Nicking incision enzyme nucleic acid isothermal amplification and rapid detection kit of vibrio parahaemolyticus

A technology of hemolytic Vibrio and constant temperature amplification, which is applied in the determination/inspection of microorganisms, methods based on microorganisms, biochemical equipment and methods, etc. Specific, specific effects

Inactive Publication Date: 2012-04-04
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned method either has the problems of being time-consuming, or having expensive applied instruments, and being difficult to implement. Therefore, it is necessary to develop another method capable of effectively detecting Vibrio parahaemolyticus and its corresponding kit.

Method used

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  • Nicking incision enzyme nucleic acid isothermal amplification and rapid detection kit of vibrio parahaemolyticus
  • Nicking incision enzyme nucleic acid isothermal amplification and rapid detection kit of vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: to the detection of Vibrio parahaemolyticus standard bacterial strain

[0049] Make the nicking endonuclease nucleic acid constant temperature amplification kit of Vibrio parahaemolyticus according to the following formula:

[0050] 1) Template pretreatment reaction solution:

[0051]Each 23μL contains 2.5μL 10×Taq Platinum buffer II, 1.0μL 2.5mmol / L dNTP, 1.0μL 10μmol / L forward primer, 1.0μL 10μmol / L reverse primer, 1.0μL 2.5U / μL Taq Platinum DNAPloymerase and 16.5μL wxya 2 O (sterilized double distilled water).

[0052] The forward primer SEQ ID NO: 1 described therein:

[0053] 5-GGCCATAATTCGAA GCTCTTC GCTACTTCACCATTGACGGCTAC-3;

[0054] Reverse primer: SEQ ID NO: 2:

[0055] 5-TTCGCCAAAATCTAA GCTCTTC TCACAACGCTGACGGATAACG-3;

[0056] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0057] 2) NEMA constant temperature amplification reaction solution:

[0058] Each 46 μL contains 5...

Embodiment 2

[0081] Embodiment 2: to the detection of vibrio vulnificus standard bacterial strain

[0082] 1) Template pretreatment reaction solution:

[0083] Each 23μL contains 2.5μL 10×Taq Platinum buffer II, 1.0μL 2.5mmol / L dNTP, 1.0μL 10μmol / L forward primer, 1.0μL 10μmol / L reverse primer, 1.0μL 2.5U / μL Taq Platinum DNAPloymerase and 16.5μL wxya 2 O (sterilized double distilled water).

[0084] The forward primer SEQ ID NO: 1 described therein:

[0085] 5-GGCCATAATTCGAA GCTCTTC GCTACTTCACCATTGACGGCTAC-3;

[0086] Reverse primer: SEQ ID NO: 2:

[0087] 5-TTCGCCAAAATCTAA GCTCTTC TCACAACGCTGACGGATAACG-3;

[0088] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0089] 2) NEMA constant temperature amplification reaction solution:

[0090] Each 46 μL contains 5 μL 10×NEBuffer 3, 1.0 μL 2.5 mmol / L dNTP, 1.0 μL 10 μmol / L forward primer, 1.0 μL 10 μmol / L reverse primer, 0.5 μL 10 mg / mL BSA, 4 μL DMSO and 33.5 μL ddH 2 O...

Embodiment 3

[0113] Example 3: Detection of food samples suspected of being infected with Vibrio parahaemolyticus

[0114] The food sample was subjected to DNA extraction and amplification detection according to the method described in Example 1, and the product was detected with a universal nucleic acid amplification rapid detection plate. Results The quality control area C showed a red band, and the detection area T also had a red band, indicating that the sample to be tested was infected by Vibrio parahaemolyticus.

[0115] The primers, probes and kits of the present invention can also detect Vibrio parahaemolyticus on other samples.

[0116]

[0117]

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PUM

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Abstract

The invention relates to primers and probes for isothermally amplifying and rapidly detecting vibrio parahaemolyticus. The sequence of the forward primer is SEQ ID NO:1, and the sequence of the reverse primer is SEQ ID NO:2; the sequence of the probe P1 is SEQ ID NO:3; and the sequence of the probe P2 is SEQ ID NO:4. Meanwhile, the invention also provides a kit containing the sequences of the primers and the probes and used for isothermally amplifying and rapidly detecting vibrio parahaemolyticus as well as an application method. In the kit provided by the invention, the primers and the probes are designed according to the conservative gene sequence of a strain to be detected so as to ensure the specificity of the detection method. The method provided by the invention has similar sensitivity to the PCR detection method, an expensive PCR instrument is not required, and only an ordinary water bath pot is required. The result is not necessary to observe by using the gel electrophoresis method, only a universal nucleic acid amplification product rapid detection plate is required, and the method is simple, rapid, safe and free of pollution and is especially applicable to food detectioninstitutions.

Description

technical field [0001] The invention belongs to the technical field of detection of pathogenic microorganisms, and in particular relates to a prescription kit for rapid detection of bacterial samples using a nicking endonuclease constant temperature amplification (NEMA) technology and an application thereof. Background technique [0002] Vibrio parahemolyticus (Vibrio Parahemolyticus) is a halophilic bacterium mainly found in seafood, such as cuttlefish, sea fish, sea shrimp, sea crab or jellyfish; and preserved foods with high salt content, such as pickles, cured meat Wait. The bacterium has a strong survival ability and can survive for more than 1 month on rags and cutting boards, and can survive in seawater for 47 days. If you accidentally eat food containing this bacterium, it will often cause food poisoning. Clinically, acute onset, abdominal pain, vomiting, diarrhea and watery stool are the main symptoms. Vibrio parahaemolyticus food poisoning mostly occurs in coasta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/63
Inventor 姜英辉雷质文王妍婷王建广房保海尼秀媚马维兴张健祝素珍
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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