RPA primer and probe for detecting Listeria monocytogenes and detection method

A technology for mononucleosis and Listeria, which is applied in the field of molecular biology detection, can solve the problems of long detection time, long detection cycle, non-specific amplification, etc., and achieve the effect of being easy to promote and use, and exempting investment

Inactive Publication Date: 2018-05-15
SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the traditional culture method has low requirements for experimental equipment and strong operability, the detection cycle is long and the detection efficiency is low, which cannot meet the requirements of rapid detection of samples in the current detection work.
However, immunological detection methods have non-specific reactions, although they have high sensitivity, they lack specificity.
The disadv

Method used

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  • RPA primer and probe for detecting Listeria monocytogenes and detection method
  • RPA primer and probe for detecting Listeria monocytogenes and detection method
  • RPA primer and probe for detecting Listeria monocytogenes and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Selection of target genes and design and screening of RPA primers and probes

[0039] Use bioinformatics knowledge and related analysis software to analyze common virulence genes of Listeria monocytogenes, such as hly, inl, actA, etc., and compare these genes of Listeria monocytogenes with other microorganisms For comparison analysis, actA was used as the target gene to be selected, and sequence fragments with higher specificity were selected. According to the RPA design requirements for primers and probes, design specific primers and probes, and then compare the sequences of the primers and probes with the species with high homology of Listeria monocytogenes to select the specificity High primers and probes. Then compare and analyze the primers and probes to be selected in the same process, and select a combination with high specificity and high amplification efficiency. The sequences of the primers and probes obtained by screening are shown respectively (SEQ ID No.1...

Embodiment 2

[0045] Establishment of RPA method for Listeria monocytogenes

[0046]Inoculate Listeria monocytogenes CICC 21632 into the nutrient broth, place it on a shaker, and culture overnight according to the optimum growth temperature of each bacterium, draw 1mL of the culture solution and drop it into a 1.5mL centrifuge tube, 12 000r / Centrifuge for 2 min, discard the supernatant, add 500 μL sterile saline, suspend and mix well, centrifuge at 12 000 r / min for 1 min, discard the supernatant, add sterile saline repeatedly, centrifuge again, and discard the supernatant. Add 50 μL of normal saline, put in a water bath at 100°C for 10-15min, centrifuge at 12000r / min for 1min after cooling down to room temperature, and store the supernatant at -20°C for later use.

[0047] Using the DNA of Listeria monocytogenes CICC 21632 as a template, set a blank control at the same time, utilize the RPA primers and probes in Example 1 to carry out RPA amplification, and use a fluorescence collection de...

Embodiment 3

[0051] Specific test analysis of the established RPA method

[0052] The standard strains used in this example were all purchased by Guangzhou Huankai Microbiology Co., Ltd., and the specific information is shown in Table 1.

[0053] Table 1 Strains used for RPA specificity analysis

[0054]

[0055] The strain DNA in Table 1 was used as the RPA reaction template, and the RPA primers and probes in Example 1 were used for RPA amplification.

[0056] The RPA amplification system is as follows: add 29.5uL of rehydration buffer, 11.2uL of deionized water, 2.1uL of upstream and downstream primers, 0.6uL of probe, and 2uL of template to the RPA reaction tube containing lyophilized enzyme powder, and finally add Magnesium acetate solution 2.5uL.

[0057] RPA reaction conditions: Mix the above RPA reaction system well, and amplify at 39°C for 15 minutes.

[0058] Amplification results such as figure 2 and image 3 As shown, it can be known from the results that only the ampli...

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Abstract

The invention provides a RPA primer and a probe for detecting Listeria monocytogenes and a detection method. The RPA primer and the probe are designed based on a Listeria monocytogenes actA gene, andprimer sequences are shown in SEQ ID No.1, 2 and 3. The probe length is 45-52bp, a position where is 30bp apart from 5' terminal in the middle of the probe is modified by dSpacer, thymidine at doublesides of the dSpacer molecule are respectively replaced by a fluorescence group FAM and a quenching group BHQ1, a 3' terminal of the probe is modified by a blocking group C3Spacer, and the sequence isshown in SEQ ID No.4. Compared with the prior art, detection by the detection method can be completed within 15 minutes, in order to rapidly detect whether Listeria monocytogenes exists in a sample;the established RPA method has good specificity and high sensitivity, special apparatuses are not needed, and the method has important application values in the fields of disease monitoring, clinic diagnosis, foodstuff safety monitoring, and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a primer, a probe and a detection method for detecting Listeria monocytogenes. Background technique [0002] Listeria monocytogenes is one of the most important foodborne pathogens and a zoonotic pathogen. After infection, the main manifestations are sepsis, meningitis and mononucleosis. It exists widely in nature. Listeria monocytogenes in food is dangerous to human safety. The bacteria can still grow and reproduce in an environment of 4°C. It is one of the main pathogenic bacteria that threaten human health in refrigerated food. Therefore, It is of great significance to develop a rapid detection method for Listeria monocytogenes. [0003] The current detection methods for Listeria monocytogenes are traditional culture methods, immunological methods and PCR methods. Although the traditional culture method has low requirements on experimental equipment an...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 刘小青陈晶黄建飞陈国培严琼英肖承荣李日升蔡琳兰全学杨国武
Owner SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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