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Preparation method of swine fever virus nucleic acid standard substance

A swine fever virus and standard substance technology, which is applied in the field of preparation of swine fever virus nucleic acid standard substances, can solve the problems of restricting the accuracy of detection results, pollution, RNase pollution, unreported, etc., and achieves increased reliability and safety. High performance and wide application range

Inactive Publication Date: 2010-12-08
CHINA INST OF VETERINARY DRUG CONTROL
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of technology detects swine fever virus by detecting virus-specific nucleic acid fragments, which has the advantages of high sensitivity and rapid detection; but it is also susceptible to kit quality, nucleic acid extraction, transcription efficiency, nucleic acid contamination, RNase contamination, etc. , the experimental results often have large errors due to the influence of reagent quality, personnel experience and ability, experimental facilities, and the environment, which seriously restricts the accuracy of the test results. Therefore, it is urgent to use standard materials as objective and fair scales for Quality control of detection kit products, reference of scientific research and laboratory diagnosis, and capability comparison between laboratories
However, due to the high quality requirements of CSFV nucleic acid standard substances, the preparation procedures, inspection and calibration methods are strict, and the relevant preparation procedures and calibration methods have not been reported so far.

Method used

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  • Preparation method of swine fever virus nucleic acid standard substance
  • Preparation method of swine fever virus nucleic acid standard substance
  • Preparation method of swine fever virus nucleic acid standard substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Select the 5' non-coding region (5'UTR) of the conserved region of the classical swine fever virus gene as the amplification target region.

[0057] 2. Design and synthesis of primers: A1: 5'-AGT ACA GGG TAG TCG TCA GTG GTT CG-3'; A2: 5'-CTG CTG TAC ATG GCA CAT GGA GTT G-3'; provided by Shanghai Yingjun Biotechnology Co., Ltd. Synthesized by the company, made up to 0.25μM with DEPC water, and stored at -20°C for future use.

[0058] 3. Amplification of the target sequence: Extract the total RNA of the virus liquid (tissue) with the TRIzol Reagent kit (purchased from Invitrogen); take 10 μl of RNA, add 2 μl of Oligo (dT) and 8 μl of reverse transcription solution (5×Buffer 4 μl, 2 μl DTT, 1 μl dNTP, 0.5 μl reverse transcriptase, 0.5 μl RNasin), reverse transcription at 42°C for 60 min and 70°C / 15 min to obtain total cDNA; take 10 μl cDNA, add 29 μl H 2 O, pre-denaturation at 95°C for 5min, add 11µl PCR solution (10×Buffer 5µl, A1 1µl, A2 1µl, dNTP 2µl, Taq enzyme 2µl...

Embodiment 2

[0063] RNA copy number determination

[0064] The prepared RNA sample was diluted 10 times and 100 times, and the absorbance value measurement results were: 10 times dilution, OD260=0.44406, the concentration was 35.5248 μg / ml; 100 times dilution, OD260=0.03481, the concentration was 3.4827 μg / ml; Calculated from the nucleic acid sequence, the molecular weight of the single-stranded template is MW=669.40kDa; after calculation, the copy number of RNA molecules per tube is: 3.11×10 12 Copies / 20 μl.

Embodiment 3

[0066] Inspection and determination of standard substance of classical swine fever virus nucleic acid

[0067] 1. Uniformity test: 10 samples were randomly selected from the samples, and the RNA copy number per unit volume of each sample was detected. The results are shown in Table 1. After statistical analysis, there was no significant difference among the samples, indicating that the batch of samples had good uniformity.

[0068] Table 1 RNA sample copy number (×10 12 copy / 20μl)

[0069]

[0070] 2. Stability test: Samples were randomly selected every other week for testing, and the test results after 11 weeks were not significantly different from the test results in the first month, indicating that the sample has good stability. It is speculated that its stable storage period is 5 years above.

[0071] 3. Collaborative calibration: Distribute the samples to relevant domestic institutions, and let each unit detect the RNA copy number per unit volume of the sample recei...

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Abstract

The invention relates to a preparation method of a swine fever virus nucleic acid standard substance. The swine fever virus nucleic acid standard substance is prepared through the following technical steps: (1) selecting a gene conservation area as an amplification target area; (2) designing and synthesizing a primer; (3) carrying out sequence amplification; (4) carrying out cloning and identification; (5) carrying out in vitro transcription; (6) sub-packaging; (7) determining RNA copy number; (8) inspecting; (9) carrying out collaborative calibration; and (10) setting value. The swine fever virus nucleic acid standard substance prepared by utilizing the preparation method has no infection, a wide range of applications, good uniformity, high stability and accurate set value of the copy number, and can be used for quality control, contrast between scientific research and laboratory diagnosis and capacity contrast among laboratories of RT-PCR or fluorescent quantitative PCR diagnostic reagents.

Description

technical field [0001] The invention relates to a method for preparing a standard substance of classical swine fever virus nucleic acid, and belongs to the technical fields of standard substance technology and veterinary biological products. Background technique [0002] Classical Swine Fever (CSF) is a highly contagious and fatal swine infectious disease caused by Classical Swine Fever Virus (CSFV), which occurs in many countries and regions in the world. Industrial production has brought serious economic losses. The World Organization for Animal Health (OIE) lists swine fever as a statutory notifiable disease, and my country classifies it as a first-class animal infectious disease. [0003] With the development of molecular biology detection technology, RT-PCR, fluorescent quantitative PCR and other detection technologies have gradually been widely used in the detection of swine fever virus. This type of technology detects swine fever virus by detecting virus-specific nu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 戴志红关孚时王在时蒋卉李翠
Owner CHINA INST OF VETERINARY DRUG CONTROL
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