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NASBA detection method for identifying 1/2c serotype of listeria monocytogenes

A detection method, Listeria monocytogenes technology, applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve rapid detection, high sensitivity, and strong specificity

Active Publication Date: 2018-02-23
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, NASBA technology has been widely used in the detection of viruses and bacteria at home and abroad. In my country, NASBA has begun to be used as the national standard detection method for avian influenza virus. There are researches on NASBA detection methods. Most of them use electrophoresis to directly detect amplification results or combine ELISA. , while molecular beacon-based real-time NASBA methods for the detection of foodborne pathogens have rarely been reported.

Method used

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  • NASBA detection method for identifying 1/2c serotype of listeria monocytogenes
  • NASBA detection method for identifying 1/2c serotype of listeria monocytogenes
  • NASBA detection method for identifying 1/2c serotype of listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Specific verification of NASBA detection method

[0045]Using the RNA of 36 strains of Listeria monocytogenes (including 13 serotypes, including 4 strains of 1 / 2c serotype) and 24 strains of non-Listeria monocytogenes as templates, the specificity of the established NASBA reaction system was evaluated . Three parallel samples were set up for each sample, and NASBA water was used as a blank control. The reaction result is judged by the Ct value, greater than 35 is negative, and less than 35 is positive. The test results are shown in Table 2. The results showed that the 4 strains of Listeria monocytogenes serotype 1 / 2c could produce obvious fluorescent amplification signals, while the other non-1 / 2c serotype strains had no fluorescent signals, and the reaction was negative , indicating that the established real-time NASBA reaction has good specificity.

[0046] Table 2 Experimental strains and NASBA reaction detection results

[0047]

[0048]

example 2

[0049] The detection limit determination of example 2NASBA reaction system

[0050] (1) Determination of detection limit of total RNA

[0051] Take 1 mL of FSIS 57115 serotype 1 / 2c strain of Listeria monocytogenes cultured to the logarithmic phase to extract total RNA, measure the concentration of total RNA with a nucleic acid concentration analyzer, and then use RNase-free ddH 2 O Perform serial 10-fold serial dilutions for real-time NASBA reactions. The test results are shown in Table 3. When the concentration of Listeria monocytogenes is in the range of 13.8ng / μL~13.8fg / μL, the NASBA detection system can obtain credible positive results, and the fluorescent signal is still strong at this time. However, when the RNA concentration continued to decrease, an effective Ct value could not be obtained, and the result was judged to be negative. The detection limit of this reaction system for total RNA of Listeria monocytogenes serotype 1 / 2c was 13.8 fg / μL.

[0052] Table 3 RNA t...

example 3

[0058] Example 3NASBA reaction system detects live bacteria, heat-killed bacteria and VBNC bacteria in chicken samples

[0059] Cultivate Listeria monocytogenes 1 / 2c serotype strain FSIS 57115 to the logarithmic phase, take 1mL of the bacterial liquid, wash the bacterial cells twice with sterile saline and resuspend, adjust the cell concentration of the resuspended bacterial liquid to N× 10 7 CFU / mL, which is to be tested as a live bacteria sample. The heat-killed bacteria were prepared by heat inactivation in a boiling water bath for 15 minutes, and it was confirmed by plate counting that there were no colonies in the bacteria liquid sample. Viable but non-culturable (VBNC) bacteria were induced by low temperature.

[0060] Take 40g of frozen chicken, and use the national standard method to determine that the sample to be contaminated does not contain Listeria monocytogenes. Three different states of Listeria monocytogenes were used to artificially pollute chicken, includi...

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Abstract

The invention discloses an NASBA detection primer and an NASBA detection method for identifying the 1 / 2c serotype of listeria monocytogenes, belonging to the field of biotechnology. A forward primer sequence is 5'-GAAATACATTTTGGTCTAG-3', a reverse primer sequence is 5'-aattctaatacgactcactatagggATTACTGGGAATAACTTGA-3', a sequence of a molecular beacon probe for detecting the 1 / 2c serotype of listeria monocytogenes is 5'-JOE-CGATCGAGGAAATGCTTATGCAGATATTACGATCG-DABCYL-3'. The detection method comprises the following steps: extracting RNA of listeria monocytogenes, carrying out a real-time NASBA reaction, and carrying out qualitative analysis. The constructed primer and molecular beacon have high specificity and sensitivity for the identification of the 1 / 2c serotype strain of the listeria monocytogenes, for the detection method, the operation steps are simple, the detection time is short, real-time qualitative analysis can be carried out, and meanwhile, living bacteria and dead bacteria can be effectively distinguished.

Description

technical field [0001] The invention relates to a NASBA detection method for identifying Listeria monocytogenes 1 / 2c serotype, belonging to the field of biotechnology. Background technique [0002] Among the 13 serotypes of L. monocytogenes, serotypes 1 / 2a, 1 / 2b, 1 / 2c, and 4b were the most frequently isolated from food and clinical cases. According to reports, most of the food-borne Listeria monocytogenes distributed in China are 1 / 2c serotype. Compared with other serotypes, 1 / 2c serotype strains can survive in room temperature, nutrient-rich or low-nutrient, alkaline and other foods. Processing and sales live under common environmental conditions and produce substances that contaminate food, such as biofilms, which can easily lead to foodborne listeriosis. Therefore, it is very important to establish a fast, effective and accurate serotype identification method for Listeria monocytogenes. necessary. [0003] When detecting live bacterial infection, due to the stability of...

Claims

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Application Information

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IPC IPC(8): C12Q1/6865C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/6865C12Q1/689C12Q2531/143C12Q2563/107
Inventor 陆兆新陶婷婷别小妹吕凤霞张充
Owner NANJING AGRICULTURAL UNIVERSITY
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