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Specific primer and detection method thereof

A specific and molecular detection technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as difficult to quickly and effectively identify pathogenic bacteria, cumbersome procedures, and strong experience. Achieve the effect of remarkable detection effect, strong specificity and accuracy, and wide application value

Active Publication Date: 2019-06-11
BEIJING AGRO BIOTECH RES CENT
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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

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  • Specific primer and detection method thereof
  • Specific primer and detection method thereof
  • Specific primer and detection method thereof

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Embodiment 1

[0033] The design and synthesis of embodiment 1 primer

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

Embodiment 2

[0054] According to Example 1, the inventors aimed at the unique and highly conserved srlE gene of the bacterial species Pectinbacterium carotobacter subsp. 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 were 60.47°C, 59.53°C, and the GC content was 52.38 %. This example is to verify the subspecies-level specific detection of the primer.

[0055] Using a spin-column bacterial genomic DNA extraction kit (purchased from Beijing Tiangen Company), the DNA of the following bacterial species was extracted. Using the published 117 strains of Pectinbacterium carotosa (41 strains of Pco, 48 strains of Pcb and 28 strains of Pcc) 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 of celery in the region. Acta Phytopathology. 201...

Embodiment 3

[0061] The sensitivity detection of embodiment 3 primers to Pco pathogen genome

[0062] Dilute the initial template DNA solution of Pco subspecies reference strain BCS7 with sterile ultrapure water to concentrations of 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL , 9 gradients such as 10fg / μL and 1fg / μL, carry out PCR amplification, evaluate the sensitivity of this primer to Pco genome detection, wherein the reaction system of PCR, reaction condition and gel electrophoresis condition are identical with embodiment 2, obtain as follows image 3 The electropherogram shown. The results show that the electrophoresis target band 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 primer pair for detecting pectobacterium carotovorum Pco subspecies and a detection method of the primer. The primer pair comprises an upstream primer srlE-F1:5'-TGACTGTGGTGGAACACTTCG-3' and a downstream primer srlE-R1:5'-CAGCCAGTGATAAACCAACCG-3', and a detection method for P.carotovorum subsp.odoriferum subspecies in the pectobacterium carotovorum is established based onthe primer. The primer pair and the method provided by the invention have the advantages of being high in accuracy, high in specificity, high in sensitivity, simple, convenient and quick to operate in the detection process and the like when being used for detecting the P.carotovorum subsp.odoriferum subspecies of the pectobacterium carotovorum, and a base is established for the development of early diagnosis of soft rot of field vegetables and pathogenic bacterium monitoring technique.

Description

technical field [0001] The invention belongs to the field of plant protection, and in particular relates to rapid, sensitive and specific molecular identification of soft rot pathogenic strains isolated from susceptible plants, belongs to the field of detection of pathogenic microorganisms of crop diseases, and more specifically relates to specific detection primers for plant bacterial soft rot pathogens and its detection method. Background technique [0002] Pectobacterium carotovorum (Pc) is a plant soft rot pathogen that is ubiquitous under natural conditions and has a wide host range and geographical distribution (Ma B, Hibbing ME, Kim HS, et al. Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium 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 in the world (Da...

Claims

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Application Information

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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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