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Primer and probe sequences for LAMP-LFD detection of Plecoglossus altivelis glugeasis

A LAMP-LFD and probe sequence technology, which is applied in the field of primers and probe sequences for LAMP-LFD detection of A. japonica, achieves the effects of clear and obvious detection results, improved detection speed, and obvious results.

Active Publication Date: 2020-04-17
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports at home and abroad on the application of this technology to the detection of sweetfish gramelus

Method used

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  • Primer and probe sequences for LAMP-LFD detection of Plecoglossus altivelis glugeasis
  • Primer and probe sequences for LAMP-LFD detection of Plecoglossus altivelis glugeasis
  • Primer and probe sequences for LAMP-LFD detection of Plecoglossus altivelis glugeasis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Establishment of a method for the detection of Glycerus ayumi by LAMP-LFD technique

[0036] 1. Primer design: Design according to the published β-tubulin gene of Gleuria sweetfish in NCBI (GenBank accession number: MN811004), where the primer sequences are as follows:

[0037] Gpl-F3: 5'-GGAAGGTGCTGAACTCATAG-3',

[0038] Gpl-B3: 5'-TCAGCATTCAACTGACCAG-3',

[0039] Gpl-FIP: 5'-ACGGTTCAACCACAGTATCAGATGGGTACTGGTGCTGGAATG-3',

[0040] Gpl-BIP: 5'-GAGGCACTCTACAATATCTGCTCAACCGCTCATAACAAGTGATACT-3',

[0041] Gpl-LF: 5'-ACGAACACATCATCCTATCAGG-3',

[0042] Gpl-LB: 5'-TGCCTAATCCTGGTTATGCC-3',

[0043] Probe Gpl-HP: 5'-CTTGTTGAAAATGCTGACG-3',

[0044] Among them, the 5' end of Gpl-FIP is labeled with biotin; the 5' end of probe Gpl-HP is labeled with fluorescein isothiocyanate.

[0045] 2. Sample DNA extraction: Dissect fresh sweetfish, extract white heterogeneous tumors from organs such as liver or gonads, rupture them, and obtain and purify the spores of G. ayutus. Take ...

Embodiment 2

[0053] Specificity Determination of LAMP-LFD Detection of G. sweetfish Using Primers and Probes of the Invention

[0054] Using the designed specific primers and probes, the samples of shrimp tissue samples positive for Gleua sweetfish, Neobenedenia melini, Cryptocaryon stimuli, Enterocystis hepatica, Anisakis peizi, and Palace Genomic DNA of lipid nematode, Vibrio anguillarum, Pseudomonas ayucidida, and healthy sweetfish liver tissue (without Ayumus DNA) were used as templates, and LAMP was carried out according to steps 3 and 4 of the above-mentioned Example 1 - LFD reaction, to verify the specificity of primers and probes, use double distilled water without DNA of A. sweetfish as a negative control. The result is as figure 1 with figure 2 As shown, using the electrophoresis method ( figure 1 ) and LFD ( figure 2 ) can only amplify the target bands from the genomic DNA samples of A. sweetfish, and other samples have no amplified bands, indicating that the use of the pr...

Embodiment 3

[0056] Using the primers and probes of the present invention to measure the sensitivity of the LAMP-LFD detection of T. ayumi (Tubulin β-tubulin gene plasmid standard is used as a template)

[0057] The method of step 2 of the above-mentioned embodiment 1 is used to obtain the standard substance of the β-tubulin gene plasmid of the sweetfish Glycerus japonica, and carry out a 10-fold gradient dilution, and the selected dilution factor is 10 of the original concentration of the plasmid standard substance. -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 As a template, the LAMP-LFD reaction was carried out according to the steps 3 and 4 of the above-mentioned Example 1, and the sensitivity of the primers and probes was verified, and the double distilled water without the DNA of A. sweetfish was used as a negative control. The result is as image 3 with Figure 4 As shown, the sensitivity of the LAMP-LFD detection using the primers and probes provided by the present invention to carry...

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Abstract

The invention discloses primer and probe sequences for LAMP-LFD detection of Plecoglossus altivelis glugeasis. Three pairs of primer sequences and one probe sequence of LAMP are designed according tobeta-tubulin genes of the Plecoglossus altivelis glugeasis, the primers of the three pairs of LAMP are Gpl-F3 and Gpl-B3; Gpl-FIP, Gpl-BIP; and Gpl-LF, Gpl-LB, and the probe sequence is Gpl-HP, and the nucleotide sequences of the primers and the probe are represented by SEQ NO.1 to NO.7. The visual detection of the Plecoglossus altivelis maculatus is realized by using the primers and the probe through an LAMP reaction system amplification step, an LAMP reaction product probe hybridization step and an LFD detection step. The primer and probe sequences have the advantages of high rapidness, highspecificity, high sensitivity and simple instrument requirement, facilitation of the early diagnosis and the early detection of the Plecoglossus altivelis glugeasis, and meeting the requirements of the grassroots detection mechanism and the on-site epidemic source detection.

Description

technical field [0001] The invention relates to the technical field of parasite diagnosis, in particular to a sequence of primers and probes for detection of LAMP-LFD of G. sweetfish. Background technique [0002] Ayu gruela is a kind of microsporidia obligately parasitic in the body of sweetfish, which was first identified and named by Japanese scholars. After being infected with this worm, a heterogeneous cyst will be formed in the body cavity of sweetfish, which contains a large number of spores. After the spores are released, they can cause the infiltration of mononuclear cells such as lymphocytes and macrophages, resulting in sublethal infection and destruction. The body surface and appearance of sweetfish not only intensifies the consumption of feed, leading to a substantial increase in the cost of fish farming, but also the shape of infected fish is poor, and the market value is reduced. [0003] In my country, large-scale and intensive high-density artificial breedi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6893C12Q1/6844C12Q2531/113C12Q2537/1376C12Q2565/625
Inventor 陈炯周前进
Owner NINGBO UNIV
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