LAMP (loop-mediated isothermal amplification) detection primer group and kit for burkholderia gladioli and preparation method of freeze-dried microspheres

A technology of Holder's bacteria and detection kits, applied in biochemical equipment and methods, microbial determination/inspection, drying, etc., can solve the problems of low specificity and false positives, aerosol pollution, strong subjective factors, etc. , to achieve the effect of high specificity, easy to distinguish, and not easy to false positive

Pending Publication Date: 2022-05-24
河南冠宇仪器有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are mainly 1 methods for interpreting the results of the ring-mediated constant temperature amplification method: turbidity method, which can be used to observe whether there is white flocculent precipitate with the naked eye after the reaction, and determine the test result. The subjective factors are strong and the sensitivity is low.
2. Chromogenic method, add nucleic acid dyes such as SYBR GREEN, HNB, calcein, etc. after the reaction, and display different colors under ultraviolet light or naked eyes. The result is easy to judge, but it needs to be opened after the reactio

Method used

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  • LAMP (loop-mediated isothermal amplification) detection primer group and kit for burkholderia gladioli and preparation method of freeze-dried microspheres
  • LAMP (loop-mediated isothermal amplification) detection primer group and kit for burkholderia gladioli and preparation method of freeze-dried microspheres
  • LAMP (loop-mediated isothermal amplification) detection primer group and kit for burkholderia gladioli and preparation method of freeze-dried microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1LAMP primer design

[0046] The conserved sequence 16S-23S rRNA unique to Burkholderia gladiolus was screened out by reviewing literature and comparison, and taking this as the target gene, using the online primer design software (https: / / primerexplorer.jp / lamp3.0.0 / index .html) Design LAMP primers, and after analysis and optimization, the final primer sequences are shown in Table 1:

[0047] Table 1. LAMP primer sequences for detection of Burkholderia gladiolus

[0048]

Embodiment 2

[0050] The present embodiment provides a freeze-dried microsphere for detecting Burkholderia gladiolus, and its preparation method includes:

[0051] (1) Preparation of lyophilization protective agent: 70% trehalose, 8% threonine, and 25% PEG20000 are mixed according to a volume ratio of 2:3:6 to obtain a lyophilized protective agent.

[0052] (2) Construct an amplification reaction system:

[0053] The total volume of the LAMP detection and amplification system constructed in this example is 15 μL, and the detection primer set shown in Table 2 is mixed with the LAMP reaction base liquid shown in Table 2 to obtain an amplification reaction system.

[0054] Table 2. Amplification reaction system

[0055]

[0056]

[0057] Among them, the buffer is 100mmKCl, 100mm (NH 4 ) 2 SO 4 , 100mmMgSO 4 , 1% triton-100;

[0058] The developer is phenol red, cresol or neutral red, preferably phenol red.

[0059] The pH adjusting agent is Tris-HCl or NaOH, preferably, the pH adj...

Embodiment 3

[0061] Example 3 Establishment of LAMP detection reaction system

[0062] The total volume of the LAMP detection reaction system constructed in this example is 26 μL, which specifically includes:

[0063] The freeze-dried microspheres prepared in Example 2 were added to 26 μL of water for reconstitution, and the template DNA (2 μL) extracted from the sample to be tested was added to the reconstituted reaction system, and the reaction was carried out at 60 to 65 ° C for 50 to 70 min, LAMP amplification was performed; in this example, the amplification was performed at 63° C. for 60 min.

[0064] Result judgment:

[0065] (1) Visual method: directly observe the color change to determine the LAMP amplification and the presence or absence of Burkholderia gladiolus in the sample, and then determine whether the test sample contains oryzae acid toxin. When effective amplification occurs in the reaction system, the reaction system changes from red to bright yellow, and when no effec...

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Abstract

The invention discloses a burkholderia gladioli LAMP (loop-mediated isothermal amplification) detection primer group, a kit and a preparation method of freeze-dried microspheres, and belongs to the field of nucleic acid detection. The detection primer group is designed aiming at 16S-23S rRNA conserved sequences of burkholderia gladioli, and comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB. The preparation method of the freeze-dried microspheres containing the detection primer group comprises the following steps: mixing the detection primer group with an LAMP reaction base solution to obtain an amplification reaction system; and mixing the amplification reaction system with a freeze-drying protective agent, dropwise adding into liquid nitrogen, quickly freezing into balls, and freeze-drying. According to the detection method, whether the burkholderia gladioli exists or not can be simply and efficiently detected by utilizing the freeze-dried microspheres, so that whether a detection sample contains the rice yeast acid toxin or not is judged, the specificity is high, and false positive does not occur easily.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a LAMP detection primer set of Burkholderia gladiolus, a kit and a preparation method of freeze-dried microspheres. Background technique [0002] Burkholderia gladiolus is a gram-negative brevis bacilli with blunt ends, no dentate spores, and flagella, which are widely distributed in nature. Burkholderia gladiolus is facultatively anaerobic and easily grows on food surfaces. Burkholderia gladiolus can produce rice yeast acid and cause food poisoning. Burkholderia gladiolus is not resistant to high temperature and is easily killed by high temperature, but the "rice yeast acid" it produces The toxin is heat-resistant and cannot be destroyed by ordinary cooking methods. Due to the large scale of food poisoning of Burkholderia gladiolus, the high morbidity and mortality rate, and the lack of specific detoxification measures at present. Therefore, the sensitivity and rapidit...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/04C12N15/11F26B5/06
CPCC12Q1/6844C12Q1/689F26B5/06C12Q2531/119C12Q2537/1376C12Q2563/103Y02A50/30
Inventor 许世伟许远邵俊影吴镇宇王欣林圣博
Owner 河南冠宇仪器有限公司
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