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Primer pair, kit and method for detecting and identifying Pectobacterium bacteria

A pectin bacillus and primer pair technology, applied in the biological field, can solve the problems of low specificity, prone to false positive or false negative detection, low versatility, etc., and achieve the effect of accurate and sensitive inspection and quarantine

Active Publication Date: 2021-04-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the shortcomings of the existing PCR detection of Pectobacterium bacteria with low specificity or low versatility, and the detection is prone to false positives or false negatives, and the purpose is to provide a method that can accurately identify and quarantine Pectobacterium bacteria Methods

Method used

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  • Primer pair, kit and method for detecting and identifying Pectobacterium bacteria
  • Primer pair, kit and method for detecting and identifying Pectobacterium bacteria
  • Primer pair, kit and method for detecting and identifying Pectobacterium bacteria

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Effect test

Embodiment 1

[0032]Designed for general specific primers for the Pectobacterium strand 23S rRNA gene:

[0033]Download Pectobacteriaum in Pectobacteriacea, Pectobacteriacea, Pectobacteriacea, Pectobacteriacea, Pectobacteriacea, DICKEYA, Brenneria, and Lonsdalea, Erwinia and Pantoea in Owenia, Kosakonia in E. coli, and Klebsiella are complete or Close to complete (>The 2600 bp) 23S rRNA gene sequence, the nucleic acid sequence is collected with the MUSCLE program in Mega5 software, and the 23S rRNA gene of Typical P.carotovorum Typical strain NCPB 312 (= CFCC 10814) is a reference sequence, and the BioEdit software is displayed. Sequence Difference nucleotide site (figure 1 ), With 20 to 30 nucleotides as modules, scan sequences, found that there are multiple nucleotide differences in the Pectobacterium straighteners in the bacterial 23S rRNA gene sequence, which is a variety of nucleotide differences, which are sequences for these Pectobacterium specific conserved area sequences. , Design primers w...

Embodiment 2

[0041]Comparison primers were specific to PECTOBACTERIUM bacteria for 23Specf / 23SPECR, PEC-1F / PEC3R and PCC3F / PCC3R.

[0042]Each primer nucleotide sequence is as follows:

[0043]Positive primer 23specf: 5'-cggtaagctgattructgaagcgttatac-3 ',

[0044]Reverse primers 23SPECR: 5'-TGTGTACGGACTATCCCCCTTACTACT-3 ';

[0045]Positive primers PEC1-F: 5'-gtgcaagcgttaatcgaatg-3 ',

[0046]Reverse primers PEC1-R: 5'-ctctacaagactctcctgtcagtttt-3 ';

[0047]Positive primer PCC3F: 5'-gggattcgaaaaattactggctg-3 ',

[0048]Reverse primers PCC3R: 5'-gcttttctttcatcaacca-3 '.

[0049]Positive primer 23Specf and reverse primer 23SpeCr correspond to the nucleotide sequence of the 23S rRNA gene of Pectobacterium and other near-belly bacteria. The 23S rRNA gene sequence is displayed with the identical nucleotide of the primer corresponding to the nucleotide of the primer, and the nucleotide different from the corresponding nucleotide is shown in the form of a nucleotide (figure 1 ).figure 1 Showing reverse primers 23SPECR h...

Embodiment 3

[0055]The comparative primer was used to detect the sensitivity of the Pectobacterium bacteria in 23SPECF / 23SPECR and Y1 / Y2.

[0056]Each primer nucleotide sequence is as follows:

[0057]Positive primer 23specf: 5'-cggtaagctgattructgaagcgttatac-3 ',

[0058]Reverse primers 23SPECR: 5'-TGTGTACGGACTATCCCCCTTACTACT-3 ';

[0059]The forward primer Y1: 5'-ttaccgacgccgagctgtgggct-3 ',

[0060]Reverse primers Y2: 5'-caggaagatgtcgttatcgcgagt-3 '.

[0061]The Pectobacterium Carotovorum CFCC 10814 genomic DNA with a concentration of 40 ng / μl was diluted in 10 times series, and each of the 1 μl of DNA solution was a template, 23 JPECF and 23SPECR were used in 25 μl of reaction system, and Shanghai Hoheng Biotechnology is limited. The 2 × HIEFF PCP MASTER MIX produced by the company was carried out by the amplification procedure, and the PCR product (266 bp) was detected by agarose gel electrophoresis, showing the detection of the minimum concentration of 400 Fg / μL (Figure 6 ).

[0062]The Pectobacterium C...

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PUM

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Abstract

The invention discloses a primer pair for detecting and identifying Pectobacterium bacteria and a using method thereof. The primer comprises a forward primer 23SPecF and a reverse primer 23SPecR. The method for detecting and identifying the Pectobacterium bacteria comprises the following steps of: performing Polymerase Chain Reaction (PCR) on the 23SPecF and the 23SPecR by using the primer pair, wherein a 266bp nucleic acid fragment can be only amplified from a sample containing the genome DNA of the Pectobacterium bacteria. Due to that the Pectobacterium bacteria have seven copied 23SrRNA genes, the detection sensitivity of the primer pair for performing the PCR on the 23SPecF and the 23SPecR is seven times that of a single-copy gene primer, and the Pectobacterium bacteria can be detected more sensitively.

Description

Technical field[0001]The present invention relates to the field of biotechnology, and more particularly to primers, kits and methods for detecting and identifying the bacteria of Pectobacterium.Background technique[0002]Enterobacteriaceae Bacteria Bacteria Bacteria By producing pectinase, degrading pectin in plant cell walls, destroying plant cells, leading to plant cell death and tissue to softening, plant soft rot, in order to harm numerous vegetables and Grain crops, ornamental flowers and forests. The Pectobacterium and Dickeya are a wide range of bacterial hosts, including phytors, Sakko, Lily, Umbrella, Aski and Gramcut, etc., which caused plant soft rot to affect the production of crops and Quality, causing serious economic losses, threatening agricultural production and food security.[0003]At present, Pectobacterium has been identified 18 species. DICKEYA has been identified 11 species. The Pectobacterium and Dickeya are the most types of strains are plant pathogens, resulti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12Q1/04C12R1/01
CPCC12Q1/689C12Q1/686C12Q2565/125C12Q2563/107
Inventor 张健男马媛媛李斌安千里
Owner ZHEJIANG UNIV
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