DNA vs RNA based studies of nitrogen removal bacteria genes via qPCR

• Autorzy: Banach-Wiśniewska Anna , Gamoń Filip , Ziembińska-Buczyńska Aleksandra

Identyfikatory

DOI

10.24425/aep.2021.136444

Warianty tytułu

PL

DNA i RNA-zależna analiza genów bakterii przemian azotowych metodą qPCR

• Języki publikacji: EN

Abstrakty

EN

Improvements in water quality requires the removal of nitrogen compounds from wastewater. The most promising and cost-effective methods for this purpose are biological ones based on activated sludge microorganisms such as nitrifiers, denitrifiers, and anammox bacteria. Due to the most of the nitrogen removal bacteria are uncultivable in a laboratory, the application of the molecular tools is required to investigate microorganisms involved in the nitrogen removal. In case of this study for the analysis of relative genes abundance of nitrogen removal bacteria, quantitative PCR (qPCR) based on bacterial DNA and qPCR preceded by reverse transcription (RT-qPCR) based on bacterial mRNA as a template, were used with specific bacterial functional genes (amoA, nrxA, nirS, nirK, hzo). Samples from four anammox sequencing batch reactors (SBRs) were analyzed, while the nitrogen removal process and bacteria growth were supported by biomass immobilization and nanoparticles addition. There were statistically significant differences between results obtained in the case of mRNA and DNA (p<0.05). Statistically significant positive correlations were found between results obtained with those two approaches. In case of mRNA analysis, positive results were obtained only for hzo, amoA and partly for nirS genes, despite additional purification and removal of inhibitors from samples prior to reaction.

PL

Z uwagi na to, że większości bakterii przemian związków azotowych nie można wyizolować w postaci czystych kultur, do ich zbadania konieczne jest zastosowanie metod biologii molekularnej. Jedną z najczęściej stosowanych w tym celu jest ilościowa reakcja łańcuchowa polimerazy (ang. Quantitative Polimerase Chain Reaction, qPCR). Celem eksperymentu było porównanie wyników analizy wybranych genów funkcyjnych bakterii przemian związków azotowych przy pomocy metody qPCR wykonanej na matrycy DNA i RNA (po odwrotnej transkrypcji). Względna liczebności genów funkcyjnych analizowana była z zastosowaniem metody qPCR (na matrycy DNA) oraz RT-qPCR (ang. Reverse Transcription-qPCR) (na matrycy RNA). Analizę przeprowadzono w oparciu o geny: amoA, nrxA, nirS, nirK i hzo. Próbki osadu czynnego pobrano z czterech sekwencyjnych reaktorów porcjowych, w których proces usuwania azotu i wzrostu bakterii wspomagano za pomocą immobilizacji biomasy i dodatkiem nanocząstek. Wykazano statystycznie istotne różnice między wynikami uzyskanymi w przypadku badań mRNA i badań opartych na DNA (p<0,05). Wyniki uzyskane za pomocą zastosowanych narzędzi biologii molekularnej (qPCR, RT-qPCR) były skorelowane pozytywnie. W przypadku analizy opartej na mRNA pozytywne wyniki uzyskano tylko dla genów hzo, amoA i częściowo dla genów nirS, pomimo dodatkowego oczyszczania i usuwania inhibitorów z próbek przed reakcją. Należy podkreślić, że w zależności od matrycy zastosowanej w qPCR (bakteryjne DNA lub cDNA zsyntetyzowane z bakteryjnego mRNA w procesie odwrotnej transkrypcji) uzyskane wyniki mogą wskazywać na różne informacje naukowe. Pomimo znaczących różnic pomiędzy wynikami otrzymanymi za pomocą dwóch metod, obliczone współczynniki korelacji Spearmana wskazują na wzajemne powiązanie pomiędzy otrzymanymi wynikami oraz powiązania ekologiczne pomiędzy bakteryjnymi genami przemian związków azotowych.

Słowa kluczowe

EN

qPCR; RT-qPCR; nitrogen removal bacteria; relative gene abundance

PL

qPCR; RT-qPCR; bakterie usuwające azot; względna obfitość genów

• Wydawca: Institute of Environmental Engineering, Polish Academy of Sciences
• Czasopismo: Archives of Environmental Protection
• Rocznik: 2021
• Tom: Vol. 47, no. 1
• Strony: 19--25
• Opis fizyczny: Bibliogr. 39 poz., tab., wykr.

Twórcy

autor

Banach-Wiśniewska Anna

• Silesian University of Technology, Faculty of Power and Environmental Engineering, Environmental Biotechnology Department, Gliwice, Poland

autor

Gamoń Filip

• Silesian University of Technology, Faculty of Power and Environmental Engineering, Environmental Biotechnology Department, Gliwice, Poland

autor

Ziembińska-Buczyńska Aleksandra

• Silesian University of Technology, Faculty of Power and Environmental Engineering, Environmental Biotechnology Department, Gliwice, Poland

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Uwagi

Opracowanie rekordu ze środków MNiSW, umowa Nr 461252 w ramach programu "Społeczna odpowiedzialność nauki" - moduł: Popularyzacja nauki i promocja sportu (2021).