Primer for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of anisakis simplex/anisakis paasche and probe sequence

A LAMP-LFD, simple Anisakis technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve obvious results, clear and obvious detection results, and simple operation.

Active Publication Date: 2017-10-13
NINGBO UNIV
View PDF5 Cites 2 Cited by
  • 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 simple Anisakis / Anisakis peizii

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of anisakis simplex/anisakis paasche and probe sequence
  • Primer for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of anisakis simplex/anisakis paasche and probe sequence
  • Primer for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of anisakis simplex/anisakis paasche and probe sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Establishment of a method for detection of Anisakis simplex / Anisakis peizii by LAMP-LFD technique

[0036] 1. Primer design: Design according to the coding sequence of Anisakis simplex / Anisakis paizii rDNA-ITS2 (GenBank accession number: KU991876) published in NCBI, where the primer sequences are as follows:

[0037] AniITS-F3: 5'-GAATTGCAGACACATTGAGC-3'

[0038] AniITS-B3: 5'-ATAGTAGATTCGGTGTTGACG-3',

[0039] AniITS-FIP: 5'-GTAATTCGACCCTCAGCCAGACACTAAGAATTCGAACGCACA-3',

[0040] AniITS-BIP: 5'-CTGTGAAGCATTCGGCAAGCAACCGCTCGTCATATTGTC-3',

[0041] AniITS-LF: 5'-TGCCATCGGGAATGAACC-3',

[0042] AniITS-LB: 5'-TTGCTGTTGTGTTGTTGGTG-3',

[0043] Probe AniITS-HP: 5'-GGTGAACTGTCTTCACGGTT-3',

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

[0045] 2. Sample DNA extraction: Each individual worm body of each worm isolate was treated with 70% ethanol. After removing the etha...

Embodiment 2

[0052] Specificity Determination of Simple Anisakis / Anisakis paizii LAMP-LFD Detection Using Primers and Probes of the Invention

[0053] Using the designed specific primers and probes, the anisakis simplex, anisakis peizi, sperm whale anisakis, typical anisakis, ceca nematode, gray moray Genomic DNA from lipid nematodes, lipid nematode fischeri, needle Ascaris, neodeepidensis, acanthocephala, neobenedenia mezei, human roundworm, Ascaris suum, haemonchus contortus, and Caenorhabditis elegans As a template, LAMP-LFD reaction was carried out according to step 3 and step 4 of the above-mentioned Example 1 to verify the specificity of primers and probes, and double distilled water was used 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 band from the genomic DNA samples of Anisakis simplex / Anisakis paizii, and other samples have no amplified bands, indica...

Embodiment 3

[0055] Sensitivity Determination of Simple Anisakis LAMP-LFD Detection Using Primers and Probes of the Invention

[0056] Using the method of step 2 of the above-mentioned embodiment 1 to extract the genomic DNA of simple Anisakis L3, carry out 10-fold gradient dilution, select 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 : The simple Anisakis genome was used as a template, and the LAMP-LFD reaction was carried out according to steps 3 and 4 of the above-mentioned embodiment 1 to verify the sensitivity of the primers and probes, and double distilled water 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 is 10% of that of a single parasite genome DNA extract. -4 times. Since the total volume of Anisakis simplex L3 genomic DNA extraction solution is 20 µL, 2 µL was taken as a template for LAMP reaction. Therefore, the sensitivity of LAMP-L...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a primer for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of anisakis simplex / anisakis paasche and a probe sequence. The primer is characterized by comprising three pairs of LAMP primers (AniITS-F3 and EtaompA-B3, AniITS-FIP and AniITS-BIP, and AniITS-LF and AniITS-LB), and a probe (AniITS-HP); a nucleic acid sequence is shown in SEQ NO. 1 to NO. 7. The primer and the probe have the advantages that by performing LAMP reaction system amplification step, probe hybridization LAMP reaction product detection step and LFD detection step, the visualized detection of the anisakis simplex / anisakis paasche can be realized; the detection speed is high, the whole detection process is only 50min, the time is shortened, the labor intensity is decreased, the reliance on instrument equipment is small, and the primer and the probe are more suitable for the detection of base organizations and field infectious focuses.

Description

technical field [0001] The invention relates to a primer and a probe sequence for detecting anisakis, in particular to a primer and a probe sequence for detecting anisakis simplex / anisakis paizii LAMP-LFD. Background technique [0002] Anisakis ( Anisakis spp.) usually parasitizes in the viscera and muscle tissue of bony fishes, and can cause allergic symptoms when people eat raw, semi-finished or pickled fish, leading to anisakiasis. With the popularity of eating fresh fish fillets, the incidence of anisakis disease is increasing year by year. In 2013, the "List of Quarantine and Epidemic Diseases of Imported Animals of the People's Republic of China" listed anisakiasis as a second-class infectious disease, which restricted the import and export of seafood to a certain extent (http: / / www.aqsiq. gov.cn). Anisakiasis infection is mainly associated with Anisakis simplex ( A. simplex ) related. Studies have shown that Anisakis has three sister species, namely Anisakis si...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2565/625
Inventor 陈炯周前进
Owner NINGBO UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap