High-throughput micro-fluidic LAMP chip for detecting livestock respiratory tract pathogens and detection method

A technology of pathogenic bacteria and respiratory tract, applied in the field of high-throughput microfluidic LAMP chips, can solve the problems of difficult clinical diagnosis, difficult clinical diagnosis, low sensitivity, etc., and meet the requirements of rapid clinical detection, shortened amplification reaction time, and sensitive sex high effect

Pending Publication Date: 2021-03-23
GUANGXI VETERINARY RES INST
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  • Application Information

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Problems solved by technology

The main respiratory pathogens of domestic animals include Mannella hemolytica, Pasteurella multocida, Mycoplasma, Cryptobacter pyogenes, Klebsiella pneumoniae, etc. Clinical respiratory diseases often appear in the form of mixed infection. The etiological research It is becoming more and more specialized and complicated, which brings great difficulties to clinical diagnosis
The traditional diagnostic method is the method of bacterial culture, but it is time-consuming and has low sensitivity, which is difficult to meet the needs of clinical diagnosis. Therefore, it is extremely important to quickly detect pathogens of livestock respiratory diseases
[0004] The Chinese invention patent application with the publication number CN110016512A discloses a multiplex fluorescent quantitative PCR detection kit and method for simultaneously detecting three bovine respiratory pathogens, which are aimed at Mycobacterium bovis, Mycoplasma bovis, Klebsiella bovis, and for three bovine respiratory pathogens. The detection of respiratory pathogens has high accuracy, but the method is complicated
[0005] At present, there is no simple, rapid and accurate detection method and chip for respiratory pathogens that can be applied to any poultry

Method used

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  • High-throughput micro-fluidic LAMP chip for detecting livestock respiratory tract pathogens and detection method
  • High-throughput micro-fluidic LAMP chip for detecting livestock respiratory tract pathogens and detection method
  • High-throughput micro-fluidic LAMP chip for detecting livestock respiratory tract pathogens and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 preparation material

[0062] 1) Reagent

[0063] Bacterial Genomic DNA Extraction Kit was 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. Mannella hemolyticus, Pasteurella multocida, Mycoplasma, Cryptobacter pyogenes, and Klebsiella pneumoniae are preserved by our laboratory.

[0064] 2) Extraction of pathogen genome

[0065] Bacterial genome DNA extraction reagents were used to extract the DNA of Mannella hemolyticus, Pasteurella multocida, Mycoplasma, Cryptobacter pyogenes, and Klebsiella pneumoniae. DNA samples were stored at -20°C for future use.

[0066] 3) Preparation of LAMP chip

[0067] There are 11 sample detection wells in the A and B reaction areas on the left and right sides of the LAMP chip, and the volume of each well is 2 μL. After the optimized LAMP primers were mixed a...

Embodiment 2

[0068] Embodiment 2 Detection method of LAMP chip

[0069] 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

[0070] Example 3 LAMP chip specificity test

[0071] After mixing the DNA of Mannella hemolyticus, Pasteurella multocida, Mycoplasma, Cryptobacterium pyogenes, and Klebsiella pneumoniae with the LAMP amplification reagent, add it to the LAMP chip reaction tank, and amplify the microfluidic at a constant temperature. Amplify at 65°C for 50 min in the analyzer to determine the specificity of the LAMP chip.

[0072] result:

[0073] Using the DNA of Mannella hemolyticus, Pasteurella multocida, Mycoplasma, Cryptobacterium pyogenes, and Klebsiella pneumoniae as templates, the LAMP amplification reagent was used for detection, and the results were as follows: figure 2 shown. Depend on figure 2 It can be seen that the corresponding detection indicators of Mannella haemophilus, Pasteurella multocida, Mycoplasma, Cryptobacter pyogenes, and Klebsiella pneumoniae and the LAMP amplification results of the positive control reaction tank were positive, and the fluorescence curve had a ...

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Abstract

The invention discloses a high-throughput micro-fluidic LAMP chip for detecting livestock respiratory tract pathogens and a detection method, and belongs to the technical field of biochips. The LAMP chip comprises an LAMP amplification reagent, a DNA template, an LAMP quality control positive control, an LAMP quality control negative control, a detection chip, a chip probe and a microfluidic LAMPchip analyzer, wherein the detection chip is arranged in the isothermal amplification microfluidic LAMP chip analyzer; and the chip probe comprises nucleotide sequences as shown in SEQ ID NO: 1-23. The LAMP chip is used for detecting specific nucleic acid fragments of mannheimia haemolytica, pasteurella multocida, mycoplasma, arcanobacterium pyogenes and klebsiella pneumoniae. The LAMP chip provided by the invention can complete detection within 50 minutes, has a detection limit of 1.5x10<-2> ng/mu L, can monitor main livestock respiratory tract pathogens in real time, has the characteristicsof simple operation, rapid detection, high sensitivity and strong specificity, and brings convenience to detection of the livestock respiratory tract pathogens.

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 pathogenic bacteria in the respiratory tract of livestock. 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. It has strong specificity, high sensitivity, and responsiveness. With the advantages of short time and wide detection range, it can realize high-throughput and rapid detection of nucleic acids, which has an important impact on multidisciplinary basic research, especially in grassroots veterinary and animal husbandry fields where resources and technologies are relatively short. Diagnosis is of great importance. [0003] In recent years, with the intensive and large...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6837C12Q1/04C12N15/11C12R1/35C12R1/22C12R1/01
CPCC12Q1/689C12Q1/6844C12Q1/6837C12Q2531/119
Inventor 贺会利李军吴翠兰潘艳彭昊冯世文钟舒红介百飞胡帅谢永平庞彬辉马春霞李常挺
Owner GUANGXI VETERINARY RES INST
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