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High-throughput micro-fluidic LAMP chip for detecting various original bacteria of goat epidemic diseases and detection method

A detection chip, high-throughput technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA / RNA fragments, etc., can solve the problem of inability to accurately detect goats, difficult to meet clinical diagnosis, difficult to meet existing needs, etc. problems, achieve the effects of shortening the amplification reaction time, rapid detection, and prevention of epidemics and outbreaks

Pending Publication Date: 2021-10-08
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disease pathogens of goats include Brucella, Peste des petits ruminants virus, mycoplasma, goat pox virus, sheep mouth disease virus, foot-and-mouth disease, E. Tends to specialization and complexity, causing great difficulties for clinical diagnosis
The traditional detection method is the method of pathogen isolation and culture, but it is time-consuming, low sensitivity, and difficult to meet the needs of clinical diagnosis. Therefore, it is extremely important to quickly detect the pathogens of major goat diseases
[0004] The Chinese invention patent application with the publication number CN111996287A discloses a visual detection kit for aphthus virus LAMP, which has a detection effect for aphthus virus, but the detection of pathogenic bacteria is single, and it cannot be comprehensively and accurately detected for goats, and it is difficult to meet the existing needs
The Chinese invention patent application with the publication number CN102367492A discloses a goat pox virus and sheep pox virus double PCR detection kit and detection method, which is aimed at the detection of goat pox virus and sheep pox virus dual virus, with high accuracy, but it cannot detect goat pox virus. conduct a comprehensive test
[0005] At present, there is no simple, fast, accurate and high-throughput detection method and chip for the main disease pathogens of goats

Method used

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  • High-throughput micro-fluidic LAMP chip for detecting various original bacteria of goat epidemic diseases and detection method
  • High-throughput micro-fluidic LAMP chip for detecting various original bacteria of goat epidemic diseases and detection method
  • High-throughput micro-fluidic LAMP chip for detecting various original bacteria of goat epidemic diseases and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 preparation material

[0064] 1) Reagent

[0065] Viral DNA / RNA Genome Extraction Kit and Bacterial Genome DNA Extraction Kit were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; The amplification reaction kit was purchased from Rongyan Biotechnology (China) Co., Ltd.; other chemical reagents were domestic analytical reagents. Brucella (BM) vaccine and Peste des petits ruminant virus (PPRV) vaccine were purchased from Chongqing Aolong Biological Products Co., Ltd. Mycoplasma ovis pneumoniae (MO), goat pox virus (GV) and oropharynx virus (ORFV) were purchased from our laboratory save.

[0066] 2) Extraction of pathogen genome

[0067] The RNA or DNA of BM, PPRV, MO, GV and ORFV were extracted by bacterial genomic DNA and viral DNA / RNA genome extraction kits, respectively. RNA is reverse transcribed into cDNA. DNA and cDNA samples were stored at -20°C for later use.

[0068] 3) Probe design

[0069] According to the pathogenic gene seq...

Embodiment 2

[0113] The detection method of embodiment 2LAMP chip

[0114] The LAMP reaction system is 13 μL of LAMP amplification reagent and 13 μL of DNA, totaling 26 μL. When testing samples, take out the LAMP chip and let it return to room temperature, use a pipette to draw 26 μL of the prepared nucleic acid amplification reaction system and inject it into the main channel of the microfluidic LAMP chip to fill the chip, and fix the chip after adding the sample on the On a low-speed centrifuge, centrifuge for 30s, and then place the chip in a constant temperature amplification microfluidic LAMP chip analyzer for detection. During the LAMP reaction, the fluorescence value in the reaction tank is collected in real time. The reaction tank that produces a typical fluorescence jump amplification curve is positive amplification, otherwise it is negative amplification.

Embodiment 3

[0115] Example 3 LAMP chip specificity test

[0116] Mix the DNA / cDNA of BM, PPRV, MO, GV and ORFV with the LAMP amplification reagent, add it into the LAMP chip reaction tank, amplify in the constant temperature amplification microfluidic analyzer at 65°C for 20-50min, and measure the LAMP chip specificity.

[0117] result:

[0118] Using the DNA / cDNA of BM, PPRV, MO, GV and ORFV as templates, using LAMP amplification reagents for detection, the results are as follows figure 2 shown. Depend on figure 2 It can be seen that the corresponding detection indicators of BM, PPRV, MO, GV and ORFV and the LAMP amplification results of the positive control reaction tank were positive, and the fluorescence curve had a typical "S" shape, while the negative control and blank control were negative, and the microfluidic The specific amplification results of the microfluidic chip were consistent with expectations, and there were no false positive results, indicating that the microfluid...

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Abstract

The invention discloses a high-throughput micro-fluidic LAMP chip for detecting various original bacteria of goat epidemic diseases and a detection method, and belongs to the technical field of biological chips. The LAMP chip comprises an LAMP amplification reagent, a DNA template, a quality control primer positive control PC, a quality control primer negative control NC and a chip probe of goat plague pathogenic bacteria; a detection chip is arranged in an isothermal amplification micro-fluidic LAMP chip analyzer; and the chip probe comprises nucleotide sequences as shown in SEQ ID NO1-27, and is used for detecting specific nucleic acid fragments of brucella melitensis, peste des petits ruminants virus, mycoplasma ovipneumoniae, goatpox viruses and orf virus. According to the invention, detection can be completed within 20-50 minutes; furthermore, the detection limit is 1.5*10<-2> ng / mu L; main pathogenic bacteria of goats can be monitored in real time; the method has the characteristics of simplicity in operation, rapidness in detection, high sensitivity and high specificity; and convenience is brought to detection of the main pathogenic bacteria of the goats.

Description

technical field [0001] The invention relates to the technical field of biochips, in particular to a high-throughput microfluidic LAMP chip and a detection method for detecting various original bacteria of goat blight. Background technique [0002] Microfluidic chip technology uses the principle of loop-mediated constant temperature amplification (LAMP) to integrate the sample detection process into a chip of several square centimeters, which can detect more than a dozen pathogens at the same time. Compared with the traditional PCR detection method, LAMP It has the advantages of low equipment requirements, strong specificity, high sensitivity, short reaction time, and wide detection range. [0003] With the intensive and large-scale development of the goat breeding industry, brucellosis, Peste des petits ruminants, mycoplasma pneumonia, goat pox, and sheep sores develop rapidly and are highly contagious, and are the most harmful and widespread to the breeding industry. Impor...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/70C12Q1/6837C12Q1/6844C12Q1/04C12N15/11C12R1/01C12R1/35
CPCC12Q1/689C12Q1/701C12Q1/6837C12Q1/6844C12Q2531/119C12Q2565/629
Inventor 潘艳贺会利冯世文李军彭昊胡帅谢永平马春霞钟舒红
Owner GUANGXI VETERINARY RES INST
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