Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae on the basis of dual RAA-LFD technology

A technology for hemolytic Vibrio and Vibrio cholerae, applied in the field of molecular biology technology detection, to achieve the effects of simple result judgment, easy operation and high sensitivity

Pending Publication Date: 2021-12-03
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no dual RAA-LFD method has been developed for the simultaneous detection of V. parahaemolyticus and V. cholerae

Method used

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  • Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae on the basis of dual RAA-LFD technology
  • Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae on the basis of dual RAA-LFD technology
  • Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae on the basis of dual RAA-LFD technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 designs double RAA-LFD reaction primer

[0053] Download the specific gene (toxR gene) of Vibrio parahaemolyticus (toxR gene) and the specific gene (Ompw gene) sequence of Vibrio cholerae from GenBank as the target site of primer design, use DNAMAN to find its conserved sequence, use RAA primer design principle to use Primer Premier 5 designed primers, respectively labeled the 5' ends of primers toxR-F and toxR-R with biotin (Biotin) and carboxyfluorescein (6-FAM), and labeled the 5' ends of primers Ompw-F and Ompw-R Digoxin (DIG) and Biotin (Biotin). The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the primer sequences are shown in the table below:

[0054]

[0055] Double RAA-LFD detection was performed, and false positives were found.

[0056] The RAA reaction was performed according to the manual (RAA nucleic acid amplification kit, Jiangsu Qitian) with slight improvements. The reaction system (50μL) is as follows: ...

Embodiment 2

[0063] The screening of embodiment 2 double RAA-LFD reaction primer combination

[0064] In order to verify the amplification efficiency of the improved primers, the unimproved primers and the improved primers were combined in pairs, and the double RAA-LFD method was used to detect the best primer combination. The combinations are: toxR-F / R and Ompw-F / R, M-toxR-F / R and Ompw-F / R, toxR-F / R and M-Ompw-F / R, M-toxR-F / R and M-Ompw-F / R.

[0065] The RAA reaction was operated with Example 1 according to the instructions.

[0066] The result is as figure 2 As shown, after using the improved primers (toxR-F / R and M-Ompw-F / R, M-toxR-F / R and M-Ompw-F / R) to amplify, the test strips all showed Correct positive signal (negative control amplifies normally and does not affect positive amplification results). However, the brightness difference between the two T-lines after amplification using primers M-toxR-F / R and M-Ompw-F / R is relatively large. Therefore, primers toxR-F / R and M-Ompw-F / ...

Embodiment 3

[0068] The optimization of the double RAA-LFD reaction time of embodiment 3

[0069] The preparation of the system is the same as in Example 2, and the amplification time of RAA is optimized.

[0070] After incubating the reaction tubes at 37°C for 10 min, 15 min, 20 min, 25 min, and 30 min, 10 μL of the amplification product was taken for test strip detection. The result is as image 3 As shown, the brightness of the two detection lines of the test strip changes with the increase of the reaction time. As the reaction time increases, the brightness of the bands on the T1 detection line gradually increases. When the reaction time is 10 minutes, there is no band on the T1 detection line. When the reaction time is 20 minutes, the brightness of the bands on the T1 detection line is stable. . On the contrary, the brightness of the strips on the T2 detection line decreases with the increase of the reaction time, and the longer the reaction time, the darker the brightness of the s...

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Abstract

The invention discloses a specific primer combination, a kit and a method for simultaneously detecting vibrio parahaemolyticus and vibrio cholerae on the basis of a dual RAA-LFD technology, and belongs to the field of rapid detection of food-borne pathogenic bacteria. The primer is high in specificity, the vibrio parahaemolyticus and the vibrio cholerae can be accurately detected from other vibrios and other pathogenic bacteria, sensitivity is high, genome sensitivity reaches 1fg, and the sensitivity of pure bacterial liquid is 10<4> CFU/mL and 10<3> CFU/mL respectively, and the influence of dimer is reduced. Detection can be completed by reacting for 15 minutes at a relatively low reaction temperature (37 DEG C) and detecting for 5 minutes through a test strip, so that the problems that the detection period of the vibrio parahaemolyticus and the vibrio cholerae is long, dependence on instruments and equipment is high, difficulty for simultaneously detecting the vibrio parahaemolyticus and the vibrio cholerae is high and the like are solved. The invention has low requirements on equipment, and has good application prospects in field detection, aquaculture point detection and other on-site detection.

Description

technical field [0001] The invention belongs to the field of molecular biology technology detection, and in particular relates to an improved primer, a kit and a detection method for simultaneous detection of Vibrio parahaemolyticus and Vibrio cholerae based on double RAA-LFD technology. Background technique [0002] Vibrio parahaemolyticus and Vibrio cholerae are the main pathogenic bacteria in aquatic products. Vibrio parahaemolyticus is widely found in aquatic animals such as fish, shrimp, and shellfish. Since Fujino and others first isolated it from sardines in 1950, Vibrio parahaemolyticus has been considered to be the cause of global aquatic product poisoning. important reason. In China, food poisoning incidents caused by Vibrio parahaemolyticus rank first among diseases caused by food-borne pathogens. There are more than 200 serogroups of Vibrio cholerae, and Vibrio cholerae of O1 and O139 serotypes can cause cholera. Vibrio cholerae is especially prevalent in poor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6804C12N15/11C12R1/63
CPCC12Q1/689C12Q1/6844C12Q1/6804C12Q2600/16C12Q2521/507C12Q2537/143C12Q2565/625C12Q2563/131
Inventor 孙晓红李达容赵勇
Owner SHANGHAI OCEAN UNIV
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