Primer for detecting metapneumovirus, metapneumovirus kit and preparation method thereof

An avian metapneumovirus and detection kit technology, applied in the biological field, can solve the problems of avian metapneumovirus disease detection, prevention and control restrictions, etc., and achieve a simple pretreatment process, less time-consuming, good repeatability and stability. Effect

Inactive Publication Date: 2021-04-30
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In terms of detection of avian metapneumovirus nucleic acid, the detection method used by the International Organization for Animal Health currently includes RT-PCR method, and the laboratory can also use real-time fluorescence quantitative reverse transcription PCR (realtime RT-PCR), fluorescent ring-mediated reverse transcription isothermal amplification Pathogen identification by RT-LAMP technology, the detection minimum nucleic acid concentration of these methods generally cannot be lower than 1800 copies / μl, which has great limitations on the detection, prevention and control of avian metapneumovirus disease

Method used

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  • Primer for detecting metapneumovirus, metapneumovirus kit and preparation method thereof
  • Primer for detecting metapneumovirus, metapneumovirus kit and preparation method thereof
  • Primer for detecting metapneumovirus, metapneumovirus kit and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] The preparation of embodiment 1 detection card

[0049] 1. Preparation of gold nanoparticles

[0050] (1) Treatment of the container used for nano-gold: boil 2M sulfuric acid for 3 times, place it at room temperature, and then boil 3 times with ultrapure water. Return to room temperature and set aside.

[0051] (2) Boil 155g±5g ultrapure water until boiling, stir and add 4.95ml of chloroauric acid (1μg / μl), stir well until boiling, then slowly add 6.3ml of 1% sodium citrate, at this time the solution turns purple, boil Afterwards, the solution was cooled to room temperature and stored at 4°C until use. The particle size of the colloidal gold particles in the prepared nano-gold solution is 10-40nm.

[0052] 2. Nanogold-labeled avidin

[0053] (1) Add 1ml nano-gold solution to 10μl 0.2M K 2 CO 3 , adjust the pH to 7.0.

[0054] (2) Add 40 μl of avidin with a concentration of 10 mg / ml into 1 ml of gold nanometer solution, and mix slowly.

[0055] (3) 10 μl of 20% B...

Embodiment 2

[0064] Example 2 Amplification and detection of viral nucleic acid

[0065] 1. Sample collection and nucleic acid extraction

[0066] (1) Sample collection and processing

[0067] Chicken throat test: Use a sterile cotton swab to collect chicken throat mucus, and soak the cotton swab in a pre-cooled PBS solution with a pH value of 7.0. A part of the mixture was taken out and centrifuged at 800 rpm / min at 4°C for 10 min, and the supernatant was kept for later use, and stored at ultra-low temperature.

[0068] Chicken tissue samples: collect chicken turbinates, lungs or trachea and grind them separately (the ratio of tissue samples to grinding solution is 1:4, and the grinding solution is pre-cooled PBS). After grinding, centrifuge the mixture at 800rpm / min at 4°C for 10 minutes, and keep the supernatant Stand-by, stored at ultra-low temperature.

[0069] Chicken blood sample: Collect chicken blood with a sterile syringe, place it at 37°C for 2 hours, then place it at 4°C for...

Embodiment 3

[0084] The selection of embodiment 3 nano-gold labeled avidin concentration

[0085] According to the method of Example 1, 30 μl, 40 μl, and 50 μl of avidin solutions with a concentration of 10 mg / ml were respectively labeled with gold nanoparticles, and detection cards were prepared to obtain detection cards with different concentrations of avidin. Then according to the method of Example 2, the gradient dilution (10 -1 -10 -10 ) of different concentrations of aMPV-N plasmids (the fragment of the N gene of avian metapneumovirus type C is constructed on the T vector to obtain the recombinant plasmid) after amplification, and then added to the above-mentioned detection cards with different concentrations of avidin for detection.

[0086] The results showed that the detection card made with 50 μl avidin detected 10 -6 Concentration plasmid produces barb phenomenon, can not react ( figure 2 ). The detection card made with 40μl avidin can detect 10 -8 Concentration plasmid ( ...

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Abstract

The invention provides a primer for detecting metapneumovirus. Nucleic acid detection of the metapneumovirus is carried out by specifically amplifying an N gene of C-type metapneumovirus. The invention further provides an metapneumovirus detection kit, visualization of a detection result is achieved through RTPCR amplification, nanogold tracing and double signal amplification, the kit is used for metapneumovirus detection, the detection lower limit can be greatly reduced, and meanwhile the specificity, sensitivity, accuracy and repeatability of virus detection are effectively improved. When the kit is used for detecting metapneumovirus, the pretreatment process of a sample is simple, consumed time is short, and a large number of samples can be detected simultaneously.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to nucleic acid detection and application of avian metapneumovirus. Background technique [0002] Avian metapneumovirus (aMPV) was first reported in South Africa in 1978. aMPV can cause acute, highly contagious upper respiratory tract infections and decreased egg production in poultry such as turkeys and chickens. The virus was first isolated and reported in my country in 1999, which confirmed the occurrence of aMPV in my country. In the process of epidemiological investigation on local breeder chicken farms in many provinces and regions of our country, it was found that the infection rate of aMPV can be as high as 100%. [0003] Avian metapneumovirus belongs to the family Paramyxoviridae and the genus Metapneumovirus in the subfamily Pneumoviridae. It is a non-segmented single-stranded negative-sense RNA virus with a length of about 14 kb. It consists of 8 genes and mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/6804C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q1/6804C12Q2563/137C12Q2563/155C12Q2565/625C12Q2563/131C12Q2531/113
Inventor 朱珊珊刘爵韦莉王菁侯磊全荣王丹姜海军石蕊寒宋江伟
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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