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A kind of specific primer and detection method thereof

A primer pair and molecular detection technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of difficult to quickly and effectively identify pathogenic bacteria, cumbersome procedures, and strong experience. Achieve the effect of remarkable detection effect, high sensitivity and wide application value

Active Publication Date: 2022-06-24
BEIJING AGRO BIOTECH RES CENT
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to detect and identify the different subspecies of Pectinbacterium carotosa Pc in the prior art mainly based on physiological, biochemical and conventional molecular biology characteristics, such as ITS-RFLP, MLSA multi-site sequence analysis, the method is time-consuming and procedural It is cumbersome and empirical, and it is difficult to quickly and effectively identify pathogenic bacteria, and timely monitor and control the spread and prevalence of pathogenic bacteria when diseases occur. A specific detection primer and detection method for pectinobacillus carotosa subspecies Pco , using the detection primers and detection method described in the present invention can simply and quickly detect the Pco strain in pectinobacillus carotovii Pc, with high accuracy, strong specificity, high sensitivity, easy operation, short detection time and reliable results

Method used

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  • A kind of specific primer and detection method thereof
  • A kind of specific primer and detection method thereof
  • A kind of specific primer and detection method thereof

Examples

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Effect test

Embodiment 1

[0033] Design and Synthesis of Primer of Example 1

[0034] This example provides primer design and screening procedures. The details of the srl operon were first reported in Escherichia coli and involved in the utilization and metabolism of sorbitol; this operon was also found in Erwinia amylovora, a pear blight pathogen, and was associated with the pathogenicity of the pathogen (Aldridge P, Metzger M , Geider K. Genetics of sorbitolmetabolism in Erwinia amylovora and its influence on bacterial virulence. MolGenet Genomics. 1997;256:611-619). The operon consists of six genes (srlAEBDMR). Since all and only Pco strains have the srl operon, this operon consists of six genes. Since it is not clear which of the genes can design specific primers, the inventors designed 2 pairs of primers with different sequences for each gene according to the sequences of these six genes, and a total of 12 pairs of primers were designed (Table 1, respectively in order are SEQ ID NOs: 1 to 24); ...

Embodiment 2

[0054] According to Example 1, the inventors used the online primer design software Oligo Calc: Oligonucleotide Properties Calculator (http: / / biotools.nubic.northwestern.edu / OligoCalc.html), designed a pair of specific primers srlE-F1 / srlE-R1 with strong specificity and high sensitivity to Pco bacteria, the Tm of the upstream and downstream primers are 60.47 ℃, 59.53 ℃, and the GC content is 52.38 %. This example is to verify the subspecies-level specific detection of the primers.

[0055] The DNA of the following bacterial species was extracted using a spin-column bacterial genomic DNA extraction kit (purchased from Beijing Tiangen Company). Using the published 117 strains (41 Pco, 48 Pcb and 28 Pcc strains) of Pectinobacter carotii collected from different suburban counties and hosts in Beijing in Table 2 (Tian Yu, Ma Yali, He Fuxin, et al. Beijing Identification and pathogenicity analysis of bacterial soft rot pathogens of regional celery. Chinese Journal of Phytopatholo...

Embodiment 3

[0061] Example 3 Sensitivity detection of primers to Pco bacteria genome

[0062] Dilute the initial template DNA solution of Pco subspecies reference strain BCS7 with sterile ultrapure water to the concentrations of 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, respectively , 9 gradients of 10fg / μL and 1fg / μL, etc., carry out PCR amplification, and evaluate the sensitivity of this primer to the detection of Pco genome, wherein the PCR reaction system, reaction conditions and gel electrophoresis conditions are the same as in Example 2, and the following results are obtained: image 3 Electropherogram shown. The results show that the target band of electrophoresis can be observed at concentrations above 10pg / μL, indicating that the method of the present invention has better sensitivity.

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Abstract

The invention discloses a pair of Pco The primer of subspecies and detection method thereof, said primer pair comprises upstream primer srlE-F1: 5'-TGACTGTGGTGGAACACTTCG-3' and downstream primer, srlE-R1: 5'-CAGCCAGTGATAAACCAACCG-3', established on the basis of said primer Pectobacter carota P. carotovorum subsp. odoriferum Subspecies detection method. Primers and methods provided by the present invention are effective against Pectinbacterium carota P. carotovorum subsp. odoriferum The detection of subspecies has the advantages of high accuracy, strong specificity, high sensitivity, and the detection process is simple and fast, which lays the foundation for the early diagnosis of field vegetable soft rot and the development of monitoring technology for pathogens.

Description

technical field [0001] The invention belongs to the field of plant protection, in particular to the rapid, sensitive and specific molecular identification of soft rot pathogenic strains isolated from susceptible plants, belonging to the field of crop disease pathogenic microorganism detection, and more particularly to plant bacterial soft rot pathogen-specific detection primers and its detection method. Background technique [0002] Pectobacterium carotovorum (Pc) is a ubiquitous, host range and geographically distributed plant soft rot pathogen under natural conditions (Ma B, Hibbing ME, Kim HS, et al. Host range and molecular phylogenies of the soft rot enterobacterial generaPectobacterium and Dickeya. Phytopathology. 2007;97(9):1150-1163.). The pathogen can infect plants in the field and during storage, causing serious economic losses, and is listed as one of the top ten important plant pathogenic bacteria worldwide (Davidsson PR, Kariola T, Niemi O, et al. Pathogenicity...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
Inventor 谢华李晓颖陈昌龙田宇
Owner BEIJING AGRO BIOTECH RES CENT
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