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Primer and fluorescent probe for detecting anaerobic denitrifying bacteria

An anaerobic denitrification and fluorescent probe technology is applied in the field of primers and fluorescent probes for detecting anaerobic denitrification bacteria, which can solve the problems of long cycle, lack of specific detection, and complicated method operation.

Inactive Publication Date: 2007-12-12
HARBIN INST OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that there are no specific primers and specific probes for detecting anaerobic denitrifying bacteria at present, and the existing lack of specific detection methods have low counting accuracy, cumbersome method operations, and high costs. , the problem of long period, providing a kind of primer and fluorescent probe for detecting anaerobic denitrification bacteria

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  • Primer and fluorescent probe for detecting anaerobic denitrifying bacteria

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specific Embodiment approach 1

[0007] Embodiment 1: A pair of conservative specific primers and a specific fluorescent probe are designed for the 16S rDNA gene sequence fragment of anaerobic denitrifying bacteria. The gene sequences of the primers and fluorescent probe are as follows:

[0008] Upstream primer (FP): 5'-ATGCGTAGCCGACCTGAGA-3'

[0009] Downstream primer (RP): 5'-CGTCAGACTTTCGTCCATTGC-3'

[0010] Fluorescent probe: 5'-CGGGAGGCAGCAGT-3'

[0011] The 5' end of the fluorescent probe is labeled with the reporter fluorescent group FAM, and the 3' end is labeled with the quencher fluorescent group TAMRA-MGB.

[0012] The synthesis of primers and fluorescent probes was done by a bioengineering company.

specific Embodiment approach 2

[0013] Specific embodiment two: this embodiment is used to prepare the DNA standard template of anaerobic denitrifying bacteria:

[0014] (1) After injecting 1ml of sewage into the centrifuge tube, shake it in an ultrasonic instrument for 5 minutes, then centrifuge it in a centrifuge at 3000rpm / min for 2 minutes, and get the supernatant; add 2 times the volume of 10×PBS to the supernatant to wash, Shake in a sonicator for 5 minutes, then centrifuge in a centrifuge at 12,000 rpm / min for 5 minutes, remove the supernatant; add 100 μl of 10×PBS buffer solution to the centrifuge tube, shake in a sonicator for 5 minutes; extract with a small amount of DNA bacteria Kit (purchased from Shanghai Huashun Biological Engineering Co., Ltd.) to extract DNA;

[0015] (2) Perform PCR amplification on the DNA extracted in the previous step: establish a PCR reaction system (20 μl): template (DNA) 20 ng, Taq DNA polymerase (purchased from Dalian Bao Biological Engineering Co., Ltd.) 0.3 U, 4 kin...

specific Embodiment approach 3

[0017] Specific embodiment three: this embodiment is used to detect the fluorescence quantitative detection sensitivity of anaerobic denitrifying bacteria and draw a standard curve:

[0018] (1) Measure the concentration of pGEM-T-ADNB stock solution and convert it into copy number. The conversion result shows that the copy number content in pGEM-T-ADNB stock solution is 1.302×10 8 copies / μl. Conversion formula: copy number (copies / μl) = [C (μg / ul) × 10 -6 ×6.02×10 23 ] / [660×(vector + target fragment)bp]

[0019] (2) Drawing a standard curve: the pGEM-T-ADNB stock solution was diluted 10 times according to the copy number, and the dilution ratios were 0 and 10 respectively. 1 、10 2 、10 3 、10 4 and 10 5 , Fluorescence RT-PCR assay was carried out on each concentration dilution, and water was set as a negative control at the same time.

[0020] Fluorescence RT-PCR reaction system (25 μL): 5×real time buffer 5.0 μl, dNTP (10 mmol / L) 0.75 μl, Mg 2+ (250mmol / L) 0.5μl, upst...

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Abstract

The invention discloses a kind of primer and fluorescent probe that are used for anaerobic denitrifying bacteria detection, relating to primer and fluorescent probe that are used for microbe detection. It overcomes shortcomings of long detection time, smaller detection result compared to correct result, complicate operation and high detection price existed in current quantitative determination method. The gene order of upstream primer is 5'-ATGCGTAGCCGACCTGAGA-3'and gene order of downstream primer is 5'-CGTCAGACTTTCGTCCATTGC-3'. The gene order of fluorescent probe is 5'-CGGGAGGCAGCAGT-3'; the report fluorophor marked by 5'end in fluorescent probe is FAM, and the quenching fluoropho marked by 3' end is TAMR- MGB. The invention provides specific primer and probe for anaerobic denitrifying bacteria detection, and it is characterized by accurate bacteria counting, simplified operation, reduced cost, shortened detection period and practical meaning in production.

Description

technical field [0001] The invention relates to a primer and a probe for detecting bacteria, in particular to a primer and a fluorescent probe for detecting anaerobic denitrifying bacteria. Background technique [0002] Anaerobic denitrifying bacteria (ADNB) is a general term for a class of bacteria that perform anaerobic denitrification reactions, which reduce nitrate and nitrite by inducing the production of nitrate reductase and nitrite reductase. In the wastewater biological treatment of biological denitrification and denitrification, denitrification and desulfurization, and various processes related to anaerobic denitrification bacteria, the number of anaerobic denitrification bacteria is an important indicator of biomass and its activity. . [0003] Existing detection methods for anaerobic denitrifying bacteria include the most probable number (MPN)-Griess, Duchenne tubule method, etc., and they all use conventional culture methods, which are cumbersome to operate, hi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/52
Inventor 马放魏利姚杰
Owner HARBIN INST OF TECH
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