Comparison of five methods for isolation of DNA from Mycoplasma cynos

Sakmanoğlu, Aslı; Sayın, Zafer; Uçan, Uçkun Sait; Pinarkara, Yasemin; Uslu, Ali Uğur; Erganiş, Osman

doi:10.1016/j.mimet.2017.07.003

Cited by 5 publications

(9 citation statements)

References 20 publications

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“…Agar plate scraping and agar plugs yielded low or undetectable DNA concentrations. These findings were inconsistent with a previous study that obtained enough DNA of M. cynos from agar plugs using the same DNA extraction kit 21 . Therefore, our next attempt was to grow mycoplasmas in broth cultures (6 mL) incubated overnight, but DNA quantities were below ONT recommendations, likely because of slow growth and small volume of broth (supplementary file 4).…”

Section: Discussioncontrasting

confidence: 99%

“…The literature evaluating the potential impact of DNA extraction workflows on subsequent whole genome sequencing of mycoplasmas is limited if not absent. For other bacteria such as E. coli , solid-phase extraction kits, as the ones investigated in this study, were preferred over salting-out kits for sequencing purposes 15,21 . Based on performance and user experience, we recommend the solid-phase DNA extraction method 1 for long-read NGS-based workflow of respiratory mycoplasmas.…”

Section: Discussionmentioning

confidence: 99%

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Development of a long-read NGS workflow for improved characterization of fastidious respiratory mycoplasmas

Framst

D`Andrea

Baquero

et al. 2022

Preprint

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Mycoplasmas are respiratory pathogens in humans and animals and due to their fastidious nature, they have been historically underdiagnosed. Lack of standardised diagnostic, typing and antimicrobial susceptibility testing makes clinical management and epidemiological studies challenging. This study aimed to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of respiratory mycoplasmas using a rapid long-read sequencing platform. Critical aspects of whole genome sequencing were explored using fastidious respiratory Mycoplasma (M. felis and M. cynos) isolated from animals including: (i) four solid and liquid-based media based on a specialized formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for sequencing purposes, and (iii) two de novo assembly platforms as key components of a bioinformatic pipeline including Flye and Canu assemblers. DNA quality and quantity compatible with long-read sequencing requirements were obtained with culture volumes of 160ml in modified Hayflick’s broth incubated for 96 hours. The other three culture approaches investigated did not meet the DNA quality criteria required for long-read sequencing. The use of bead-beating bacterial cell lysis in the extraction protocol resulted in smaller fragments and shorter reads compared to enzymatic lysis methods. Overall, Flye generated more contiguous assemblies than the Canu assembler. This novel study provides a step-by-step sequencing workflow including mycoplasma culture, DNA extraction and de novo assembly approaches for the characterization of highly fastidious respiratory mycoplasmas. This workflow will provide diagnosticians, epidemiologists, and researchers with a more comprehensive tool than the laborious conventional methods for a complete genomic characterization of respiratory mycoplasmas.

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development of a long-read NGS workflow for improved characterization of fastidious respiratory mycoplasmas

Framst

D`Andrea

Baquero

et al. 2022

Preprint

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“…The cycling conditions started with an initial denaturation step at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 20 s and elongation at 72 °C for 1 min. For M. cynos, the primers mix contained 20 µL of the forward primer (5′-GTG GGG ATG GAT TAC CTC CT-3′) and the reverse primer (5′-GAT ACA TAA ACA CAA CAT TAT AAT ATTG-3′) at 10 µM and 10 µL of the probe (5′-TCT ACG GAG TAC AAG TTA CAA TTC ATT TTA GT-3′) at 10 µM [33]. The cycling conditions were as follows: an initial denaturation step at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 30 s, primer annealing at 50 °C for 20 s and elongation at 72 °C for 1 min.…”

Section: B Bronchiseptica and M Cynos Qpcrmentioning

confidence: 99%

“…1300 (1040-3622) 33 meningoseptica, Fusobacterium sp., Methylotenera sp. and Escherichia-Shigella sp.…”

Section: Total Cell Count (Cells/µl) Macrophages (%) Neutrophils (%) mentioning

confidence: 99%

Analysis of the lung microbiota in dogs with Bordetella bronchiseptica infection and correlation with culture and quantitative polymerase chain reaction

Fastrès

Taminiau

et al. 2020

Vet Res

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Infection with Bordetella bronchiseptica (Bb), a pathogen involved in canine infectious respiratory disease complex, can be confirmed using culture or qPCR. Studies about the canine lung microbiota (LM) are recent, sparse, and only one paper has been published in canine lung infection. In this study, we aimed to compare the LM between Bb infected and healthy dogs, and to correlate sequencing with culture and qPCR results. Twenty Bb infected dogs diagnosed either by qPCR and/or culture and 4 healthy dogs were included. qPCR for Mycoplasma cynos (Mc) were also available in 18 diseased and all healthy dogs. Sequencing results, obtained from bronchoalveolar lavage fluid after DNA extraction, PCR targeting the V1-V3 region of the 16S rDNA and sequencing, showed the presence of Bb in all diseased dogs, about half being co-infected with Mc. In diseased compared with healthy dogs, the β-diversity changed (P = 0.0024); bacterial richness and α-diversity were lower (P = 0.012 and 0.0061), and bacterial load higher (P = 0.004). Bb qPCR classes and culture results correlated with the abundance of Bb (r = 0.71, P < 0.001 and r = 0.70, P = 0.0022). Mc qPCR classes also correlated with the abundance of Mc (r = 0.73, P < 0.001). Bb infection induced lung dysbiosis, characterized by high bacterial load, low richness and diversity and increased abundance of Bb, compared with healthy dogs. Sequencing results highly correlate with qPCR and culture results showing that sequencing can be reliable to identify microorganisms involved in lung infectious diseases. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article'

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“…Continued refinement of the modified Hayflick's media could support faster and even more abundant growth of M. cynos and M. felis. In addition, we have some concerns about the reliability of assessing M. cynos and M. felis growth based on OD measurements because some strains may form clumps in liquid culture; therefore, we used an additional qualitative growth indicator based on media colour change [25,26]. Further studies are needed to investigate other methods for M. cynos and M. felis growth assessment.…”

Section: Discussionmentioning

confidence: 99%

Development of a long-read next generation sequencing workflow for improved characterization of fastidious respiratory mycoplasmas

et al. 2022

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Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick’s broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4–5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.

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Comparison of five methods for isolation of DNA from Mycoplasma cynos

Cited by 5 publications

References 20 publications

Development of a long-read NGS workflow for improved characterization of fastidious respiratory mycoplasmas

Development of a long-read NGS workflow for improved characterization of fastidious respiratory mycoplasmas

Analysis of the lung microbiota in dogs with Bordetella bronchiseptica infection and correlation with culture and quantitative polymerase chain reaction

Development of a long-read next generation sequencing workflow for improved characterization of fastidious respiratory mycoplasmas

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