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Zooplankton rrnL gene amplification primers and screening method, application and application method thereof

A technology for zooplankton and gene amplification, applied in biochemical equipment and methods, special data processing applications, microbial measurement/inspection, etc., can solve the harsh PCR reaction conditions, poor amplification ability of crustacean groups, difficult sequencing, etc. problems, to achieve the effect of improving the success rate of PCR, mild conditions, and reducing non-specific amplification

Active Publication Date: 2015-03-11
南京易基诺环保科技有限公司
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Problems solved by technology

[0005] Aiming at the problems of existing COI primers such as high degeneracy, harsh PCR reaction conditions, poor amplification ability for crustaceans, and difficulty in sequencing on a high-throughput sequencing platform, the present invention provides a zooplankton rrnL gene amplification Primers and their screening methods and applications and application methods, the mitochondrial ribosomal gene rrnL is a relatively conservative gene in the mitochondrial genome of animals, and is one of the potential barcodes for species identification. The present invention is designed based on the rrnL gene sequence of epizooplankton groups 5 upstream primers and 4 downstream primers, the combination of upstream and downstream primers can amplify PCR products ranging in length from 190bp to 390bp, the degeneracy of primers is low, the success rate of PCR is high, and the length of PCR products can be compatible with two A high-throughput sequencing platform that can be used for species identification, biodiversity analysis, etc.

Method used

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  • Zooplankton rrnL gene amplification primers and screening method, application and application method thereof
  • Zooplankton rrnL gene amplification primers and screening method, application and application method thereof
  • Zooplankton rrnL gene amplification primers and screening method, application and application method thereof

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Embodiment 1

[0043] (1) Sequence processing of metazoan plankton rrnL gene

[0044] Download all zooplankton rrnL gene sequences from NCBI database and BLOD database, a total of 1558 sequences, including 176 rotifer sequences, 642 copepod sequences, and 740 cladoceran sequences, and remove the duplications among them by bioinformatics software Sequences, so that there are no more than 3 sequences for each species. After screening, 44 rotifer sequences, 132 copepod sequences, and 109 cladocera sequences were finally obtained. Use the software mafft-win to perform sequence comparison on the above sequences respectively, take the shortest sequence as the standard, remove the parts of other sequences that are not aligned at both ends to make all sequences equal in length, and then delete the occurrence frequency of each site that is less than 10%. Base, and then output the conserved sequences of rotifers, cladocerans, and copepods respectively, and put together the conserved sequences of rotif...

Embodiment 2

[0053] Zooplankton samples were collected in Zhushan Bay (120.036°E, 31.373°N) and Miaogang (120.461°E, 31.002°N) in Taihu Lake, Jiangsu Province. The collected samples were stored in 90% alcohol on site, and then brought to the experiment The laboratory carried out species identification and separation, and a total of 7 species of rotifers were collected (Brachionus calyxae, Brachionus cucurbitus, Trilimidae longus, Brachioperus schizopoda, Cysticus anteriore, Caucus Brachionus rotifers and Rotifers sp.) 4 species of Cladocera (Daphnia fuzzy, Diaphragm pellucidum, Daphnia diaphyllum and Daphnia chinensis) and 5 species of copepods (Daphnia globosa, DNA of a single zooplankton was extracted from Daphnia spp., Daphnia dextritus, Daphnia sp. and Daphnia mammoth sp.). The extracted zooplankton DNA was amplified by PCR with rrnL primers and COI primers respectively. When amplifying with rrnL primers, select the combination of ZoorrnL_80F and ZoorrnL_400R primers with the longest a...

Embodiment 3

[0055] Filter 30L of lake water with a zooplankton net (160μm pore size), concentrate the lake water to 50mL and then filter it with a filter membrane with a pore size of 50μm to remove water, according to The E.Z.N.A. Water DNA Kit (OMEGA, CHINA) kit standard operation was used to extract filter membrane DNA, and use rrnL primers and reagents in Example 2 to perform PCR amplification on filter membrane DNA. The PCR amplification product was recovered by tapping the gel with the MinElute Gel Extraction Kit (Qiagen, USA), and was recovered by Qubit TM dsDNA HS Assay Kits were used for quantification, and the purified PCR product was analyzed for fragment size using Bioanalyser 2100 (Agilent Technologies, USA). The analysis results were as follows: Figure 5 shown. It can be seen from the figure that the amplification of environmental DNA with primers combined with ZoorrnL_80F and ZoorrnL_400R will have different fragment sizes, and the size distribution is between 321bp and ...

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Abstract

The invention discloses zooplankton rrnL gene amplification primers and a screening method, application and an application method thereof, and belongs to the biotechnical field. According to common zooplankton rrnL gene sequences, 9 PCR (polymerase chain reaction) amplification primers are designed, comprising 5 upstream primers and 4 downstream primers; the primers have wide coverage and high amplification efficiency for zooplankton, and PCR products of the primers are compatible with a high-throughput sequencing platform; after the upstream primers and the downstream primers are combined in pairs, products with the lengths of 190-390 bp can be amplified; by a multi-primer combination method, nonspecific amplification can be reduced and the success rate of PCR can be improved; the PCR products can be widely used for performing species identification, biodiversity analysis and other researches through Barcode sequences.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a zooplankton rrnL gene amplification primer and a screening method, application and application method thereof. Background technique [0002] DNA barcoding (DNA-barcoding) is currently one of the most powerful species identification techniques, especially playing an important role in the identification of unknown species. With the improvement of next-generation sequencing throughput and the reduction of sequencing costs, DNA barcoding technology has begun to be more and more applied in the investigation of environmental biodiversity. At present, species identification based on DNA barcodes relies on the amplification process of PCR, which first amplifies DNA fragments that can distinguish species, and then identifies species based on sequence differences. At present, the DNA barcode used to identify animals is mainly the mitochondrial cytochrome oxidase I (COI) gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G06F19/22
CPCC12Q1/6888C12Q2600/156
Inventor 张效伟杨江华
Owner 南京易基诺环保科技有限公司
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