and the results were illustrated in dedicated melting curves. Results: All melting curves of gene amplification had an equal melting point. In all dilutions, the observed melting temperatures shown in the melting curves of gene amplification were equal to 83.79°C for ITS-gene, 74.2°C for phop gene, 76.49°C for sap-gene, 78.2°C for mvaA gene, 79.57°C for 16srRNA-gene, 74.83°C for mupA gene, and 76.6°C for mecA gene. Conclusions: The MCA based on real time-PCR for identifying staphylococcal species and antibiotic resistance is a highly effective method with high sensitivity and specificity.

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Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers

Cited by 26 publications

References 26 publications

Active trachoma cases in the Solomon Islands have varied polymicrobial community structures but do not associate with individual non-chlamydial pathogens of the eye

Active trachoma cases in the Solomon Islands have varied polymicrobial community structures but do not associate with individual non-chlamydial pathogens of the eye

Active Trachoma Cases in the Solomon Islands Have Varied Polymicrobial Community Structures but Do Not Associate with Individual Non-Chlamydial Pathogens of the Eye

Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

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