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

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...

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 reaction, which is very easy to cause aerosol Pollution

3. Fluorescence detection method. By adding fluorescent dye SYBR GREEN to the reaction system, the fluorescence changes in the system can be observed in real time through a fluorescent PCR instrument to generate a fluorescence curve, and the amplification results can be observed through the curve. This method is closed-tube detection to eliminate gas Sol pollution, but due to the characteristics of SYBR GREEN dye, it can combine with any double-stranded DNA and generate fluorescence, which is low in specificity and prone to false positives

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