Bombyx mori cytoplasmic polyhedrosis virus RT-LAMP detection primer group and detection kit thereof

A plastid polyhedrosis and kit technology, which can be used in DNA/RNA fragments, recombinant DNA technology, and microbial determination/inspection, etc., can solve problems such as complicated operation, no silkworm plastid polyhedrosis virus, and difficulty in popularization and application. , to achieve the effect of high detection sensitivity, easy to popularize and apply in a large range, and high sensitivity

Active Publication Date: 2013-11-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method is cumbersome to operate and requires special PCR equipment, so it is difficult to popularize and apply in production practice
At present, there is no relevant technical report on the kit for detecting silkworm plasmopolyhedrosis virus by using the LAMP method

Method used

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  • Bombyx mori cytoplasmic polyhedrosis virus RT-LAMP detection primer group and detection kit thereof
  • Bombyx mori cytoplasmic polyhedrosis virus RT-LAMP detection primer group and detection kit thereof
  • Bombyx mori cytoplasmic polyhedrosis virus RT-LAMP detection primer group and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] S1. Artificial propagation and purification of silkworm plasmopolyhedrosis virus

[0039] Biological material: Bombyx mori cytoplasmic polyhedrosis virus is subcultured by the Sericulture Biotechnology Laboratory of South China Agricultural University through conventional artificial mulberry leaves (only to illustrate the method of the present invention, and the silkworm cytoplasmic polyhedrosis virus used in other laboratory related experiments can also be used. virus); Proteinase K, β-mercaptoethanol, CTAB (cetyltrimethylammonium bromide), EDTA (disodium ethylenediaminetetraacetic acid), Triton X-100, isopropanol (analytical grade), NaOH , KCl , Na 2 HPO 4 12H 2 O, HCl (analytical pure), and glycerin were purchased from Beijing Dingguo Biotechnology Co., Ltd.; TRIZOL Reagent was purchased from Yingwei Chuangjin Biotechnology Co., Ltd. 1000×SYBR Green I chromogenic solution was purchased from Yingwei Chuangjin Biological Co., Ltd.; the constant temperature m...

Embodiment 2

[0064] Different concentrations of BmCPV genomic dsRNA were used to detect the sensitivity of RT-LAMP reaction.

[0065] S1. Artificial propagation and purification of silkworm plasmopolyhedrosis virus

[0066] The biological material, inoculation method, collection method and purification method are the same as in Example 1.

[0067] S2. Extraction of Genome dsRNA of Bombyx mori Plasmopolyhedrosis Virus

[0068] The method for extracting the genome dsRNA of the silkworm plasmopolyhedrosis virus is the same as in Example 1.

[0069] The BmCPV dsRNA with a measured concentration of 50 ng / μL was serially diluted 10 times. The operation steps of the 10-fold gradient dilution are as follows: in a good operating environment, take 8 PCR tubes, add 9 μL of sterilized deionized water to each PCR tube, and cover the lid to avoid cross-contamination of each gradient , and dilute one by one from high to low. Take 1 μL of the standard with an initial concentration of 50ng / μL and add i...

Embodiment 3

[0087] Bombyx mori plasmopolyhedrosis virus RT-LAMP detection kit assembly:

[0088] Pack Bst DNA polymerase, reaction solution, stabilization solution, chromogenic solution, positive control solution, 5pmol / μL outer primer CPVF2 / CPVB2, 40pmol / μL inner primer CPVFI2 / CPVBI2, reverse transcriptase, and DEPC water into 9 reagents respectively In the tube, the formula of the reaction solution is: each 1L reaction solution contains 35-45 mM Tris-HCl with pH 8.8, 15-25 mM potassium chloride, 15-20 mM magnesium sulfate, 15-20 mM ammonium sulfate, 0.15~0.25% Tween 20, 2.5~3.0 mM dNTPs and 1.5~1.8 M betaine. Described stabilizing liquid is glycerol; Described chromogenic liquid is SYBR Green I or EvaGreen; Described positive control liquid is the recombinant plasmid that contains the 5th fragment nucleotide partial sequence of Bombyx mori plasmopolyhedrosis virus genome, the preparation of recombinant plasmid The method refers to the conventional method in the field. The posit...

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Abstract

The invention discloses a bombyx mori cytoplasmic polyhedrosis virus RT-LAMP detection primer group and a detection kit thereof. The kit comprises the detection primer group, a BstDNA polymerase, a reaction solution, a stabilization solution, a coloring solution and a positive contrast solution, the detection primer group comprises CPVF2, CPVB2, CPVFI2 and CPVBI2 primers, and the nucleotide sequences of the CPVF2, CPVB2, CPVFI2 and CPVBI2 primers are represented by SEQIDNO:1-4 respectively. The accurate and rapid detection of the bombyx mori cytoplasmic polyhedrosis virus can be realized based on the kit. The kit has the advantages of high detection sensitivity, strong specificity, rapid and accurate detection, suitableness for the operation of a small amount of samples, and simple operation, provides a technical basis for the early-stage detection and prevision inspection of the bombyx mori midgut polyhedrosis in the production, and is of great practical significance.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis and identification, and in particular relates to a primer set and a detection kit for RT-LAMP detection of silkworm plasmotype polyhedrosis virus. Background technique [0002] Bombyx mori plasmopolyhedrosis is also called silkworm midgut imputosis, and the pathogen is silkworm plasmopolyhedrosis virus ( Bombyx mori cytoplasmic polyhedrosis virus , referred to as BmCPV), is the type species of the Reoviridae Pyropolyhedrosis virus genus. The BmCPV virion is icosahedral and has a unique single-layer capsid structure, and its genome is composed of 10 segments of double-stranded RNA. At present, it is divided into 9 strains according to the shape of its polyhedron and the part formed in the cell, and the full sequence of the dsRNA gene of the BmCPV-I strain has been determined. BmCPV infects silkworm midgut epithelial cells through feeding on silkworms, causing pathological changes in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 刘吉平杨吉龙
Owner SOUTH CHINA AGRI UNIV
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