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Primer and probe sequences for LAMP-LFD detection of Vibrio vulnificus

A technology of LAMP-LFD and Vibrio vulnificus, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of detection sensitivity and detection speed, and achieve short detection time and high detection efficiency. High sensitivity and improved detection speed

Active Publication Date: 2015-08-26
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] 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 Infectious spleen and kidney necrosis virus (ISKNV), Listonia eelensis ( Listonella anguillarum ), but there is no relevant report on the application of this technology in the diagnosis and detection of Vibrio vulnificus in China, even if foreign literature (Thanai Surasilp a, Siwaporn Longyant a, Sombat Rukpratanporn b, Pattarin Sridulyakul a, Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene, Molecular and Cellular Probes, 2011, 25: 158-163), but its detection sensitivity and detection speed are far inferior to the present invention

Method used

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  • Primer and probe sequences for LAMP-LFD detection of Vibrio vulnificus
  • Primer and probe sequences for LAMP-LFD detection of Vibrio vulnificus
  • Primer and probe sequences for LAMP-LFD detection of Vibrio vulnificus

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Establishment of a method for detection of Vibrio vulnificus by LAMP-LFD technique

[0040] 1. Primer design: Design according to the coding sequence of the Vibrio vulnificus outer membrane protein gene TolC (GenBank accession number: DQ296643) published in NCBI, where the primer sequences are as follows:

[0041] TolC-F3: 5'-TCTTGAAGCCACTTATCGC-3',

[0042] TolC-B3: 5'-CAGCATCAACATCCAGTACA-3',

[0043] TolC-FIP: 5'-TACCAACATCAAAGCCCGCTTttttCGTGCGTATGAGCAATCT-3',

[0044] TolC-BIP: 5'-GATGCCACTCGTCGCCTTttttGCAGTACACTCAAGATGTAGTT-3',

[0045] TolC-LF: 5'-GGTTGCTTCTAATGCTGAACG-3',

[0046] TolC-LB: 5'-AACCTATCGAATGCACGCT-3', the 5' end of TolC-FIP is biotinylated;

[0047] Simultaneously, design a set of probes, can specifically bind to LAMP amplification product, be used for LFD detection, sequence is as follows:

[0048] TolC-HP: 5'-GGTACTCGTACTATTGTGGAT-3', the 5' end is labeled with fluorescein isothiocyanate.

[0049] 2. Sample DNA extraction: using water boili...

Embodiment 2

[0056] Specificity Determination of Vibrio vulnificus LAMP-LFD Detection Using Primers and Probes of the Invention

[0057] Using the designed specific primers and probes, Vibrio vulnificus ATCC 27562, Vibrio harveyi ATCC 33866, Vibrio riverina ATCC 33809, Vibrio parahaemolyticus ATCC 33847, Vibrio rotiferus DSM 17186T, Vibrio alginolyticus Genomic DNA of bacteria ATCC 17749, Vibrio anguillarum sweetfish isolate, Aeromonas hydrophila (Ah0201), Staphylococcus aureus ATCC 6538, and Listeria monocytogenes ATCC 19115 were used as templates, according to the steps in Example 1 above 3 and step 4 for LAMP-LFD reaction, verify 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 vulnificus, and other samples have no amplified bands, indicating that the use of...

Embodiment 3

[0059] Sensitivity determination of Vibrio vulnificus LAMP-LFD detection using primers and probes of the present invention

[0060] The genomic DNA of Vibrio vulnificus was extracted by the boiling method in step 2 of the above-mentioned embodiment 1, and used as the initial concentration of the template for the LAMP-LFD reaction (equivalent to 3.7×10 9 wxya -1 ), carry out 10-fold serial dilution, respectively as templates, carry out LAMP-LFD reaction according to step 3 and step 4 of the above-mentioned Example 1, and verify the sensitivity of primers and probes, and double distilled water is 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 3.7×10 2 wxya -1 ( Figure 4 ), consistent with the sensitivity obtained by agarose gel electrophoresis detection of LAMP amplification products ( image 3 ), which is 100 times that of the co...

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Abstract

The invention discloses primers for loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) detection of a vibrio vulnificus and probe sequences. The primers are characterized by including three pairs of LAMP primers, namely TolC-F3, TolC-B3, TolC-FIP, TolC-BIP, TolC-LF and TolC-LB, and a probe TolC--HP, wherein the Van-FIP is a biotin labeling primer at 5' terminal; the probe TolC-HP is a fluorescein isothiocyanate FITC labeled probe at the 5' terminal; concrete sequences are shown in SEQ NO1-NO7 in a sequence table. The primer has the advantages of good quickness, specificity and sensitivity and simple demands on instruments; early diagnosis and detection of the vibrio vulnificus are facilitated; the demands of a basic-level detection mechanism and field epidemic focus detection can be met.

Description

technical field [0001] The invention relates to a primer and a probe sequence of Vibrio vulnificus, in particular to a primer and a probe sequence for LAMP-LFD detection of Vibrio vulnificus. Background technique [0002] Vibrio vulnificus ( Vibrio vulnificus ) is a Gram-negative halophilic Vibrio, commonly found in estuaries and seawater environments, and fish and fritillary are the main susceptible aquatic animals. At the same time, this bacterium is an important pathogen of zoonotic diseases, and people are usually infected with this bacterium by eating raw contaminated seafood, or contacting wounds with contaminated sea water or marine animals. Patients with bacterial infection mainly have clinical symptoms such as primary sepsis, trauma infection, acute gastroenteritis, and cellulitis, and the mortality rate of septic shock can be as high as 50%. In my country, Vibrio vulnificus infection mostly occurs in coastal areas and is listed as one of the eight high-risk micro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/63
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 陈炯周前进
Owner NINGBO UNIV
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