Microbial Functional Gene qPCR Quantification

Overview

In all kinds of environments in nature, there are microorganisms, and they all play a greater or lesser role in their respective "posts". Some microorganisms with special functions have attracted widespread attention due to the importance or particularity of their functions. These microorganisms with special functions are called functional microorganisms. Functional microorganisms often participate in specific geochemical cycles. The common ones are carbon cycle, Nitrogen cycle, methane cycle, sulfur cycle, etc. Common functional microorganisms include ammonia-oxidizing bacteria, ammonia-oxidizing archaea, nitrifying bacteria, nitrogen-fixing microorganisms, methanogens, sulfate-reducing bacteria, etc.

The genes that govern these functional microorganisms to perform important functions are called functional genes, such as amoA, dsrB, nxrA, nirK, mcrA, pmoA. CD Genomics has completed the detection of dozens of functional genes, and has mature detection technology and analysis experience.

These specific functional genes are the coding genes of enzymes with a specific role, and the copy number of the gene in the sample can be determined by quantifying the gene of the enzyme by qPCR.

At present, more research is mainly on the copy number information of enzymes in each reaction in the nitrogen cycle, carbon cycle, sulfur-related, and arsenic-related enzymes.

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Nitrogen cycle

Nitrogen is an essential component of nucleic acids and proteins in living organisms. The nitrogen cycle plays a key role in the existence and continuation of life, and is essentially a microbial-driven process of nitrogen transformation, utilization, and recycling. he nitrogen cycle is the sum of the processes of interconversion of N2, inorganic nitrogen compounds, and organic nitrogen compounds in nature, including ammonification, nitrification, denitrification, nitrogen fixation, etc.

Common nitrogen cycle functional genes are shown in the table below:

	Gene	Enzyme
N2—NH4+	nifH	Nitrogenase
OrgN—NH4+	ureC	Glutamate dehydrogenase
NH4+—NO2–	amoA	ammonia monooxygenase
NO2–—NO3–	nxrA	nitrite oxidoreductase
NO3–—NO2–	narG	nitrate reductase
NO2–—NO	nirS/nirK	nitrite reductase
NO—NO2	norB	nitric oxide reductase
NO2—N2	nosZ	nitrous oxide reductase
NO2–—N2	hzo	Hydrazine oxidase
NO3–—NO2–	napA	nitrate reductase
NO2–—NH4+	nrfA	c-type
NO2–—NH4+	nrfA	cytochrome nitrite reductase

Carbon Cycle

The carbon cycle is mainly a process of carbon fixation and transformation, which mainly includes carbon fixation, methane production, and methane oxidation. Autotrophic microorganisms have great environmental adaptability and carbon fixation potential. Among the five major biological carbon fixation pathways discovered so far, the Calvin cycle is the main pathway for CO2 fixation by autotrophic organisms, in which ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme in the Calvin cycle, so RubisCO and its encoding genes are used to study the structure and diversity of carbon fixing microbial communities in different ecological environments.

the natural carbon cycle. Methanogenic bacteria are a group of archaea capable of converting inorganic or organic compounds into methane and carbon dioxide by anaerobic fermentation.

The main functional genes in the carbon cycle are shown in the table below

Gene	Enzyme
cbbL/cbbM	Ribulose-l,5-bisphosphate
cbbL/cbbM	carboxylase/oxygenase
mcrA	methyl coenzyme M reduc-tase
pmoA	particulate methane monooxygenase

Sulfur-associated and arsenic-associated detection genes

Sulfur is one of the most abundant elements in nature. It exists in different forms. These different forms of sulfur can be transformed into each other under the action of microorganisms, which constitutes the geochemical cycle of sulfur.

There are three main pathways for microorganisms to act in the sulfur cycle, 1) desulfurization of sulfur-containing organic matter, 2) oxidation of reducing inorganic sulfur, and 3) reduction of sulfate. Among them, sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) are reused microorganisms that promote the sulfur cycle.

Common functional genes related to the sulfur cycle include:

Gene	Enzyme
dsrA	dissimilatory sulfite reductase
dsrB	dissimilatory sulfite reductase
soxB	Sulfur oxidation
aioA	arsenite (As(III)) oxidase genes
arrA	respiratory arsenate (As(V)) reductase genes
arsC	As(V)reductase genes
arsM	As(III) methyltransferase genes

The Principle of qPCR

Real-time quantitative PCR technology (Real-time QuantitativePCR, qPCR) refers to the addition of fluorescent groups that can specifically bind to DNA products in the PCR reaction system, and the use of fluorescent signal accumulation to monitor the entire PCR process in real time, and finally through relative quantitative or absolute quantitative methods to determine the expression level of each sample.