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Primer and probe sequence used for LAMP-LFD detection of vibrio fluvialis and application of primer and probe sequence

A LAMP-LFD, Vibrio river technology, applied in the field of detection of primers and probe sequences of Vibrio river, to achieve high sensitivity, obvious results, clear and obvious results

Inactive Publication Date: 2015-11-11
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology has been successfully applied to infectious spleen and kidney necrosis virus ( Infectious spleen and kidney necrosis virus , ISKNV), infectious myonecrosis virus ( Infectious myonecrosis virus , IMNV), Vibrio vulnificus ( Vibriovulnificus ), and Streptococcus iniae ( Strepstococcusiniae ) and other aquatic pathogenic microorganisms, there have been no reports at home and abroad that this technology has been applied to the diagnosis and detection of riverine vibrio

Method used

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  • Primer and probe sequence used for LAMP-LFD detection of vibrio fluvialis and application of primer and probe sequence
  • Primer and probe sequence used for LAMP-LFD detection of vibrio fluvialis and application of primer and probe sequence
  • Primer and probe sequence used for LAMP-LFD detection of vibrio fluvialis and application of primer and probe sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Establishment of LAMP-LFD detection method for Vibrio riverina

[0037] 1. Primer design: Design according to the coding sequence of the outer membrane protein ompU of Vibrio riverina (GenBank accession number: KC182592) published in NCBI, where the primer sequence is as follows:

[0038] VflompU-F3: 5'-TCATGGCTTACCACGGTA-3',

[0039] VflompU-B3: 5'-GGTGTAAGACGCTGCTAG-3',

[0040] VflompU-FIP: 5'-AGTTGAACCGTCATCGCTACGttttGGCCAGTTCTCTGACCTA-3',

[0041] VflompU-BIP: 5'-TCTACGCAATCGGCGACACttttCTTTGTCTTGGTCAGCGT-3',

[0042] VflompU-LF: 5'-ACGGTAAGTTGCACGTAGAG-3',

[0043] VflompU-LB: 5'-GTGAAACTAGGTGCTGGCT-3',

[0044] Probe VflompU-HP: 5'-CTCTGATAACAACAAGATGG-3',

[0045] Among them, the 5' end of VflompU-FIP is labeled with biotin, and the 5' end of the probe VflompU-HP is labeled with fluorescein isothiocyanate.

[0046] 2. Sample DNA extraction: the long-term preservation of -70 ° C Vibrio strains ( Vibriofluvialis , ATCC33809) was streaked on TCBS solid mediu...

Embodiment 2

[0053] Specificity Determination of Vibrio fluvio LAMP-LFD Detection Using Primers and Probes of the Invention

[0054] Using the designed specific primers and probes, respectively, with Vibrio riverina ATCC33809, Edwardsiella tarda MCCC235, Aeromonas hydrophila ATCC7966, Vibrio vulnificus ATCC27562, Vibrio harveyi ATCC33866, Streptococcus iniae ATCC29178, Lysobacterium Genomic DNA of Vibrio bacterium ATCC33787, Vibrio anguillarum ayu-H080701, Listeria monocytogenes ATCC19115, Pseudomonas aeruginosa ATCC9027, etc. were used as templates, and the LAMP-LFD reaction was performed according to steps 3 and 4 of the above-mentioned Example 1, and verified The specificity of primers and probes, double distilled water 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 amplify the target bands from the genomic DNA samples of Vibrio riverinae, and other samples have no amplified band...

Embodiment 3

[0056] Using the primers and probes of the present invention to carry out the sensitivity measurement of Vibrio fluvio LAMP-LFD detection

[0057] Adopt the method of step 2 of above-mentioned embodiment 1 to extract the genomic DNA of Vibrio riverina, carry out 10 times of serial dilutions, select 1.0 * 10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 1.0×10 0 cfu / mL etc. were used as templates, and the LAMP-LFD reaction was carried out according to steps 3 and 4 of the above-mentioned Example 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 , Figure 4 and Figure 5 As shown, the sensitivity of the LAMP-LFD detection using the primers and probes provided by the invention is 1.0×10 2 cfu mL -1 ( Figure 4 ), consistent with the sensitivity obtained by agarose gel electrophoresis detection of LAMP amplification products ( image 3 ), which...

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Abstract

The invention discloses a primer and probe sequence used for LAMP-LFD detection of vibrio fluvialis and application of the primer and probe sequence. The primer and probe sequence is characterized by comprising three pairs of primers of LAMP including VflompU-F3 and VflompU-B3, VflompU-FIP and VflompU-BIP and VflompU-LF and VflompU-LB and a probe VflompU-HP, and a nucleotide sequence is shown as SEQ NO1-NO7. The primers and the probe are utilized to realize visual detection of vibrio fluvialis through a step of amplification by an LAMP reaction system, a step of enabling the probe to be hybridized with an LAMP reaction product and a step of LFD detection. The primer and probe sequence has the advantages of higher quickness, specificity and sensitivity, simplicity in instrument need, conduciveness to early diagnosis and detection of vibrio fluvialis and capability of meeting detection needs of primary detection mechanisms and on-site epidemic focuses.

Description

technical field [0001] The invention relates to a primer and a probe sequence for detecting Vibrio riverina, in particular to a primer and probe sequence for detecting Vibrio riverina LAMP-LFD and an application of the primer and probe. Background technique [0002] Vibrio fluvialus is a halophilic Gram-negative bacterium that is widely distributed in rivers and near-sea estuaries. It can cause diseases in various aquatic animals such as fish, shrimp and shellfish, and is one of the main pathogenic bacteria in mariculture. At the same time, Vibrio riverine can be isolated from different water bodies, urban sewage, animal and human feces, and aquatic products. After infection, people will have severe watery diarrhea, accompanied by symptoms such as vomiting, abdominal pain, fever, and varying degrees of dehydration. . In the United States, the bacterium is also considered to be one of the important pathogens causing enterocolitis in infants. Therefore, Vibrio riverina has b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/63
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 陈炯周前进
Owner NINGBO UNIV
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