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A kind of pcr primer and method for detecting the species composition of environmental microbial arsenic oxidation gene

A technology for detecting environment and microorganisms, applied in the field of biotechnology detection

Active Publication Date: 2021-10-19
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of pcr primer and method for detecting the species composition of environmental microbial arsenic oxidation gene
  • A kind of pcr primer and method for detecting the species composition of environmental microbial arsenic oxidation gene
  • A kind of pcr primer and method for detecting the species composition of environmental microbial arsenic oxidation gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

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

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

[0031] (4) According to the PrimerMatching and PrimerDeg results 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] Bioinformatics verification of the versatility of the designed aioA primers:

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

[0035] (1) From KEGG ( https: / / www.kegg.jp / ) and NCBI Refseq ( https: / / www.ncbi.nlm.nih.gov / refseq / ) download the aioA gene sequence with full length in the database, 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 with Blastn software. The criterion for alignment was 2 maximum unmatched 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 sh...

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 a PCR primer and a method for detecting the species composition of the arsenic oxidation gene of environmental microorganisms. The forward and reverse primers of the primer pair are: forward primer: 5'-atctggggbaayracaayta-3'; reverse primer: 5'-ttcatbgasgtsagrttcat- 3'. The method comprises the following steps: using environmental DNA as a template, using the above primers to amplify the aioA gene; after the amplified product is purified, it is connected to a carrier to construct a recombinant plasmid; the recombinant plasmid is transformed into Escherichia coli competent cells, and the LB Smear bacteria on the plate and culture overnight at 37°C; single clones were picked to extract plasmids and then sequenced. After the carrier sequence was removed from the sequencing results, the species composition information was determined after comparison with the NCBI-nr database. The species composition of arsenic-oxidizing bacteria in the natural environment can be efficiently analyzed by using the aioA primers designed in this application and subsequent molecular biology methods.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and particularly 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 caused widespread concern in humans. Arsenic is ubiquitous in nature and ranks 20th in crustal abundance. Arsenic enrichment in crops and arsenic contamination in groundwater are the main pathways that lead to human arsenic exposure. Arsenic in natural water is mostly inorganic arsenic, which mainly exists in two valence states: As(V) and As(III). As(V) is usually strongly adsorbed on oxide surfaces such as iron and aluminum under most environmental conditions, which limits its mobility; however, As(III) has greater mobility due to less adsorption, and its toxicit...

Claims

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Application Information

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Patent Type & Authority Patents(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