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Primers and probe for streptococcus iniae LAMP-LFD visual detection, and application of primers and probe

A technology of LAMP-LFD and Streptococcus iniae, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as no related reports, and achieve the effect of simple operation, obvious results, and short detection time

Active Publication Date: 2014-07-30
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, this technology has been successfully applied to Taura syndrome virus (TSV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), infectious spleen and kidney necrosis Virus (Infectious spleen and kidney necrosis virus, ISKNV), Listonella anguillarum (Listonella anguillarum), but there is no relevant report on the application of this technology in the diagnosis and detection of Streptococcus iniae at home and abroad

Method used

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  • Primers and probe for streptococcus iniae LAMP-LFD visual detection, and application of primers and probe
  • Primers and probe for streptococcus iniae LAMP-LFD visual detection, and application of primers and probe
  • Primers and probe for streptococcus iniae LAMP-LFD visual detection, and application of primers and probe

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Establishment of LAMP-LFD visual detection method for Streptococcus iniae

[0039] 1. Primer design: Three pairs of primer sequences and one probe sequence for LAMP-LFD were designed according to the published gyrB gene sequence of Streptococcus iniae in NCBI (GenBank accession number: KC560771.1). The primer sequences are as follows:

[0040] gyrB-F3: 5'-GAAGATGATTCCATTACCGTTG-3',

[0041] gyrB-B3: 5'-CTAAATCTTCTCTCTACCACACC-3',

[0042] gyrB-FIP: 5'-TGAAACCTTATAACCGCCTCCGttttCTGCTGTTGAAACAGTCTTAC-3',

[0043] gyrB-BIP: 5'-GGTCTGCATGGGGTTGGTTttttAACCCGGACATCTAACTGT-3',

[0044] gyrB-LF: 5'-TACCTCCAGCATGGAGAACT-3',

[0045] gyrB-LB: 5'-TCAGTTGTTAATGCCCTCTCAA-3', the 5' end of gyrB-FIP is biotinylated;

[0046] Simultaneously, design a set of probes, can specifically bind to LAMP amplification product, be used for LFD detection, sequence is as follows:

[0047] gyrB-HP: 5'-GTTTCAGGTGGTCTGCATG-3', the 5' end is labeled with fluorescein isothiocyanate.

[0048] 2. Sa...

Embodiment 2

[0055] Application of the above-mentioned primers and probes for the visual detection of Streptococcus iniae LAMP-LFD in the preparation of a visual detection kit for Streptococcus iniae LAMP-LFD. The Streptococcus iniae LAMP-LFD visual detection kit includes a LAMP reaction system: the final concentration of each component of the reaction system is 0.2 μmol / L each for gyrB-F3 and gyrB-B3, and 1.6 μmol / L each for gyrB-FIP and gyrB-BIP , gyrB-LF and gyrB-LB each 0.4μmol / L, dNTPs1.4mmol / L, Tris-HCl (pH8.8)20mmol / L, KCl10mmol / L, MgSO 4 6.5mmol / L, (NH4) 2 SO 4 10mmol / L, Triton X-1000.1%, 8U Bst DNA polymerase large fragment (New England Biolabs) and 2μL sample template, add double-distilled water to make the total volume of the reaction system 25μl; and 20pmol of the above-mentioned LAMP reaction system gyrB-HP probe, wherein the primer sequence is shown in SEQ ID NO.1-SEQ ID NO.6 in the sequence listing, and the probe sequence is shown in SEQ ID NO.7 in the sequence listing.

Embodiment 3

[0057] Specificity determination for visual detection of LAMP-LFD using primers and probes of the invention

[0058]Using the designed specific primers and probes, respectively, with Streptococcus iniae ATCC 29178, Nocardia amberiella ATCC43993, Pseudomonas aeruginosa ATCC 9027, Vibrio alginolyticus ATCC 33787, Listeria monocytogenes ATCC 19115, Genomic DNA of Vibrio anguillarum sweetfish isolate, Staphylococcus aureus ATCC 6538, Aeromonas hydrophila ATCC 7966, Vibrio harveyi ATCC 33866, and Pseudomonas putida MCCC 1A01082 were used as templates, and the implementation was carried out as above Steps 3 and 4 of Example 1 were used for LAMP-LFD reaction to verify the specificity of primers and probes, and double distilled water was used as a negative control. The result is as figure 1 and figure 2 As shown, using the electrophoresis method ( figure 1 ) and LFD ( figure 2 ) can only be amplified from the genomic DNA sample of Streptococcus iniae to obtain the target band, a...

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Abstract

The present invention discloses primers and a probe for streptococcus iniae LAMP-LFD visual detection, and an application of the primers and the probe. According to the present invention, three pairs of LAMP primers such as gyrB-F3, gyrB-B3, gyrB-FIP, gyrB-BIP, gyrB-LF and gyrB-LB, and a probe gyrB-HP are provided, and the nucleotide sequences are represented by SEQID NO.1-EQID NO.7; and with the primers and the probe, a LAMP reaction system amplification step, a LAMP reaction product probe hybridization step and a LFD detection step are performed so as to achieve streptococcus iniae visual detection and further be used for kit preparation, wherein advantages of high rapidness, high specificity and high sensitivity are provided, instrument requirements are simple, early diagnosis and detection of streptococcus iniae can be easily achieved, and requirements of grass-roots detection facilities and field epidemic focus detection.

Description

technical field [0001] The invention relates to primers and probes for detecting Streptococcus iniae, in particular to the primers and probes for visual detection of Streptococcus iniae LAMP-LFD and the application of the primers and probes. Background technique [0002] Streptococcus iniae belongs to the class Bacilli, the order Lactobacillales, the family Streptococcus, and the genus Streptococcus. The bacterium is a round or nearly round, chain-like or double-arranged Gram-positive bacterium, without flagella, and does not form spores. It can infect a variety of wild or artificially farmed fish, including freshwater and seawater, such as freshwater porpoise , tilapia, sea bass and rainbow trout, etc., can also infect ornamental fish such as zebrafish and African cichlid. After the fish is infected with the bacteria, the main manifestation is meningoencephalitis, which has become an important cause of illness and death of farmed fish. Since the 1990s, the harm of this ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/46
CPCC12Q1/6844C12Q2531/119C12Q2565/625
Inventor 陈炯周前进
Owner NINGBO UNIV
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