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A kind of miRNA detection method and lateral flow chromatography test strip for miRNA detection

A detection method and test strip technology, applied in the field of miRNA detection, can solve the problems of poor protein stability, unable to meet the requirements of rapid and accurate detection, complicated operation and long time, etc., and achieve the effect of simple detection method.

Active Publication Date: 2022-07-29
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mass spectrometry method is accurate and quantifiable, but the operation is complicated and takes a long time. It can only be completed in a professional laboratory, and rapid detection cannot be achieved. Moreover, the stability of the protein is relatively poor. It is affected by high temperature, high pressure and various chemical reagents. The properties will change to varying degrees, so it is not suitable for the detection of deep-processed foods
Various PCR-based nucleic acid detection methods can only be qualitative but not quantitative. At the same time, due to the high sensitivity of PCR, it is impossible to distinguish between contamination and adulteration. In addition, compared with mass spectrometry methods, although the detection time of nucleic acid methods is greatly shortened , but it still takes 1-2 hours to complete
Therefore, the above method cannot meet the requirements of rapid and accurate on-site detection by market supervision, public security and other departments

Method used

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  • A kind of miRNA detection method and lateral flow chromatography test strip for miRNA detection
  • A kind of miRNA detection method and lateral flow chromatography test strip for miRNA detection
  • A kind of miRNA detection method and lateral flow chromatography test strip for miRNA detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. High-abundance, stable and species-specific lamb miRNA screening

[0048] Collect mutton samples of common varieties in my country and roasted mutton products, and use deep Small RNA sequencing technology to study the miRNA expression profiles of different samples. Screen for miRNA sequences that are consistently expressed in high abundance in different samples. At the same time, bioinformatics and other means were used to compare and analyze miRNA databases such as miRBase, miRecords, CoGeMiR, TargetScan, etc., and use software such as Blast to confirm species specificity. Finally, mutton was screened with high abundance, stability and species specificity. Sex miRNA sequence (5'-CACAAGUUAGGGUCUCAGGGA-3').

[0049] 2. S9.6 antibody-DNA-miRNA ELISA validation

[0050] Extract the miRNAs of different meats and their cooked food products, add the designed single-stranded DNA to form a DNA-miRNA hybrid complex, then add the ELISA plate that has been coated with...

Embodiment 2

[0060] 2.1. High-abundance, stable and species-specific duck miRNA screening

[0061] Duck meat samples of common varieties in my country and roasted duck meat products were collected, and deep Small RNA sequencing technology was used to study the miRNA expression profiles of different samples. Screen for miRNA sequences that are consistently expressed in high abundance in different samples. At the same time, bioinformatics and other means were used to compare and analyze miRNA databases such as miRBase, miRecords, CoGeMiR, TargetScan, etc., and use software such as Blast for species-specific confirmation. Finally, duck meat (5'-UUCUCUAGUGUAGUGGUUAUCACGUUC-3' ) high-abundance stable and species-specific miRNA sequences.

[0062] 2.2 S9.6 antibody-DNA-miRNA ELISA validation

[0063] The miRNAs of different meats and their cooked food products were extracted and added to the enzyme-linked plate with the designed single-stranded DNA immobilized to form a DNA-miRNA hybrid comple...

Embodiment 3

[0079] 3.1 Preparation of test strips for simultaneous detection of mutton and duck

[0080] Two different wavelengths of fluorescein Cy5 and Cy3 were labeled on the 5' end of the single-stranded DNA probes capable of recognizing sheep miRNA and duck miRNA, respectively, and both the 3' ends were labeled with biotin, and then immobilized on the binding pad. The T line of the test strip was immobilized with S9.6 antibody, and the C line was immobilized with streptavidin. Other preparation methods of test strips are the same as above.

[0081] 3.2. Preparation and detection of mixed meat samples

[0082] The homogenized mutton and duck were mixed at a weight ratio of 10:0, 7:3, 5:5, 3:7, and 0:10, respectively. miRNA is then extracted and added to the test strip sample pad for detection.

[0083] Experimental results:

[0084] 1. Mixed lamb and duck test results

[0085] The results are shown in Table 4. It can be seen from the results that when the sample is pure mutton, th...

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Abstract

The invention provides a miRNA detection method and a lateral flow chromatography test strip for detecting miRNA, and relates to the technical field of miRNA detection. The method includes: designing and synthesizing a complementary DNA single strand for the miRNA to be detected; based on the formation of a DNA-miRNA hybrid complex between the miRNA and the DNA single strand, the detection is performed by means of immunochromatography. The present invention provides a new method for detecting miRNA, which can realize the rapid detection of miRNA by adopting colloidal gold or fluorescent immunochromatography.

Description

technical field [0001] The invention relates to the technical field of miRNA detection, in particular to a miRNA detection method and a lateral flow chromatography test strip for detecting miRNA. Background technique [0002] Food adulteration has become one of the hot issues of current food safety. The "horse meat scandal" that broke out in Europe in 2013, in which horse meat was sold as beef, aroused widespread global concern about food fraud such as meat adulteration. In my country, the most prominent phenomenon of meat adulteration is the adulteration of mutton skewers. Among them, using duck meat to pretend to be mutton is the main means of adulteration of mutton skewers. [0003] At present, the adulteration identification of mutton skewers mainly needs to solve two problems: rapid and quantitative. At present, the identification methods of meat adulteration mainly include mass spectrometry methods based on species-specific peptides and DNA-based nucleic acid detecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/113G01N33/558G01N33/532G01N33/533
CPCC12Q1/6888G01N33/558G01N33/533G01N33/532C12Q2600/158C12Q2600/178C12Q2600/124
Inventor 陈爱亮王楠张娟谢瑞彬于文杰孙小云
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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