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Primer design suitable for lactobacillus 16SrRNA sequencing

A technology for sequence determination and lactic acid bacteria, applied in the field of genetic engineering, can solve the problems of increased sequencing costs and workload, multiple double peaks or miscellaneous peaks, and fewer nucleotides, and achieve the effect of accurate classification and identification methods

Inactive Publication Date: 2012-04-18
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although 5S rRNA is easy to analyze, there are too few nucleotides, and there is not enough genetic information for classification research; while 23S rRNA contains almost twice as many nucleotides as 16S rRNA, so analysis is very difficult
The cloning method is accurate, but it will undoubtedly increase the cost and workload of sequencing
The direct sequencing method of amplification products is fast and simple, but because the sequencing primers use universal amplification primers, the sequencing results often have more double peaks or miscellaneous peaks, which is a disadvantage that cannot be overcome by sequencing with universal amplification primers

Method used

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  • Primer design suitable for lactobacillus 16SrRNA sequencing
  • Primer design suitable for lactobacillus 16SrRNA sequencing
  • Primer design suitable for lactobacillus 16SrRNA sequencing

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Genomic DNA extraction:

[0047] Take 1.0mL of purely cultured lactic acid bacteria solution in a 1.5mL centrifuge tube, centrifuge and wash, add 0.5mL TE buffer solution, freeze completely in liquid nitrogen, take it out and put it in a 65°C water bath to thaw, and freeze and thaw 3 times repeatedly. Add 0.1 mL of 10% SDS and 10.0 μL of 10 mg / mL proteinase K, shake in a constant temperature shaker at 37°C at 200 r / min for 2 h, centrifuge at 10303 g for 10 min at room temperature, collect the supernatant and transfer it to another centrifuge tube. The supernatant and an equal volume of chloroform were centrifuged at 10303 g for 10 min, and the supernatant was transferred to another centrifuge tube for phenol-chloroform extraction twice. After precipitation with 0.1 volume of sodium acetate and 1 volume of ice isopropanol, the total DNA, then washed the precipitate twice with 70% ethanol, and redissolved for later use. figure 1 Is the extracted DNA electrophoresis patte...

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Abstract

The invention provides an amplification primer and a sequencing primer suitable for lactobacillus 16SrRNA sequencing. When a lactobacillus 16SrRNA segment is amplified by using the amplification primer, the lactobacillus 16SrRNA segment is sequenced by using the sequencing primer, and the base composition and the arrangement sequence of the 16SrRNA segment can be measured completely, accurately and quickly, so the species identification of lactobacilli can be accurately finished, wherein the sequences of the amplification primer are SEQ ID No.1 (AD27F) and SEQ ID No.2 (AD1495R), and the sequences of the sequencing primer are SEQ ID No.3 (A27F) and SEQ ID No.4 (A1495R).

Description

【Technical field】 [0001] The invention belongs to the field of genetic engineering, in particular to a primer suitable for the determination of 16S rRNA sequences of lactic acid bacteria, which can be used to amplify the 16S rRNA fragments of lactic acid bacteria and then perform sequencing, so that the base composition of the 16S rRNA fragments can be completely, accurately and quickly determined And the order of arrangement, so as to accurately complete the identification of the species of lactic acid bacteria. 【Background technique】 [0002] Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that can ferment carbohydrates to produce lactic acid with different morphological, metabolic, and physiological characteristics. Lactic acid bacteria found in nature can be divided into 43 genera in bacterial taxonomy, including 373 species and subspecies, most of which are anaerobic bacteria or facultative anaerobic bacteria. Lactic acid bacteria mainly exist in the i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 张和平孙志宏张家超刘文俊包秋华
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY