Molecular biological method for rapidly detecting and identifying tomato spotted wilf virus and primer thereof

A technology of tomato spotted wilt virus and molecular biology, which is applied in the field of molecular biology detection and identification of plant viruses, can solve the problems of false positives, time-consuming, and labor-consuming, and achieve the elimination of false positives, wide detection range, and specificity strong effect

Inactive Publication Date: 2015-06-17
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The technical problem to be solved by the present invention is to overcome the existing RT-PCR detection technology based on gene and sequence sequence design of primers, the defects of false positives that may occur, and the time-consuming and labor-consuming of cloning, sequencing and sequence comparison, real-time fluorescence quantitative The defect of expensive PCR technology, the purpose is to provide a fast, accurate, reliable and specific molecular biology method for detection and identification of tomato spotted wilt virus and its special primers

Method used

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  • Molecular biological method for rapidly detecting and identifying tomato spotted wilf virus and primer thereof

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Embodiment 1

[0034] Example 1 The special primer L3 of the present invention for rapid detection and identification of tomato spotted wilt virus and its design and the molecular biology method for detecting and identifying tomato spotted wilt virus with the special primer L3

[0035] (1) Special primer L3 and its design and synthesis

[0036] The special primer L3 is based on the RNA L fragment in the genome of Tomato spotted wilt virus (TSWV) (accession number: NC-002052) reported on NCBI (National Center for Biological Information, USA) as a template, and was designed using Primer 3 It is numbered as special primer L3, which is composed of a front primer and a back primer. The base sequence of the front primer of the special primer L3 is shown in SEQ ID NO: 1, and the back primer of the special primer L3 is The base sequence is shown in SEQ ID NO: 2, and the target product fragment length of the special primer L3 is 605bp.

[0037] The above special primer L3 was synthesized by Beijing ...

Embodiment 2

[0054] Example 2 The special primer M3 for fast and accurate detection and identification of tomato spotted wilt virus of the present invention and its design and molecular biology method for detecting and identifying tomato spotted wilt virus with the special primer M3

[0055] (1) Special primer M3 and its design and synthesis

[0056] The special primer M3 is based on the RNA M fragment in the genome of the tomato spotted wilt virus (Tomato spotted wilt virus, TSWV, accession number NC_002050) reported on NCBI as a template, using the primer designed by Primer 5, and it is numbered as the special primer M3, It is composed of a front primer and a back primer. The base sequence of the front primer of the special primer M3 is shown in SEQ ID NO: 3, and the base sequence of the back primer of the special primer M3 is shown in SEQ ID NO: 4. It is shown that the target product fragment length of the special primer M3 is 794bp.

[0057] The above special primer M3 was synthesized...

Embodiment 3

[0075] Example 3 The special primer S4 of the present invention for rapid and accurate detection and identification of tomato spotted wilt virus and its design and the molecular biology method for detecting and identifying tomato spotted wilt virus with the special primer S4

[0076] (1) Special primer S4 and its design and synthesis

[0077] Special primer S4 is based on the RNA S fragment in the genome of tomato spotted wilt virus (TSWV, accession number NC_002051) downloaded from NCBI as a template, and the primer designed by Primer5 is numbered as special primer S4, It is composed of a front primer and a back primer. The base sequence of the front primer of the special primer S4 is shown in SEQ ID NO: 5, and the base sequence of the back primer of the special primer S4 is shown in SEQ ID NO: 6. It is shown that the target product fragment length of the special primer S4 is 1277bp.

[0078] The special primer S4 mentioned above was synthesized by Beijing Sanbo Polygala Bio...

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Abstract

The invention discloses a molecular biological method for rapidly detecting and identifying tomato spotted wilf virus and a primer thereof, and belongs to the technical field of molecular biological detection and identification of plant viruses. According to the method, three pairs of primers and alkali sequences of the primers are provided, RT-PCR amplification is carried out by using one pair of the primers, and a sequence of the obtained target product is analyzed through the comparison of NCBI BLAST; the sample is confirmed to be infected by the tomato spotted wilf virus when the consistency of the sequence of the obtained target product and the tomato spotted wilf virus reported by Genbank is more than 90 percent. If RT-PCR amplification is carried out by using three pairs of primers, the sample can be confirmed to be TSWV when the fragments with the lengths of 605bp, 794bp and 1277bp are amplified respectively, and cloning, sequencing and sequence comparison are not needed, so that the detection process is simplified, and false positive results in the detection process can be avoided.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection and identification of plant viruses, in particular to a method for molecular biology detection and identification of tomato spotted wilt virus and special primers thereof. Background technique [0002] Tomato spotted wilt virus (Tomato spotted wilt virus, TSWV) is an important virus harmful to agricultural production, belonging to Bunyaviridae (Bunyaviridae), tomato spotted wilt virus (Tospovirus). The virion is a spherical virus with a diameter of 80-120 nm. Its genome consists of three single-stranded RNA molecules: S RNA encodes nucleocapsid protein (NP) and nonstructural proteins (NSs); M RNA encodes glycoprotein precursor (G1G2) and nonstructural proteins (NSm); L RNA Encodes RNA-dependent RNA polymerase (RdRp). [0003] TSWV was first discovered in Australia, and is now widely distributed in many countries and regions such as Europe, North America, South America, Asia, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/686C12Q1/70
Inventor 刘雅婷徐烨李永忠孙文涛黄亚宁
Owner YUNNAN AGRICULTURAL UNIVERSITY
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