Method for detecting vibrio parahaemolyticus through multiple cross amplification in combination with gold-nano biosensing

A technology of biosensing and cross constant temperature amplification, applied in the field of detection of Vibrio parahaemolyticus, can solve the problems of relying on reagents and complicated operation steps, and achieve the effect of fast detection speed and excellent detection sensitivity

Active Publication Date: 2017-05-31
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these constant temperature technologies require multiple enzymes to work simultaneously to achieve nucleic acid amplification, relying on expensive reagents and complicated operating steps

Method used

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  • Method for detecting vibrio parahaemolyticus through multiple cross amplification in combination with gold-nano biosensing
  • Method for detecting vibrio parahaemolyticus through multiple cross amplification in combination with gold-nano biosensing
  • Method for detecting vibrio parahaemolyticus through multiple cross amplification in combination with gold-nano biosensing

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Experimental program
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Embodiment 1

[0060] The feasibility of embodiment 1.MCDA-LFB amplification

[0061]Standard MCDA reaction system: the concentration of cross primer CP1* and CP1 is 30pmol, the concentration of cross primer CP2 is 60pmol, the concentration of displacement primers F1 and F2 is 10pmol, and the concentration of amplification primers R1, R2, D1 and D2 is 30pmol, The concentration of amplification primers C1* and C2 is 20pmol, Betain 10mM, MgSO4 6mM, dNTP 1mM, 12.5μL 10×BstDNA polymerase buffer, 10U strand displacement DNA polymerase, 1μL template, add to deionized water to 25 μL. The whole reaction was kept at 65°C for 1 hour, and the reaction was terminated at 85°C for 5 minutes.

[0062] After MCDA amplification, three detection methods are used for MCDA amplification discrimination. First, visual dyes (such as HNB reagent, naphthalene hydroxyphenol blue visual reagent) are added to the reaction mixture, and the color of the positive reaction tube changes from purple to blue. The negative r...

Embodiment 2

[0066] Embodiment 2. measure the optimal reaction temperature of MCDA technology

[0067] Under standard reaction system conditions, the DNA template and corresponding MCDA primers designed for Vibrio parahaemolyticus were added, and the template concentration was 10 pg / μl. The reaction was carried out under constant temperature conditions (60-67°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see Figure 4 . 61-64°C ( Figure 4 B-E) are recommended as the optimal reaction temperature for MCDA primers. In the follow-up verification of the present invention, 62° C. was selected as the constant temperature condition for MCDA amplification. Figure 4 It represents the temperature dynamic curve of MCDA primers designed for the detection of Vibrio parahaemolyticus against the toxR gene sequence.

Embodiment 3

[0068] Embodiment 3.MCDA-LFB detects the sensitivity of single target

[0069] After the standard MCDA amplification reaction was performed using the serially diluted genomic DNA of Vibrio parahaemolyticus, the LFB detection showed that the detection range of MCDA-LFB was 10 ng-10 fg, and red lines appeared in the TL and CL regions of LFB ( Figure 5A ). When the amount of genomic template in the reaction system was reduced to below 10fg, the LFB only appeared a red line in the CL area, indicating a negative result (Figure 5A4-A5). Figure 5A Use LFB to visually read the MCDA amplification results; Figure 5A1 to A5 indicate that the template amounts of Vibrio parahaemolyticus are 10ng, 10pg, 10fg, 1fg, and 0.1fg, and A6, A7, and A8 indicate Enterococcus faecalis (10pg), Chi Shigella template (10pg), blank control (1 microliter double distilled water).

[0070] In order to further verify the sensitivity of MCDA-LFB in detecting Vibrio parahaemolyticus, three other detection m...

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Abstract

The invention discloses a method for detecting vibrio parahaemolyticus through multiple cross amplification in combination with gold-nano biosensing. According to the method, biotin is marked at 5' end of a cross primer CP1 or CP2 in multiple cross displacement amplification, a hapten is marked at 5' end of an amplification primer C1 or C2, and amplification primers for the toxR gene of the vibrio parahaemolyticus can be visually detected by a gold-nano biosensor. The method is convenient, fast, sensitive, specific and suitable for detecting various nucleotide fragments.

Description

technical field [0001] The invention discloses a method for detecting vibrio parahaemolyticus, which belongs to the field of microbiology. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a filamentous, arc-shaped or rod-shaped Gram-negative bacteria that does not form spores. The pathogen is an important pathogen of foodborne diseases and a halophilic bacterium widely present in estuarine, coastal and marine environments. Vibrio parahaemolyticus is often isolated from seafood and clinical specimens, causing seafood-derived enteric fever with clinical symptoms of fever and diarrhea. According to WHO statistics, Vibrio parahaemolyticus is considered to be the most important factor causing seafood-borne diseases. Vibrio parahaemolyticus food poisoning is caused by eating foods containing the bacteria, mainly from seafood, such as cuttlefish, sea fish, sea shrimp, sea crab, jellyfish, and preserved foods with high salt content, such as pickle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6804C12Q1/689C12Q2531/119C12Q2563/137C12Q2563/155C12Q2563/131Y02A50/30
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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