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Fluorescent quantitative PCR primer group and kit for detecting European eel circovirus

A circovirus, fluorescence quantitative technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of lack of molecular biology diagnosis of European eel circovirus, and achieve repeatability The effect of high performance, convenient operation and simple conditions

Pending Publication Date: 2022-05-06
广州双螺旋基因技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of fluorescent quantitative PCR primer group and kit for detecting European eel circovirus, to solve the basis of the molecular biology diagnosis that lacks European eel circovirus in the prior art

Method used

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  • Fluorescent quantitative PCR primer group and kit for detecting European eel circovirus
  • Fluorescent quantitative PCR primer group and kit for detecting European eel circovirus
  • Fluorescent quantitative PCR primer group and kit for detecting European eel circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Effect of primer set on detection of European eel virus fluorescent quantitative PCR

[0032] 1. Design primers

[0033] Primers were designed with the specific target gene of European eel circovirus (the accession number of Eecv sequence in GenBank is KU951579.1), and two sets of primers were designed with primer design software PrimerExpress3.0.1, including forward primer F, reverse primer R, fluorescent probe Needle Q was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0034] European eel circovirus primer set 1:

[0035] Forward primer F: 5'GCAATGCCGCGGTGAA 3' (SEQ ID NO: 1);

[0036] Reverse primer R: 5'TGTGACGGGCGGTGTGT 3' (SEQ ID NO: 2);

[0037] Fluorescent probe Q: 5'CGTTCTCGGGTCTTG 3' (SEQ ID NO: 3);

[0038] The 5' end of the fluorescent probe Q is labeled with a fluorescent group, and the 3' end is labeled with a quencher group;

[0039] European eel circovirus primer 2 sets:

[0040] Forward primer F:: 5'CCCGGCTGTGGCAAGTC 3' (SEQ...

Embodiment 2

[0054] Embodiment 2 A kind of fluorescent quantitative PCR detection kit for detecting European eel circovirus

[0055] 1. Composition

[0056] A fluorescent quantitative PCR detection kit for detecting European eel circovirus, comprising: a group of fluorescent quantitative PCR primers (SEQ ID NO: 1-3) for detecting European eel circovirus in a set of Example 1, DNA polymerisation Enzyme, UDG enzyme, 2× reaction buffer, sealing solution, positive control and negative control.

[0057] The 2×reaction buffer is a reaction solution containing dATP, dCTP, dGTP, dUTP and the like.

[0058] The DNA polymerase is Taq enzyme, the UDG enzyme is heat-sensitive uracil-DNA glycosylase, and the sealing liquid is mineral oil.

[0059] The positive control is a plasmid DNA containing the detection target gene of European eel circovirus, and the concentration of the plasmid DNA is 10 6 copies / μL, the negative control is sterilized ultrapure water.

[0060] 2. How to use

[0061] The eel...

Embodiment 3

[0072] Embodiment 3 Sensitivity experiment

[0073] The kit described in embodiment 2 detects the positive control sensitivity of European eel circovirus:

[0074] Carry out 10-fold serial dilution of the positive control plasmid DNA, respectively with 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 Copies / μL seven gradient concentrations of DNA were used as templates and negative control (sterilized ultrapure water) to establish a detection method according to the reaction system and conditions in Example 2 to determine the sensitivity of the kit.

[0075] Such as image 3 As shown, curve 1 is 10 6 copies / μL; Curve 2 is 10 5 copies / μL; Curve 3 is 10 4 copies / μL; Curve 4 is 10 3 copies / μL; curve 5 is 10 2 copies / μL; Curve 6 is 10 1 copies / μL; Curve 7 is 10 0 copies / μL; Curve 8 is the negative control; the results show that after the positive plasmid DNA was diluted 10 times, the detection concentration of the e...

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Abstract

The invention discloses a fluorescent quantitative PCR primer group and kit for detecting European eel circovirus, the kit comprises the primer group, the primer group comprises a forward primer F, a reverse primer R and a fluorescent probe Q. The kit also comprises DNA polymerase, UDG enzyme, a 2 * reaction buffer solution, a sealing solution, a positive reference substance and a negative reference substance. The kit provided by the invention can directly detect the European eel circovirus from a complex sample, has the advantages of strong specificity, high sensitivity, good experimental repeatability and convenience and rapidness in operation, can complete detection without expensive instruments, and can be used for on-site rapid and accurate detection of the European eel circovirus.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to the European eel circovirus detection technology. Background technique [0002] European eel circovirus is a member of the genus Circovirus in the Circoviridae family and is one of the pathogens that cause viral diseases in eels. At present, reports of circoviruses have been detected in aquatic animals such as sea turtles, eels, barbs, and catfish abroad. There are few domestic studies on eel viral diseases. Some researchers have reported from 160 cases of suspected eel viral diseases that died in a large number of acute cases. The positive rate of eel circovirus was as high as 40.6%, which indicated that eel circovirus may seriously endanger the development of eel aquaculture industry. [0003] The current biological detection technology for European eel circovirus is only based on the traditional PCR detection technology. The detection process has many steps, takes a l...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/11C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2600/158C12Q2563/107C12Q2531/113C12Q2527/125C12Q2561/113
Inventor 张璜林楠许智海高金艳林雪金余嘉明林梦蝶
Owner 广州双螺旋基因技术有限公司
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