Method for quantitating anaerobic ammonia oxidizing bacteria in sediment of aquiculture environment

Abstract

The invention relates to a method for quantitating ammonia oxidizing bacteria in a sediment of an aquiculture environment by a real-time fluorescent quantitative PCR (polymerase chain reaction) technology, which belongs to the field of environmental microbial ecology. The method comprises the steps that DNA (deoxyribonucleic acid) of a sample sediment is extracted and quantitated; an HZO (hydrazine oxidordeuctase) gene specific primer is subjected to PCR amplification; standard plasmids are constructed; and a fluorescent quantitation PCR amplification curve and a standard curve are made. The quantity of the anaerobic ammonia oxidizing bacteria in a sample is calculated by comparing a detected sample Ct value with the standard curve; an obtained log value of the initial template gene copy number has a better linear relationship with the Ct value; and a regression coefficient is greater than 0.99. According to the method, an HZO gene is used for conducting quantitative analysis on the ammonia oxidizing bacteria under an anaerobic condition; the deficiencies that the specificity is not high, the coverage ratio is low and the method is inaccurate due to the fact that the a 16S rRNA (ribosomal ribonucleic acid) gene is used for quantitating in the existing method are overcome; and the method for quantitating the ammonia oxidizing bacteria in the sediment of the aquiculture environment is high in specificity, quick and accurate.

Description

technical field [0001] The invention relates to a method for quantifying anammox bacteria in aquaculture environment sediments, and belongs to the technical field of environmental microbial ecology. Background technique [0002] my country is the largest country in aquaculture and aquatic product trade, and the output of aquaculture aquatic products accounts for 70% of the world's output. The scale of intensive and high-density farming is expanding day by day. Concentrated feeding causes a large amount of feces, residual bait, and dead animal and plant corpses to accumulate in the water body, resulting in the serious excess of ammonia nitrogen and nitrite nitrogen, which are the end products of protein metabolism in the water body. The ecological environment is destroyed, and fish and shrimp diseases occur frequently, causing huge economic losses. At the same time, a large amount of aquaculture nitrogen-containing wastewater is discharged into coastal waters, causing seriou...

AI Technical Summary

Problems solved by technology

The inventors of the present invention use the characteristic enzyme HZO gene of anammox bacteria as a molecular marker, design HZO gene-specific primers, and use the method of fluorescent quantitative PCR to quantitatively detect anammox bacteria, which can overcome the existing 16S rRNA gene The detection specificity is not high, the diversity is low, and there are shortcomings of false positive amplification. It can quickly, sensitively, accurately and truly detect the number of anammox bacteria in sediments.

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