Preparation method of tomato spotted wilt virus nucleic acid standard substance

A standard substance and virus technology, which is applied in the field of preparation of tomato spotted wilt virus nucleic acid standard substance, can solve the problems of high cost, long transportation period, difficult virus positive quality control, etc. The effect of positive rate

Pending Publication Date: 2021-03-26
昆明海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These standard products are the control of serological methods, local multiplication and storage are difficult, there are problems such as long transportation cycle, high cost, difficult storage, etc. In addition, the import procedures of quarantine plant viruses are cumbersome, so the positive nature of the virus is obtained and collected. very difficult to control

Method used

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  • Preparation method of tomato spotted wilt virus nucleic acid standard substance
  • Preparation method of tomato spotted wilt virus nucleic acid standard substance
  • Preparation method of tomato spotted wilt virus nucleic acid standard substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment one, the fusion of TSWV viral coat protein and replicase gene

[0030] The specific region of the selected replicase gene is amplified with specific primers,

[0031] RdRp-F: 5'-GCAACTAACGCCACACCTGAC-3' and

[0032] RdRp-R: 5'-GGTCTCGTAACAAGTGAATCAATTATTTCTTTC-3'; to obtain the replicase gene fragment, press figure 1 As shown in the vector structure, the PCR product was cloned into the vector through SacII and SpeI, and the expression vector for in vitro transcription was obtained by transforming into Escherichia coli.

Embodiment 2

[0033] Embodiment 2, the preparation of fusion protein

[0034] The obtained plasmid DNA was digested with BsaI and linearized, and the RNA sequence was obtained by reverse transcription with the Promega reverse transcription kit. The sequence is shown as SEQ ID No.2 in the sequence listing.

Embodiment 3

[0035] Embodiment three, the detection of standard substance

[0036] The standard substance obtained in Example 2 was measured by an ultraviolet spectrophotometer, and the virus value of the standard substance was converted according to the OD value, and the initial concentration of virus was measured to the power of 80,

[0037] It is proved that the standard substance can be used for quantitative analysis of TSWV.

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Abstract

The invention discloses a preparation method of a tomato spotted wilt virus nucleic acid standard substance. The virus nucleic acid standard substance is prepared by the following steps of selecting agene conserved region as an amplification target region; designing primers and synthesizing; carrying out sequence amplification; constructing an expression vector; carrying out in-vitro transcription; measuring the concentration and diluting; performing split charging; and carrying out detection quantification. The standard substance prepared by the method has the characteristics of stability, no biological infectivity, wide application range and the like, and can be used for detecting the tomato spotted wilt virus by utilizing reverse transcription PCR and fluorescent quantitative PCR.

Description

technical field [0001] The present application relates to the field of nucleic acid standard substances, in particular, the present invention relates to a preparation method of tomato spotted wilt virus nucleic acid standard substances. Background technique [0002] Molecular biology detection is an essential part of virus detection, but the positive quality control of the detection is difficult to obtain, which is extremely unfavorable to the verification of the method and the development of the detection. At present, the standard samples of plant viruses are mainly obtained by purchasing from international authoritative or famous biological standard centers; contacting laboratories that publish research literature in research journals; purchasing positive controls equipped with commercial kits. These standard products are the control of serological methods, local multiplication and storage are difficult, there are problems such as long transportation cycle, high cost, diff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/70C12N15/10
CPCC07K14/005C12N15/70C12N2760/12022
Inventor 李旻
Owner 昆明海关技术中心
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