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Nicking endonuclease nucleic acid isothermal amplification rapid detection kit for vibrio cholerae

A technology of constant temperature amplification detection and endonuclease nucleic acid, which is used in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc., to achieve the effect of strong specificity and guaranteed specificity

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

There is no report on the test kit and detection method for detecting the above-mentioned strains by nicking endonuclease nucleic acid constant temperature amplification technology

Method used

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  • Nicking endonuclease nucleic acid isothermal amplification rapid detection kit for vibrio cholerae
  • Nicking endonuclease nucleic acid isothermal amplification rapid detection kit for vibrio cholerae

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Detection of Vibrio cholerae O1 / O139 standard bacterial strain

[0046] Make the nicking endonuclease nucleic acid constant temperature amplification detection kit of Vibrio cholerae O1 / O139 (ATCC 25872) according to the following formula:

[0047] 1) Template pretreatment reaction solution:

[0048] 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 DNA Polymerase and 16.5μL wxya 2 O (sterilized double distilled water).

[0049] The forward primer SEQ ID NO: 1 described therein:

[0050]5-TGATTATATGCTCA GCTCTTC CTGGTTCCTCAACGCTTCTGT-3;

[0051] The reverse primer sequence is SEQ ID NO: 2:

[0052] 5-GATTTCATTATCCG GCTCTTC GGCATCTGCACCTGCTTTGTA-3.

[0053] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0054] 2) NEMA constant temperature amplification reaction solution:

[00...

Embodiment 2

[0077] Embodiment 2 to the detection of Vibrio parahaemolyticus standard bacterial strain

[0078] Make the nicking endonuclease nucleic acid constant temperature amplification detection kit of Vibrio parahaemolyticus (ATCC 17802) according to the following formula:

[0079] 1) Template pretreatment reaction solution:

[0080] 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 DNA Polymerase and 16.5μL wxya 2 O (sterilized double distilled water).

[0081] The forward primer SEQ ID NO: 1 described therein:

[0082] 5-TGATTATATGCTCA GCTCTTC CTGGTTCCTCAACGCTTCTGT-3;

[0083] The reverse primer sequence is SEQ ID NO: 2:

[0084] 5-GATTTCATTATCCG GCTCTTC GGCATCTGCACCTGCTTTGTA-3.

[0085] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0086] 2) NEMA constant temperature amplification reaction solut...

Embodiment 3

[0109] Example 3: Detection of food samples not infected with Vibrio cholerae

[0110] 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 did not have a red band, indicating that the sample to be tested was not infected by Vibrio cholerae.

[0111] The primers, probes and kits of the present invention can also detect Vibrio cholerae on other samples.

[0112]

[0113]

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Abstract

The invention relates to primers and probes which are used for detecting vibrio cholerae quickly by isothermal amplification. In the primers, a sequence of a forward primer is SEQ ID No.1, a sequence of a reverse primer is SEQ ID No.2; and in the probes, a sequence of a P1 probe is SEQ ID No.3, and a sequence of a P2 probe is SEQ ID No.4. The invention also provides a kit which contains the sequences of the primers and the probes and is used for detecting the vibrio cholerae quickly by the isothermal amplification simultaneously and an application method. In the kit, the primers and the probes are designed according to a conservative gene sequence of a strain to be detected to ensure the specificity of a detection method, and the icking endonuclease nucleic acid isothermal amplification rapid detection kit has the similar sensitivity with the polymerase chain reaction (PCR) detection method, and only the ordinary water bath kettle is needed without an expensive PCR instrument. Resultsare not needed to be observed by a gel electrophoresis method, namely the results are detected by the general nucleic acid amplification rapid detection plate, so the nicking endonuclease nucleic acid isothermal amplification rapid detection kit is simple, quick, safe and pollution-free; therefore the primers and probes are particularly suitable for food detection organization.

Description

technical field [0001] The invention belongs to the technical field of detection of pathogenic microorganisms, in particular to a fast detection kit for constant temperature amplification of nicking endonuclease nucleic acid of Vibrio cholerae. Background technique [0002] Vibrio cholerae (vbrio cholera) is the pathogenic bacterium of cholera. Cholera, also known as Asian cholera or infectious cholera, is a severe and sudden acute bacterial infectious disease caused by O1 serogroup and O139 serotype Vibrio cholerae, and its hazard is second only to plague. It can cause epidemics, outbreaks and pandemics. The clinical features are severe diarrhea, vomiting, a large amount of rice swill-like excretion, water and electrolyte disorders, and peripheral circulatory failure. Severe shock may be complicated by acute renal failure. Due to the rapid spread of cholera, and the high morbidity and mortality during the epidemic, it is extremely harmful, so early, rapid and correct diag...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCY02A50/30
Inventor 祝素珍邵秀玲姜英辉李正义赵丽青尼秀媚雷质文倪鑫王建广
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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