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Method for detecting bacterial fruit blotch and xanthomonas malvacearum of melons simultaneously and special kit thereof

A kind of technology of fruit spot bacteria and angular spot bacteria, which is applied in the biological field

Active Publication Date: 2018-06-29
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no high-throughput rapid detection method for melon seeds carrying acidophilus watermelon and Pseudomonas syringae pathogenic variants of cucumber horn spot

Method used

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  • Method for detecting bacterial fruit blotch and xanthomonas malvacearum of melons simultaneously and special kit thereof
  • Method for detecting bacterial fruit blotch and xanthomonas malvacearum of melons simultaneously and special kit thereof
  • Method for detecting bacterial fruit blotch and xanthomonas malvacearum of melons simultaneously and special kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Embodiment 1, the synthesis of primer

[0104] According to the nucleotide sequence of the par gene in the AC bacteria (shown as sequence 1 in the sequence listing), primers were artificially designed and primer pair AC-2 and primer pair AC-3 were synthesized. According to the nucleotide sequence of the gap1 gene in the PSL bacterium (shown in sequence 2 in the sequence table), artificially design and synthesize primers to PSL-1, primers to PSL-2, primers to PSL-3, primers to PSL-4, primers Pair PSL-5, primer pair PSL-6 and primer pair PSL-7. Primer design principles: the length of each primer is 20-26bp, and the size of the amplified fragment is about 100bp. TM The Tm value is adjusted in the software to be 55-62°C; the upstream primers of the primer pair (i.e., primer pair AC-2 and primer pair AC-3) designed according to the AC bacteria all contain linker sequence 1 (linker sequence 1 can bind to the FAM signal gene group, thus being Kraken TM software identificati...

Embodiment 2

[0110] Embodiment 2, the screening of the primer group that detects AC bacterium and PSL bacterium

[0111] 1. Screening of primers for the detection of AC bacteria

[0112] 1. Obtaining the template

[0113] Take the bacterial suspension of AC bacteria and add sterile water to obtain a concentration of 10 8 CFU / mL of AC bacterial suspension 6 (OD 600nm is 0.15).

[0114] Take the AC bacterial suspension 6, and carry out gradient dilution with sterile water to obtain a concentration of 10 3 CFU / mL AC bacterial suspension 1, the concentration is 10 4 CFU / mL AC bacterial suspension 2, the concentration is 10 5 CFU / mL AC bacterial suspension 3, the concentration is 10 6 CFU / mL of AC bacterial suspension 4 and concentration of 10 7 CFU / mL of AC bacterial suspension 5.

[0115] 2. Obtaining the reaction mixture

[0116] 12 μL of the upstream primer of primer pair AC-2 (at a concentration of 10 mM) and 15 μL of the downstream primer of primer pair AC-2 (at a concentration o...

Embodiment 3

[0148] The test of the primer group 1 and the primer group 2 of embodiment 3, embodiment 2 screening

[0149] 1. AC bacteria and PSL bacteria are mixed in different proportions

[0150] 1. Obtaining the template

[0151] Mix the AC bacterial suspension 6 and the PSL bacterial suspension (see the first column of Table 4 for the volume ratio) to obtain the mixed bacterial suspension, that is, the template.

[0152] 2. Obtaining the reaction mixture

[0153] Mix 12 µL of the upstream primer of primer pair AC-2 (at a concentration of 10 mM), 15 µL of the downstream primer of primer pair AC-2 (at a concentration of 10 mM), 12 µL of the upstream primer of primer pair PSL-7 (at a concentration of 10 mM), and 15 µL of the upstream primer of primer pair PSL-7 -7 downstream primers (10 mM concentration) were mixed to obtain primer mixture a.

[0154] Mix 12 µL of the upstream primer of primer pair AC-3 (at a concentration of 10 mM), 15 µL of the downstream primer of primer pair AC-3 (a...

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Abstract

The invention discloses a method for detecting bacterial fruit blotch and xanthomonas malvacearum of melons simultaneously and a special kit thereof. The method comprises the following steps: adoptinga sample to be detected as a template, adopting the kit to carry out PCR (Polymerase Chain Reaction) amplification and obtaining reaction products; reading fluorescence signals of the reaction products, then judging whether the sample to be detected contains the bacterial fruit blotch and / or the xanthomonas malvacearum of the melons. The kit comprises a primer pair combination. The nucleotide sequences of 4 primers forming the primer pair combination are shown as a sequence 3 to a sequence 6 in a sequence table. Proved by experiments, on an LGC molecular detection platform, by adoption of thekit provided by the invention, whether the bacterial fruit blotch and / or the xanthomonas malvacearum of the melons are / is carried on melon seeds can be detected rapidly by high flux, so that the method and the kit can be used as a prevention technology for preventing and controlling circulation of infected seeds, also can be used as a disease monitoring and early-warning technology for the fieldplanting stage and provide a fast and accurate detection method for agricultural production and port quarantine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for simultaneously detecting melon bacterial fruit spot fungus and angular spot fungus and a special kit thereof. Background technique [0002] Bacterial fruit blotch (BFB for short) of melons, also known as bacterial fruit rot, is a devastating disease in watermelon and melon production, and it is also a worldwide quarantine disease. BFB is a typical infectious disease caused by seed-carrying bacteria. It is caused by watermelon acidophilus (Acidovorax.citrulli). It has the characteristics of fast onset, fast transmission, strong explosive power, and severe damage. Various melon crops such as zucchini and cucumber pose a serious threat to agricultural production safety. Watermelon acidophilus not only harms the fruit, but also causes cotyledons and true leaves to rot and die at the seedling stage; it mainly survives on the surface of the endosperm under the see...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6855C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6855C12Q1/689C12Q2531/113C12Q2563/107
Inventor 徐秀兰芦钰温常龙杨静静张海军
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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