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A primer pair, kit and detection method for detecting coral bleaching pathogen Vibrio alginolyticus xsbz03 and xsbz14

A technology of Vibrio alginolyticus and a detection method, applied in the biological field, can solve problems such as the collapse of the coral reef ecosystem structure, and achieve the effects of short time consumption, high accuracy and strong specificity

Active Publication Date: 2021-10-26
HAINAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, frequent outbreaks of coral diseases caused by bacterial pathogens have led to the continuous collapse of the coral reef ecosystem structure, and two-thirds of the coral reefs are now at risk of extinction

Method used

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  • A primer pair, kit and detection method for detecting coral bleaching pathogen Vibrio alginolyticus xsbz03 and xsbz14
  • A primer pair, kit and detection method for detecting coral bleaching pathogen Vibrio alginolyticus xsbz03 and xsbz14
  • A primer pair, kit and detection method for detecting coral bleaching pathogen Vibrio alginolyticus xsbz03 and xsbz14

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Experimental program
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Effect test

Embodiment 1

[0038] 1. Primer design

[0039] In order to distinguish coral pathogenic strains XSBZ03 and XSBZ14 from other closely related strains, thereby improving the accuracy of disease diagnosis, the applicant of the present invention screened the target gene detected by PCR in the present invention through bioinformatics analysis and a large number of experiments , and designed a pair of suitable primers according to the target gene. The GenBank accession number of the target gene is: MH702378, which is a sequence in the whole genome of Vibrio alginolyticus XSBZ14. The primers designed according to the sequence can amplify XSBZ03 and XSBZ14, However, it has no amplification effect on other bacterial strains mentioned in this application.

[0040] Primer F1 (SEQ ID NO: 1): 5'-GCGCACTCAAAAAAACCTATCAA-3',

[0041] Primer R1 (SEQ ID NO: 2): 5'-GCACCAGTCTCACCACGC-3'.

[0042] 2. Template preparation

[0043] Vibrios such as XSBZ03 and XSBZ14 were isolated and purified in TCBS medium, ...

Embodiment 2

[0052] Sensitivity test of detection kit

[0053] XSBZ03 genome detection limit ( figure 2 ): According to the method of Example 1, title 2, DNA was extracted, and its concentration was measured by a nucleic acid measuring instrument. The initial concentration of the XSBZ03 genome was 17 ng / μL, and ten-fold gradient dilution was performed with ultrapure water, and a total of 7 gradients were diluted, respectively. 1.7ng / μL (lane 1), 170pg / μL (lane 2), 17pg / μL (lane 3), 1.7pg / μL (lane 4), 170fg / μL (lane 5), 17fg / μL (lane 6), 1.7fg / μL (swimming lane 7), ultrapure water (swimming lane 8), DNA marker500 (swimming lane 9) The results showed that the detection limit of the primer for XSBZ03 genome was 170fg / μL.

[0054] XSBZ14 genome detection limit ( image 3 ): The initial concentration of XSBZ14 is 20ng / μL, making a ten-fold serial dilution with ultrapure water, and a total of 7 gradients are diluted, 2ng / μL (lane 1), 200pg / μL (lane 2), 20pg / μL (lane 3) , 2 pg / μL (lane 4), 20...

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Abstract

The invention relates to a primer pair, a kit and a detection method for detecting vibrio alginolyticus XSBZ03 and XSBZ14, the pathogens of coral bleaching. The primer pair of the present invention includes an upstream primer F1 and a downstream primer R1; the sequences of the upstream and downstream primers F1 and R1 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2. The kit of the present invention includes upstream primer F1, downstream primer R1, 2×PCR Master Mix, ultrapure water, positive control, and negative control. The kit of the present invention can quickly detect the coral bleaching pathogen Vibrio alginolyticus XSBZ03 and XSBZ14 at the same time, and has strong specificity, high sensitivity, accurate detection and short time-consuming, so the present invention can diagnose the coral bleaching disease caused by XSBZ03 and XSBZ14 The method has high accuracy, and the invention has guiding significance for assessing vibrio diseases of corals in sea areas of my country and formulating effective disease control measures.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer pair, a kit and a detection method for detecting the coral bleaching pathogen Vibrio alginolyticus, and more specifically, the present invention relates to a method for rapidly detecting the coral bleaching pathogen of the coral pathogenic strain at the same time Primer pair, kit and detection method of Vibrio alginolyticus XSBZ03 and XSBZ14. Background technique [0002] Coral reefs are a very special type of ecosystem in the ocean. They maintain high biodiversity and primary productivity. They are known as tropical rain forests in the ocean" and "oasis in the blue desert". They have always been important life forms. Support systems. However, the high frequency of coral diseases caused by bacterial pathogens has led to the continuous disintegration of the structure of coral reef ecosystems, and now two-thirds of coral reefs are at risk of extinction. [0003] The bleaching s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 谢珍玉杨思悦龙昊章翔符亚楠顾玲
Owner HAINAN UNIV
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