Primers and probes and applications of primers and probes for visual detection of Streptococcus iniae LAMP-LFD

A LAMP-LFD, Streptococcus iniae technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as no related reports, and achieve simple operation, high convenience, and early diagnosis. and the detection effect

CN103952472BActive Publication Date: 2016-06-08NINGBO UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2016-06-08

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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.
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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...

Examples

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, TritonX-1000.1%, 8UBstDNA polymerase large fragment (NewEngland Biolabs) and 2μL sample template, add double distilled water to make the total volume of the reaction system 25μl; and 20pmol gyrB-HP probe used in conjunction with the above LAMP reaction system , 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 li...

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 ATCC29178, Nocardiella amberi ATCC43993, Pseudomonas aeruginosa ATCC9027, Vibrio alginolyticus ATCC33787, Listeria monocytogenes ATCC19115, Vibrio anguillarum Genomic DNA of fish isolates, Staphylococcus aureus ATCC6538, Aeromonas hydrophila ATCC7966, Vibrio harveyi ATCC33866, and Pseudomonas putida MCCC1A01082 were used as templates, and carried out according to steps 3 and 4 of the above-mentioned Example 1 LAMP-LFD reaction was used to verify the specificity of primers and probes, and double distilled water was used as a negative control. The result is as figure 1and 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, and other samples ...