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PCR primers and method for detecting composition of environmental microorganism arsenic oxidation gene species

A technology for detecting environment and arsenic oxidation, which is applied in the field of biotechnology detection to achieve efficient analysis

Active Publication Date: 2019-09-13
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In view of the defect that the amplification length of the existing primers is more than 600bp, the primary purpose of the present invention is to provide a pair of new PCR primers for amplifying the aioA gene, and to design the PCR product with a length of less than 500bp using the conserved domain of the molybdenum coenzyme encoding the aioA protein Primers

Method used

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  • PCR primers and method for detecting composition of environmental microorganism arsenic oxidation gene species
  • PCR primers and method for detecting composition of environmental microorganism arsenic oxidation gene species
  • PCR primers and method for detecting composition of environmental microorganism arsenic oxidation gene species

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Experimental program
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Effect test

Embodiment 1

[0027] The design of aioA gene PCR primers comprises the following steps:

[0028] (1) From KEGG (https: / / www.kegg.jp / ) and NCBI Refseq ( https: / / www.ncbi.nlm.nih.gov / refseq / ) database to download the full-length aioA protein sequence and the corresponding gene sequence;

[0029] (2) Muscle software (http: / / www.drive5.com / muscle) compared the aioA protein sequence and gene sequence to determine the gene sequence of the molybdenum coenzyme conserved domain.

[0030] (3) With the gene sequence encoding the conserved domain of the molybdenum coenzyme as the target, apply the DegePrime software ( https: / / github.com / EnvGen / DegePrime ) to design primers.

[0031] (4) According to the results of PrimerMatching and PrimerDeg of DegePrime software, select optimized primers, select forward primer (aioA-1109F1): 5'-atctggggbaayracaayta-3' (SEQ ID NO.1), reverse primer (aioA-1548R1) : 5'-ttcatbgasgtsagrttcat-3' (SEQ ID NO. 2).

Embodiment 2

[0033] Verification of the generality of the designed aioA primers by bioinformatics:

[0034] Align the primers designed in Example 1 to the full-length aioA gene sequence, and compare the effectiveness of the primers to the aioA genes of different species, the steps are as follows:

[0035] (1) from KEGG ( https: / / www.kegg.jp / ) and NCBI Refseq ( https: / / www.ncbi.nlm.nih.gov / refseq / ) database downloaded the full-length aioA gene sequence, and its species composition is shown in Table 1 and figure 1 .

[0036] Table 1. Species composition of the full-length aioA gene at the genus level

[0037]

[0038]

[0039] (2) The primer sequences were aligned to 79 full-length aioA gene sequences using Blastn software, and the alignment standard was 2 maximum mismatched bases, and the alignment similarity was greater than 90%. The ratio of aioA-62F1+aioA-518R1 to the full-length aioA gene sequence was 81.0%, and the comparison to the full-length aioA is shown in Table 2...

Embodiment 3

[0046]Extract paddy field soil microbial genomic DNA, PCR amplify aioA gene, a total of 7 paddy field soil samples, the steps are as follows:

[0047] (1) Weigh 0.25g fresh paddy field soil sample, use Powersoil TM DNA extraction kit (MOBIO company) was used to extract soil DNA samples. The quality of the extracted DNA samples was determined by horizontal electrophoresis and the concentration was determined by Nanodrop.

[0048] (2) Take 1 μl of environmental DNA as a template to amplify the aioA gene. aioA-1109F1+aioA-1548R1 were used as primers for PCR amplification. The total volume of the PCR amplification system is 50 μl, and the composition of the reaction system is: 1 μl of upstream and downstream primers (concentration 10M), 2 μl of 10×ExTag reaction solution (Shanghai Dalian Bao Biological Company), 1 μl (0.5U) of ExTag enzyme (Shanghai Dalian Bao Biological company), 2μl dNTPs (2.5mM), and 5μl sterile water. The PCR program was pre-denaturation at 94°C for 5 min...

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Abstract

The invention discloses PCR primers and method for detecting composition of environmental microorganism arsenic oxidation gene species. The forward and reverse primers of the primer pair are as follows: a forward primer: 5'-atctggggbaayracaayta-3'; and reverse primer: 5'-ttcatbgasgtsagrttcat-3'. The method comprises the following steps: using environmental DNA as a template, and amplifying aioA gene by using the primers; after the amplification product is purified, connecting the amplification product to a vector to construct a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli competent cells, smearing bacteria on an LB plate, and performing culturing overnight at 37 DEG C; and selecting monoclonal extraction plasmids, performing sequencing, removing a vectorsequence from a sequencing result, and determining species composition information after comparison with an NCBI-nr database. The aioA primers designed by the application and subsequent molecular biology means can analyze the species composition of arsenic oxidation bacteria in the natural environment efficiently.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a PCR primer and a method for detecting the composition of arsenic oxidation gene species of environmental microorganisms. Background technique [0002] Arsenic (As) is the first class I carcinogen identified by the International Agency for Research on Cancer (IARC), and its strong carcinogenicity has attracted widespread attention from humans. Arsenic is ubiquitous in nature, and its crustal abundance ranks 20th. Arsenic enrichment in crops and arsenic pollution in groundwater are the main pathways leading to human arsenic exposure. Arsenic in natural water is mostly inorganic arsenic, which mainly exists in two valence states: As(V) and As(III). Under most environmental conditions, As(V) is usually strongly adsorbed on the surface of oxides such as iron and aluminum, which limits its mobility; however, As(III) has greater mobility due to less adsorption, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q1/689C12Q2531/113
Inventor 胡敏刘传平李芳柏
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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