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System for detecting pear branch blight germs and construction method thereof

A construction method and technology of blight bacteria, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as missing and lack of detection methods, and achieve sensitive and reliable detection results, safe and efficient production The effect of sustainable development

Pending Publication Date: 2021-08-27
TARIM UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of inspection technology for pear pollen pathogens, any pollination method has a potential safety hazard of spreading pathogens during pollination
[0003] In order to block the spread of pathogens, it is necessary to detect the pathogens in pear pollen, so as to remove the pollen carrying the pathogens. At present, there is a lack of reliable detection methods to detect pear pollen branch blight bacteria, so it is urgent to develop a new method System and method for detecting pear pollen branch blight

Method used

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  • System for detecting pear branch blight germs and construction method thereof
  • System for detecting pear branch blight germs and construction method thereof
  • System for detecting pear branch blight germs and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Primer design for conventional PCR and multiplex PCR

[0039] The primers selected for the experiment in this example are PrimerE / PrimerF derived from the exopolysaccharide synthesis gene (amylovoran synthesis, ams) sequence on the chromosome of E. amylovora pathogen and two pairs of primers p29A / p29B and PEANT1 / PEANT2 derived from the pathogenic bacteria plasmid pEA29. The sequence, site, and size of the amplified bands of the three pairs of primers are shown in Table 1, the table of primers for the detection of Yali pollen amylovora pathogen. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0040] Table 1

[0041]

[0042] 2. Culture of Erwinia amylovora

[0043] The prepared LB medium was steam sterilized at 121°C and 0.1MPa for 20 minutes, then poured into a plate on an ultra-clean workbench, and allowed to cool for later use. Take the preserved Erwinia amylovora strain out of the -20°C refrigerator, and after thawing, take a ring wi...

Embodiment 2

[0065] Embodiment 2, dual PCR system establishment and sensitivity detection

[0066] Double-PCR system: Double-PCR is to add two pairs of primers to a small PCR tube for PCR amplification, and produce two products of different sizes. ddH 2 O was replaced with the bacterial solution as a blank control, and 3 μL of the amplified product was detected by 1% agarose gel electrophoresis, stained with ethidium bromide, and observed under a fluorescent imaging system. The combinations are as follows: double PCR combination 1: PrimerE / PrimerF+p29A / p29B; double PCR combination 2: PrimerE / PrimerF+PEANT1 / PEANT2, as shown in Table 4, double PCR reaction system (25 μL).

[0067] Table 4

[0068]

[0069] Combination 1 reaction program: 94°C for 3min, 94°C for 30s, 52°C for 30s; 30 cycles, 72°C for 90s, 72°C for 5min

[0070] Combination 2 reaction program sets 2 annealing temperatures, namely 52°C and 54°C. Reaction program 1 is as follows: 94°C for 3 min, 94°C for 30 s, 52°C for 30 ...

Embodiment 3

[0080] Embodiment 3, routine PCR and double PCR method detect analog sample sensitivity

[0081] 1. Preparation of simulated samples: Take 10g of healthy Yali pollen and add it to 50ml of sterilized PBS solution, shake at 180rpm at a constant temperature, shake at room temperature for 1h, filter with sterilized filter paper in an ultra-clean workbench, take 7 sterilized 1.5ml centrifuge tubes, Add 1ml of filtrate to each centrifuge tube, centrifuge at 13000rpm for 10min, discard the supernatant, pour out the supernatant as much as possible, add 900μL sterilized water to suspend, and finally add 100μL of 7 concentration gradient bacterial suspensions to each centrifuge tube, and vortex Mix well and set aside.

[0082] Sensitivity detection of simulated samples by two PCR methods According to the reaction system established above, 2 μL of simulated samples of the first 5 gradient concentrations were taken for sensitivity detection, and ddH 2 O is blank control, detected by 1% a...

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Abstract

The invention discloses a system for detecting pear branch blight germs. The system comprises a first primer pair, a second primer pair and a third primer pair, the first primer pair, the second primer pair and the third primer pair are firstly used for conventional PCR, and the first primer pair and the second primer pair are then used for dual PCR; the nucleotide sequence of the first primer pair is as shown in SEQ ID NO. 1; the nucleotide sequence of the second primer pair is as shown in SEQ ID NO. 2; and the nucleotide sequence of the third primer pair is as shown in SEQ ID NO.3. According to the system for detecting the pear branch blight germs, the high-sensitivity pear branch blight germs detection system is established by using two molecular methods of conventional PCR and duplex PCR, the detection result is sensitive and reliable, the sterile and safe commercial pear pollen is ensured, and the propagation and diffusion of the bergamot pear branch blight germs through the pollen are cut off.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a system for detecting pear pollen branch blight bacteria and a construction method thereof. Background technique [0002] Pear is a fruit-bearing crop strictly outcross-pollinated. In recent years, the prevention and control of branch blight of Korla fragrant pear (apple) in Xinjiang has been severe, the pollination of bees has been restricted, and the fragrant pear industry has been seriously threatened. UAV pollination is an efficient spray pollination technology widely promoted at present. However, due to the lack of pear pollen pathogen inspection technology, any pollination method has a safety hazard of spreading pathogens during pollination. [0003] In order to block the spread of pathogens, it is necessary to detect the pathogens in pear pollen, so as to remove the pollen carrying the pathogens. At present, there is a lack of reliable detection methods to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q1/6895C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 冯宏祖郝海婷王兰羊坚谢伟
Owner TARIM UNIV
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