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Method for high-throughput detection of pathogenic bacteria through combination of multiple PCR (Polymerase Chain Reaction) and colloidal gold test strip

The technology of colloidal gold test paper and pathogenic bacteria is applied in the field of food safety, which can solve the problems of time-consuming and low sensitivity, and achieve the effects of simple, convenient and quick operation, high sensitivity and strong specificity.

Pending Publication Date: 2022-05-24
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current common PCR amplification technology usually reads the results by gel electrophoresis, which has the disadvantages of time-consuming and low sensitivity

Method used

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  • Method for high-throughput detection of pathogenic bacteria through combination of multiple PCR (Polymerase Chain Reaction) and colloidal gold test strip
  • Method for high-throughput detection of pathogenic bacteria through combination of multiple PCR (Polymerase Chain Reaction) and colloidal gold test strip
  • Method for high-throughput detection of pathogenic bacteria through combination of multiple PCR (Polymerase Chain Reaction) and colloidal gold test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] In this embodiment, the used nanomaterial colloidal gold is prepared through the following steps:

[0032] Soak a 250 mL conical flask, magnetic stir bar in sodium hydroxide solution (1 M) for at least 15 minutes, rinse with plenty of deionized water, and then rinse with milli Q ultrapure water. Put 99.9 mL milli Q ultrapure water into a conical flask, and add 100 µl of 0.1 M HAuCl 4 solution to make the final HAuCl 4 The concentration is 0.1mM, mix well, stir and heat to boiling with an intelligent magnetic heating stirrer, quickly add 1 mL of 1% sodium citrate solution, maintain heating for about 8 min, and cool at room temperature to obtain colloidal gold, which is stored at 4°C for later use.

[0033] In the present embodiment, the test strips used are prepared by the following methods:

[0034] The test strip includes a bottom plate, a sample pad, a nitrocellulose membrane and an absorbent pad attached to the bottom plate in sequence; the bottom plate (D70019), t...

Embodiment 2

[0043] like figure 1 As shown in the middle (B and C), when there are three target bacteria in the sample at the same time, the PCR product can amplify the product (the PCR product contains thiol, biotin, fluorescein and digoxigenin at the same time), and the detection line 1 is packaged The coated anti-biotin antibody simultaneously captures AuNPs by capturing the biotin on the PCR product, that is, a red band appears on the detection line 1; the anti-fluorescein antibody coated on the detection line 2 simultaneously captures the AuNPs by capturing the fluorescein on the PCR product, That is, a red band appears on detection line 2; the anti-digoxigenin antibody coated on detection line 3 captures AuNPs by capturing digoxigenin on the PCR product, that is, a red band appears on detection line 3. When there is no target bacteria in the sample, PCR cannot amplify products with sulfhydryl, biotin, fluorescein and digoxigenin, and the antibody coated on the detection line cannot ...

Embodiment 3

[0049] Specific detection: take the concentration as 10 8 CFU / mL of Listeria monocytogenes ( L. monocytogenes ), Escherichia coli O157:H7 ( E. coli O157:H7), Salmonella typhimurium ( S. typhimurium ), Staphylococcus aureus ( S. aureus ), Vibrio parahaemolyticus ( V. parahemolyticus ) and Pseudomonas aeruginosa ( P. aeruginosa ) was detected with reference to Example 2 above, and the detection specificity of the method of the present invention was evaluated by gel electrophoresis.

[0050] Image 6 is the detection specificity map of the present invention for detecting pure cultured bacterial liquid; agarose gel electrophoresis ( Image 6 Middle B) The results show that when there is only one target bacteria, one target band appears; when there are two target bacteria, two target bands appear; when there are three target bacteria, three target bands appear, but there are no target bacteria when there are no target bacteria. The destination band appears. The test s...

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Abstract

The method comprises the following steps: respectively designing a pair of upstream and downstream primers according to specific gene segments of three different pathogenic bacteria, modifying sulfydryl at the 5'end of the upstream primer, and respectively modifying biotin, fluorescein and digoxin at the 5 'end of the downstream primer; the method comprises the following steps: washing three pathogenic bacterium samples with PBS, re-suspending in the PBS, boiling for 8-15 minutes, cooling to room temperature, centrifuging, respectively taking supernatant DNA, and carrying out multiplex PCR amplification to obtain PCR products; and combining a PCR product with colloidal gold, closing, dropwise adding onto a test strip, and observing a result with naked eyes. According to the method, the sensitivity of detecting the food-borne pathogenic bacteria is improved by utilizing a PCR amplification technology, and the method has the advantages of high-throughput detection, high sensitivity, strong specificity, short time consumption and the like, and is suitable for detecting the pathogenic bacteria.

Description

technical field [0001] The invention belongs to the technical field of food safety, and relates to nanotechnology and biotechnology. Specifically, it particularly relates to a method for high-throughput detection of pathogenic bacteria in combination with multiplex PCR and colloidal gold test strips, which utilizes multiplex PCR amplification technology to improve the sensitivity of detection of food-borne pathogens. Background technique [0002] With the development of the food industry, food safety has become one of the focus issues of the food industry and all walks of life, among which the production of food-borne pathogens is an important cause of food safety problems. Foods caused by Listeria monocytogenes ( L. monocytogenes ), Escherichia coli O157:H7 ( E.coli O157:H7) and Salmonella typhimurium ( S. typhimurium ) and other microorganisms caused a significant increase in the incidence of diseases. The key to the detection technology of foodborne pathogens is to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11G01N33/543G01N33/52G01N33/58G01N33/68C12R1/01C12R1/42C12R1/19C12R1/445C12R1/63C12R1/385
CPCC12Q1/689C12Q1/686G01N33/54313G01N33/52G01N33/582G01N33/68C12Q2600/16C12Q2537/143C12Q2563/107C12Q2527/125C12Q2565/401Y02A50/30
Inventor 白艳红杜娟赵电波刘佳蕾刘楷李宗双栗俊广相启森刘骁
Owner ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY