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      Comparison of microbial molecular diagnosis efficiency within unstable template metagenomic DNA samples between qRT-PCR and chip-based digital PCR platforms

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          Abstract

          Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain ( Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor ( greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject’s skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

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          Most cited references16

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          Commensal Staphylococci Influence Staphylococcus aureus Skin Colonization and Disease

          Commensal organisms that constitute the skin microbiota play a pivotal role in the orchestration of cutaneous homeostasis and immune competence. This balance can be promptly offset by the expansion of the opportunistic pathogen Staphylococcus aureus , which is responsible for the majority of bacterial skin infections. S. aureus carriage is also known to be a precondition for its transmission and pathogenesis. Recent reports suggest that skindwelling coagulase negative Staphylococci (CoNS) can prime the skin immune system to limit the colonization potential of invaders, and they can directly compete through production of antimicrobial molecules or through signaling antagonism. We review recent advances in these CoNS colonization resistance mechanisms, which may serve to aid development of pharmacologic and probiotic intervention strategies to limit S. aureus skin colonization and disease.
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            Optimisation of methods for bacterial skin microbiome investigation: primer selection and comparison of the 454 versus MiSeq platform

            Background The composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols.. Methods DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME. Results There was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms). Conclusions The results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-0927-4) contains supplementary material, which is available to authorized users.
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              Host-pathogen interactions between the skin and Staphylococcus aureus.

              Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection. Copyright © 2011 Elsevier Ltd. All rights reserved.
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                Author and article information

                Journal
                Genomics Inform
                Genomics Inform
                GNI
                Genomics & Informatics
                Korea Genome Organization
                1598-866X
                2234-0742
                December 2023
                29 December 2023
                : 21
                : 4
                : e52
                Affiliations
                [1 ]Department of Microbiology, College of Science & Technology, Dankook University, Cheonan 31116, Korea
                [2 ]Department of Bioconvergence Engineering, Dankook University, Jukjeon 16890, Korea
                [3 ]R&I Center, COSMAX BTI, Seongnam 13486, Korea
                [4 ]Center for Digital Health, Medical Science Research Institute, Kyung Hee University Medical Center, Kyung Hee University College of Medicine, Seoul 02447, Korea
                [5 ]Department of Pediatrics, Kyung Hee University College of Medicine, Seoul 02447, Korea
                [6 ]HuNbiome Co., Ltd, R&D Center, Seoul 08503, Korea
                Author notes
                [* ]Corresponding author: E-mail: kyudong.han@ 123456gmail.com

                Dongwan Kim, Junhyeon Jeon, and Minseo Kim contributed equally to this work.

                Author information
                http://orcid.org/0009-0003-1504-8795
                http://orcid.org/0000-0002-6549-3243
                http://orcid.org/0009-0001-9279-1289
                http://orcid.org/0000-0002-7283-0949
                http://orcid.org/0000-0002-4060-5018
                http://orcid.org/0009-0006-1684-7128
                http://orcid.org/0000-0003-1628-9948
                http://orcid.org/0000-0001-6791-2408
                Article
                gi-23068
                10.5808/gi.23068
                10788361
                38224719
                48ce34b8-dee1-43ea-aebe-b990a575671b
                (c) 2023, Korea Genome Organization

                (CC) This is an open-access article distributed under the terms of the Creative Commons Attribution license( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 August 2023
                : 3 October 2023
                : 11 October 2023
                Categories
                Application Note

                Genetics
                digital pcr,molecular diagnosis,quantitative real-time pcr,staphylococcus aureus
                Genetics
                digital pcr, molecular diagnosis, quantitative real-time pcr, staphylococcus aureus

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