RPA primer, detection system and method for identifying swine-derived components

A pig-derived, systematic technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of insufficient accuracy and stability of test results, prone to false positives, not suitable for rapid Detection and other problems, to achieve the effect of rapid identification and detection, easy results, and lower detection costs

Active Publication Date: 2022-01-25
SOUTH CHINA AGRI UNIV
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Problems solved by technology

At present, DNA constant temperature amplification technology has been reported in the detection of animal-derived components. For example, Chinese patent CN104988233A discloses a LAMP detection method using mitochondrial DNA to identify dog ​​meat from beef and mutton. However, LAMP amplification technology is likely to cause crossover. Reaction and non-specific amplification, prone to false positives, resulting in insufficient accuracy and stability of test results
Chinese patent CN107385079A discloses a PCR and RPA amplification method for detecting cattle, sheep, chicken, duck and pork components, as well as a primer set and a kit. RPA (Recombinase Polymerase Amplification) constant temperature amplification technology is used for rapid detection, but it needs to be combined with Nucleic acid electrophoresis is used to achieve discrimination, the operation is complicated, and it is not suitable for on-site rapid detection

Method used

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  • RPA primer, detection system and method for identifying swine-derived components
  • RPA primer, detection system and method for identifying swine-derived components
  • RPA primer, detection system and method for identifying swine-derived components

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The design of embodiment 1RPA primer and the optimization of reaction

[0058] 1. Design and screening of porcine-specific constant temperature amplification primers (RPA primers)

[0059] The present invention downloads the mitochondrial DNA sequences of 9 different breeds of pigs (Sus scrofa) and 9 different animal species from the GenBank database, and the information of the species involved is shown in Table 1. Using the Cluster W method in the MegAlign software, the homology comparison analysis of the mitochondrial DNA of 9 breeds of pigs was carried out, and the homology comparison analysis was also carried out with the mitochondrial DNA sequences of other 9 different animal species.

[0060] Table 1 Species information involved in RPA primer design

[0061]

[0062] On the basis of the comparative analysis results, a region that is conserved within the mitochondrial DNA of pig species and that differs greatly from the mitochondrial DNA sequences of other nine...

Embodiment 2

[0074] Example 2 Construction and optimization of the RPA-CRISPR / Cas12a detection system

[0075] The present invention obtains a highly specific crRNA probe for the amplified target gene by designing and screening the amplified target region of the RPA primer described in Example 1, the sequence of which is 5'-UAAUUUCUACUAAGUGUAGAUUUGGCCUGUGUGUAUAAAUA-3'( SEQ ID NO.3); at the same time, a fluorescently labeled ssDNA-reporter was designed with the sequence 5' FAM-TTTTTT-3'BHQ (SEQ ID NO.4), and the RPA-CRISPR / Cas12a detection system was constructed . The ssDNA-reporter has a FAM reporter group and a BHQ quencher group.

[0076] Using the purified amplified target product as a template, the crRNA concentration, Cas12a enzyme concentration, ssDNA-reporter concentration and reaction time in the CRISPR / Cas12a reaction system were optimized. The specific optimization conditions are shown in Table 3:

[0077] table 3

[0078]

[0079] By reacting under different conditions one b...

Embodiment 3

[0087] Example 3 The specificity verification of the RPA-CRISPR / Cas12a detection system

[0088] In order to verify the specificity of the RPA-CRISPR / Cas12a detection system constructed in the present invention, the present invention extracted the DNA of cattle, pigs, chickens, ducks, rats, mice, rabbits, prawns and sheep respectively as detection objects, and the constructed The specificity of the RPA-CRISPR / Cas12a detection system was verified. The DNA extraction, RPA-CRISPR / Cas12a detection system and reaction conditions were the same as those in Example 1, and the fluorescence value was measured at the end point by a real-time fluorescent PCR instrument. The specificity verification results of the RPA-CRISPR / Cas12a detection system are shown in Figure 2. It can be seen that the RPA-CRISPR / Cas12a detection system only produced obvious green fluorescence visible to the naked eye for pig DNA samples, but for the other 8 species such as cows and chickens. None of the species ...

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Abstract

The invention discloses a RPA primer, detection system and method for identifying swine-derived components. The RPA primer and the crRNA probe are high in specificity, and an identification method constructed based on the RPA primer and the crRNA probe has dual specificity, does not perform non-specific identification on other species, and is accurate in detection result. Meanwhile, the swine-derived component identification method disclosed by the invention is simple and convenient to operate, short in required time, accurate in result and applicable to on-site rapid detection on the swine-derived components by front-line detection personnel. Besides, the detection method disclosed by the invention is combined with a CTAB method to extract sample nucleic acid, the detection limit of the swine-derived components doped in beef products can reach 0.001% (w / w), and the method is suitable for identifying and detecting the swine-derived components in various meat processed products, and is wide in application range and high in reliability.

Description

technical field [0001] The invention belongs to the technical field of food inspection. More specifically, it relates to an RPA primer and detection system and method for identifying pig-derived components. Background technique [0002] Traditional sensory identification methods have been unable to meet the identification needs of succulent varieties. [0003] At present, qualitative detection methods for animal species in meat products mainly focus on the detection of metabolites, nucleic acids and proteins. Among them, nucleic acid-based molecular biological detection methods have become a research hotspot in the identification of meat species due to their high sensitivity and specificity. Among all the detection techniques of animal-derived components, there are mainly detection techniques such as PCR-RFLP analysis, PCR-RAPD analysis, nucleic acid probe hybridization, and PCR. The testing standards for pig-derived components released in China also mainly focus on con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2527/101C12Q2563/107C12Q2521/507C12Q2521/119C12Q2521/327C12Q2527/125Y02A50/30
Inventor 雷红涛赵岗沈兴王锦
Owner SOUTH CHINA AGRI UNIV
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