Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof

A Rift Valley fever virus and standard sample technology, applied in the field of molecular biology, can solve the problems of shortened inspection process time limit and difficulty in ensuring laboratory quality control, and achieve good uniformity, strong stability, and good purity

Inactive Publication Date: 2015-03-04
薛芳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the increase of import and export trade, the time limit of the inspection process is shortened, and it is difficult to ensure the quality control of the laboratory

Method used

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  • Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof
  • Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof
  • Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the preparation of standard sample

[0025] 1. Synthetic sequence: use the software Clone Manager 7.0 to download the publicly released sequence from GenBank, perform homology analysis and primer matching, and analyze and determine the fitted viral sequence: CCC CAA TAA AGT GAA AAT TCC TGA GAC ACA TG GCA TCA GGG CTC GGA AGC AAT GTA AGG GGC CTG TGT GGA CTT GTG CAA CAT CAG ATG ATG CAA GGA AGT GGA ACC AAG GCC ATT TTG TTA CAA AGT TTG CCC TCA TGC TAT GTG AGT TCA CCT CTC CCA AAT GGT GGC CAC TGA TCA TTA GGG GAT GTT CAA TGT TTA, the sequence synthesis is entrusted to the company; in order to facilitate the in vitro transcription of the viral nucleic acid sequence into RNA, a T7 promoter is added before each sequence, and the T7 promoter sequence is 20bp: TAA TAC GAC TCA CTA TAG GG;

[0026] 2. Clone the synthetic sequence bluntly into the pUC19 plasmid and identify it by enzyme digestion and gel purification: clone the sequence into the pUC19 plasmid, analyze th...

Embodiment 2

[0048] Embodiment 2: the homogeneity inspection of standard sample

[0049] Samples were randomly selected for homogeneity analysis according to the random sampling method:

[0050] 1. Uniformity in bottle

[0051] Randomly select 1 bottle, add 200 μL deionized water, take three parts, each part is 50 μL, numbered 101-103, each part is measured three times, the results are shown in Table 2; the standard deviation of the quality of the sample estimate (STDEV) = 0.001, Purity sample estimation standard deviation (STDEV) = 0.002, three sets of quality and purity data were analyzed by variance, there was no significant difference in quality and purity, which met the uniformity requirements,

[0052] Table 2 Experimental results of homogeneity in the bottle (mass unit: μg)

[0053]

[0054] 2. Uniformity between bottles

[0055] 20 bottles, numbered 201-220, were selected by simple random sampling method, and each bottle was tested 3 times. Measurement sequence: first time...

Embodiment 3

[0060] Embodiment 3: the stability check of standard sample

[0061] Samples were randomly selected for stability analysis according to the random sampling method:

[0062] 1. short-term stability

[0063] Freeze-dried samples were randomly selected and stored at -20°C, 0°C, 4°C, 25°C, and 37°C for 2 weeks (20110314-20110328). 8 bottles were placed at each temperature, a total of 40 bottles, numbered from 301-350, the quality and purity of each bottle were measured 3 times, and the integrity was observed by electrophoresis. Referring to the statistical analysis requirements of GB / T 15000.3-2008 on sample stability, SPSS 20.0 software was used to analyze the variance of the data, and the inspection level P <0.05 means significant difference;

[0064] The 5 sets of data (Table 4) were tested for homogeneity of variance, P Figure 5-7 ), while the 25°C and 37°C groups had deletions in the plasmid bands ( Figure 8-9 ), suggesting that storage at a temperature below 4°C fo...

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Abstract

The invention discloses a preparation method of a Rift Valley fever virus nucleic acid molecule characteristic standard sample, which comprises the following steps: synthesizing the sequence, cloning the vector, connecting the target segment and vector, converting the plasmid, extracting the recombinant plasmid, freeze-drying, preserving and the like to obtain the Rift Valley fever virus nucleic acid molecule characteristic standard sample. The standard sample prepared by the invention comprises virus characteristic sequence information and is suitable for the virus and content level in the analytical sample. The standard sample disclosed by the invention has the advantages of favorable uniformity, high stability and high purity, and can be stored for a long time. The preparation method is simple in process, can provide a standard sample for Rift Valley fever virus detection research, medical research and the like to implement comparison among different laboratory results, thereby ensuring the quality control on the laboratory. The preparation method provides a standard sample for quick accurate detection on Rift Valley fever virus by quarantine inspection mechanisms, import and export trade and other enterprises.

Description

technical field [0001] The invention relates to a molecular characteristic standard sample of Rift Valley fever virus nucleic acid and a preparation method thereof, belonging to the field of molecular biology. Background technique [0002] Rift Valley fever virus (RVFV) belongs to the genus Phleboviridae of the Bunyaviridae family, and is mainly transmitted by mosquitoes. The virus can cause an acute hemorrhagic zoonotic disease with high morbidity and mortality. After human infection with Rift Valley fever, the main manifestations are fever, fatigue, headache, muscle pain, joint pain, sometimes nausea, vomiting, and some eye diseases, meningitis or hemorrhagic fever. In the past 10 years, the disease has been more prevalent in Africa. In 2006-2007, 684 cases (155 deaths) were reported in Kenya, 114 cases (51 deaths) were reported in Somalia; 290 cases (117 deaths) were reported in Tanzania in 2007, Sudan 228 cases were reported; 418 cases (7 deaths) were reported in Madag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/63C12N15/66C12R1/93
CPCC12Q1/6806C12Q1/701C12Q2600/166
Inventor 薛芳李云峰孙时冯晓明
Owner 薛芳
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