Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation
| العنوان: | Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation |
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| المؤلفون: | Peer H. A. Timmers, Caroline M. Plugge, H. C. Aura Widjaja-Greefkes, Alfons J. M. Stams |
| المساهمون: | Universidade do Minho |
| المصدر: | Applied Microbiology and Biotechnology, 101(14), 5847-5859 Repositório Científico de Acesso Aberto de Portugal Repositório Científico de Acesso Aberto de Portugal (RCAAP) instacron:RCAAP Applied Microbiology and Biotechnology 101 (2017) 14 Applied Microbiology and Biotechnology |
| بيانات النشر: | Springer Nature, 2017. |
| سنة النشر: | 2017 |
| مصطلحات موضوعية: | 0301 basic medicine, Geologic Sediments, Oceans and Seas, In silico, 030106 microbiology, Population, Real-Time Polymerase Chain Reaction, DNA, Ribosomal, Sensitivity and Specificity, Applied Microbiology and Biotechnology, Microbiology, 03 medical and health sciences, AOM, Methanotrophs, Microbiologie, Computer Simulation, Anaerobiosis, 14. Life underwater, education, Gene, Phylogeny, DNA Primers, education.field_of_study, WIMEK, Science & Technology, biology, Methods and Protocols, General Medicine, 16S ribosomal RNA, biology.organism_classification, Archaea, Molecular biology, Primers, qPCR, 030104 developmental biology, Real-time polymerase chain reaction, Biochemistry, Anaerobic oxidation of methane, Primer (molecular biology), Methane, Oxidation-Reduction, ANME, Biotechnology |
| الوصف: | Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009). Research of PHATand AJMS is supported by the SIAM Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO). info:eu-repo/semantics/publishedVersion |
| وصف الملف: | application/pdf; application/octet-stream |
| اللغة: | English |
| تدمد: | 0175-7598 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::cae491935d96f4e050996240ee277483 https://hdl.handle.net/1822/47423 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....cae491935d96f4e050996240ee277483 |
| قاعدة البيانات: | OpenAIRE |
Find this article in full text from Springer Verlag. | تدمد: | 01757598 |
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