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Species-specific Quantitative PCR Primers and Its Design Method for Methylotrophic Bacillus

A methylotrophic and Bacillus technology, applied in the field of agricultural microorganisms, can solve problems such as information lag and genome data update, and achieve high accuracy and good quantitative specificity

Active Publication Date: 2020-02-21
GENLIDUO BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently commonly used primer design must first find specific sequences. The usual practice is to obtain species-specific genes from the literature and determine their practicability according to their conservation. However, the so-called "species-specific genes" obtained from the literature "Sex genes" often have relatively lagging information and have not been updated based on the growing genome data, resulting in the fact that the obtained primers may not be able to ensure their specificity in practical applications

Method used

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  • Species-specific Quantitative PCR Primers and Its Design Method for Methylotrophic Bacillus
  • Species-specific Quantitative PCR Primers and Its Design Method for Methylotrophic Bacillus
  • Species-specific Quantitative PCR Primers and Its Design Method for Methylotrophic Bacillus

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Experimental program
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Embodiment 1

[0026] Embodiment 1 is tested bacterial strain source

[0027] The methylotrophic bacillus NJAU-Z9 tested in the present invention (preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, the preservation date is April 11, 2016, and the preservation number is CGMCC NO.12342) and 6 strains Similar strains belonging to the genus Bacillus, all the bacteria are stored in the Jiangsu Key Laboratory of High-tech Research on Solid Organic Waste Recycling and Utilization, Nanjing Agricultural University (Table 1), and the whole genome sequence is uploaded to the NCBI Biotechnology Information Center with accession number NZ_CP022556.1 .

[0028] Table 1 The strains used for primer analysis and their corresponding GenBank accession numbers

[0029]

[0030]

Embodiment 2

[0031] Example 2 Primer sequence selection and cross-amplification verification test

[0032] The invention compares the whole genome sequence in the database and searches for the specific sequence for NJAU-Z9. Step 1: Compare the whole genome sequence of NJAU-Z9 (NZ_CP022556.1.) with two authoritative databases, NCBI and EzTaxon, and select genome fragments larger than 500bp that do not match. The second step: Utilize Oligo 6 software to implement primer sequence design, set the product size to be 70-250bp, and the primer length is 22 ± 2bp, design 8 pairs of primers (Table 2) in total, and pass 8 pairs of primers to NJAU- Z9 genomic DNA was amplified sequentially, and a preliminary judgment was made on the primers according to the quality of the bands ( figure 1 ). In addition, the genomic DNA of 6 strains of other similar species and the DNA of the target strain were used as primers P1-P8 to cross-amplify the templates sequentially, and a pair of reported Bacillus primers...

Embodiment 3

[0038] Construction and quantitative PCR of embodiment 3 plasmid

[0039] The fragments of 8 pairs of primer amplification products designed from the selected target gene region in the methylotrophic bacillus NJAU-Z9 genome were cloned into the pMD19-T vector (TaKaRa), and the plasmid was transformed into Escherichia coli Top10 cells, Common PCR amplification was performed to verify the amplified fragment, and sequenced by Nanjing GenScript Biotechnology Co., Ltd. The DNA concentration of the plasmid was measured using a spectrophotometer (NanoDrop 2000, Thermo Scientific Inc., USA). Use on 7500 real-time PCR system (Applied Biosystems, USA) Premix Ex TaqTM (TaKaRa) was used for gradient qPCR amplification in a 20 μl reaction system. Plasmids with different inserts were used to prepare a 10-fold dilution series (triplicate), using sterile water as a negative control. After determining the cycle threshold (CT) value for each sample, a standard curve was generated by plottin...

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Abstract

The invention discloses a Bacillus methylotrophicus specific quantitative PCR primer and a design method thereof. The design method includes the steps of: firstly comparing the complete genome sequence of bacillus methylotrophicus NJAU-Z9 in the NCBI and EzTaxon databases, selecting the genomic fragments of more than 500 bp which are not matched, using an Oligo 6 software to design primer sequences, setting a product size to be 70-250 bp and the length of the primer to be 22 plus or minus 2 bp, finally performing amplification specificity test by crossing the various strains of the same genusin sequence, adding NJAU-Z9 into soil to perform amplification specificity re-detection, and combining with the actual detection of potting soil, etc, to screen to obtain a pair of optimal bacillus methylotrophicus NJAU-Z9 quantitative PCR primers. The primer design method and designed strain-specific primer provided by the present invention can provide a quantitative PCR-specific primer design for other strains and provide a method for quantitation of bacillus methylotrophicus NJAU-Z9.

Description

technical field [0001] The invention belongs to the field of agricultural microorganisms, and relates to the design and application of quantitative PCR-specific amplification primers for a methylotrophic bacillus NJAU-Z9 with growth-promoting and disease-resistant abilities. Background technique [0002] In the study of promoting plant growth and disease resistance, a large number of beneficial microbial agents have been inoculated into the soil and rhizosphere or combined with organic fertilizers to inoculate the soil as soil improvers. However, the survival of functional strains in the soil and rhizosphere environment plays a key role in their function, thus, effective colonization is a prerequisite for functional strains to promote plant growth and improve soil microbial ecosystem. [0003] Selective media coating has been widely used to detect or isolate target microorganisms in the environment, however, this method is insensitive and time-consuming, and it is difficult ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/07
CPCC12Q1/686C12Q1/689C12Q2545/114
Inventor 沈其荣张扬李荣张楠朱成之刘红军
Owner GENLIDUO BIO TECH CO LTD
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