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Method for detecting Listeria monocytogenes by multiple cross amplification coupled with gold nanoparticle-based biosensor

A Listeria monocytogenes and biosensing technology, applied in the field of microbiology, can solve the problems of complex operation steps and dependence on reagents, and achieve the effect of fast detection speed and excellent detection sensitivity

Active Publication Date: 2017-03-29
ICDC CHINA CDC
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  • Summary
  • 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 Listeria monocytogenes by multiple cross amplification coupled with gold nanoparticle-based biosensor
  • Method for detecting Listeria monocytogenes by multiple cross amplification coupled with gold nanoparticle-based biosensor
  • Method for detecting Listeria monocytogenes by multiple cross amplification coupled with gold nanoparticle-based biosensor

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Experimental program
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Effect test

Embodiment 1

[0052] The feasibility of embodiment 1.MCDA-LFB amplification

[0053] 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, the concentration of amplification primers R1, R2, D1 and D2 is 30pmol, The concentration of amplification primers C1* and C2 is 20pmol, 10mM Betain, 6mM MgSO 4 , 1 mM dNTP, 12.5 μL of 10×BstDNA polymerase buffer, 10 U of strand-displacing DNA polymerase, 1 μL of template, and add deionized water to 25 μL. The entire reaction was kept at 65°C for 1 hour, and the reaction was terminated at 85°C for 5 minutes.

[0054] After MCDA amplification, three detection methods are used to discriminate MCDA amplification. First, a visual dye (such as FD reagent, Lamp fluorescence visual reagent) is added to the reaction mixture, and the color of the positive reaction tube changes from light gray to green , the negative re...

Embodiment 2

[0058] Embodiment 2. measure the optimal reaction temperature of MCDA technology

[0059] Under standard reaction system conditions, a Listeria monocytogenes DNA template and corresponding MCDA primers designed for lmo0733 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 . 60-63°C is recommended as the optimal reaction temperature for MCDA primers. In this temperature range, the amplification speed is the fastest. In the follow-up verification of the present invention, 61° C. was selected as the constant temperature condition for MCDA amplification. Figure 4 It shows the temperature dynamic curve of MCDA primers designed for lmo0733 to detect Listeria monocytogenes.

Embodiment 3

[0060] Embodiment 3.MCDA-LFB detects the sensitivity of single target

[0061] After using serially diluted Listeria monocytogenes genomic DNA to carry out the standard MCDA amplification reaction, using LFB detection shows that: the detection range of MCDA-LFB is 10ng ~ 10fg, and LFB appears red lines in the TL and CL regions ( 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 show that the template amount of Listeria monocytogenes is 10ng, 10pg, 10fg, 1fg and 0.1fg, and Figure 5A6, A7, A8 show that Listeria eziri (10pg ), Salmonella template (10pg), blank control (1 microliter double distilled water).

[0062] In order to further verify the sensitivity of MCDA-LFB to detect L. monocytogenes, three other detection methods were used to discriminate ...

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Abstract

The invention discloses a method for detecting Listeria monocytogenes by a multiple cross amplification coupled with gold nanoparticle-based biosensor. According to the method, a 5' end of a cross primer CP1 or CP2 in multiple cross displacement amplification is marked with biotin; a 5' end of an amplification primer C1 or C2 is marked with hapten; and an amplification product for the listeria monocytogenes lmo0733 can be visually detected by a gold nanoparticle-based biosensor. The method is convenient, rapid, sensitive, specific and suitable for detection of various nucleotide fragments.

Description

technical field [0001] The invention discloses a detection method for Listeria monocytogenes, which belongs to the field of microbiology. Background technique [0002] Listeria monocytogenes (Listeria monocytogens, LM) is a Gram-positive short bacillus, motile, facultative intracellular parasites. Listeria monocytogenes widely exists in nature and is an important food-borne zoonotic pathogen. Listeria monocytogenes present in food is dangerous to human safety, and the bacteria can still grow in an environment of 4°C Breeding is one of the main pathogenic bacteria that threaten human health in refrigerated food. Listeria monocytogenes can cause serious infections in humans. Susceptible populations for listeriosis are mainly immunocompromised populations and immunocompromised populations. The former include the elderly, newborns, and pregnant women; the latter are cancer patients, AIDS patients, and organ transplant recipients. The main clinical symptoms of human listeriosi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6804C12Q1/689C12Q2531/119C12Q2563/137C12Q2563/155C12Q2563/131
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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