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Nucleic acid, method and kit for multi-liquid phase gene chip for simultaneously detecting and identifying components of three major categories of poultry, fish and ruminants

A liquid-phase gene chip and synchronous detection technology, applied in the field of biotechnology safety detection, can solve the problems of increased detection cost, prolonged detection period, increased manpower, etc. Effect

Active Publication Date: 2019-04-12
TECH CENT OF GUANGZHOU CUSTOMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented method allows us to quickly identify different animal groups by measuring their size or coloration on microscopically small particles suspended within liquid media called droplets that contain DNA fragments from these animals. By comparing this measurement results against known standards, we may determine if there were any more than one type of bird being tested - either wild birds (piglets) or domesticated ones – without having them themselves undergo testing at all. Overall, our inventors aimed towards creating an efficient way to measure large numbers of target molecules accurately while reducing labor requirements and expenses associated with current analysis techniques.

Problems solved by technology

This patents describes two technical aspects involved in identifying animals' sources - wildlife and seafood, while minimizing costs associated with these activities by developing an effective and reliable testing system without requiring expensive laboratory facilities.

Method used

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  • Nucleic acid, method and kit for multi-liquid phase gene chip for simultaneously detecting and identifying components of three major categories of poultry, fish and ruminants
  • Nucleic acid, method and kit for multi-liquid phase gene chip for simultaneously detecting and identifying components of three major categories of poultry, fish and ruminants
  • Nucleic acid, method and kit for multi-liquid phase gene chip for simultaneously detecting and identifying components of three major categories of poultry, fish and ruminants

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Experimental program
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Effect test

Embodiment 1

[0051] Design and preparation of embodiment 1 primers and probes

[0052] Firstly, the specific target genes of poultry, fish and ruminants were screened, and the mitochondrial sequences of poultry, fish and ruminants were selected as target genes for PCR amplification through sequence retrieval and comparison. Download multiple mitochondrial genome sequences of representative species of poultry, fish and ruminants from GenBank, perform comparison analysis, select the conserved region sequences with good species specificity, and then compare the sequences between species. Finally, the 12s rRNA in the mitochondrial genome of birds, fish and ruminants was selected as the target gene for PCR amplification.

[0053] PCR amplification primers and hybridization probes in the liquid-phase gene chip reaction system are designed on the basis of the inventor’s many years of experience, with the assistance of Array Designer 4.0 and DNAMAN 7. The principle of primer design is to ensure th...

Embodiment 2 3

[0057] Example 2 Establishment and optimization of triple liquid-phase gene chip detection method

[0058] The detection process includes triple asymmetric PCR amplification and triple suspension microsphere hybridization detection. It is necessary to conduct research and a large number of experimental explorations on the amplification reaction system (the amount of various general primers and various reagents), the amplification reaction conditions (reaction temperature, time, the number of amplification cycles, etc.), as well as the hybridization reaction system and hybridization reaction conditions. Validation to establish optimal reaction conditions.

[0059] 1. In the triple asymmetric PCR amplification, the key factors such as the amount of three sets of primers, the annealing temperature and time of the PCR reaction, and the number of amplification cycles are mainly optimized. Through a large number of comparisons of the primer concentration gradient, reaction temperatu...

Embodiment 3 3

[0084] The specificity test of embodiment 3 triple liquid phase gene chip method

[0085] The genomic nucleic acid of 41 kinds of animal and plant samples was detected by the triple liquid-phase gene chip method provided in Example 2 of the present invention, including 7 kinds of ruminant and beef powder standards, 12 kinds of fish samples, 8 kinds of poultry samples and chicken Meal standards, samples of 11 other common edible and forage terrestrial and aquatic animal species, and plant protein powders (mainly soybean, wheat, and pea) were tested. The test results are detailed in Table 3 below.

[0086] Table 3 Triple liquid phase gene chip specific test results

[0087]

[0088]

[0089] The test results are consistent with the actual situation of species samples: 7 kinds of ruminants and beef powder standard products are all positive for ruminants, 12 kinds of fish samples are all positive for fish tests, and 8 kinds of poultry samples and chicken powder standard pro...

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Abstract

The invention provides a nucleic acid, method and kit for a multi-liquid phase gene chip for simultaneously detecting and identifying components of three major categories of poultry, fish and ruminants. The nucleic acid comprises universal upstream primers, universal downstream primers, and universal probes for each of the above three categories of species. The method comprises the following steps: 1) extracting an animal genomic nucleic acid from a sample; 2) performing triple PCR amplification on the nucleic acid to obtain an amplification product; 3) performing xMAP triple microsphere suspension hybridization on the amplification product, and detecting a hybridization reaction signal; and 4) according the detection signal, determining a detection result of the sample to be tested. The method of the invention realizes simultaneous detection and identification of components of three major species of poultry, fish and ruminants, avoids separate detection and identification of single species one by one for the same purpose, and significantly reduces the detection cost and the detection time; and the method can efficiently prevent missed inspection and provide technical support for preventing false fraud in food and feed doping, and has important practical value.

Description

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Claims

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Application Information

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Owner TECH CENT OF GUANGZHOU CUSTOMS
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