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Ring mediated isothermality amplification fast detecting method for norovirus

A ring-mediated isothermal and viral technology, which is applied in the field of rapid detection of norovirus ring-mediated isothermal amplification, can solve the problems of high cost, high sensitivity of immunoelectron microscopy, and narrow application range of enzyme-linked immunoassay, and achieve high sensitivity , technically specific effects

Inactive Publication Date: 2009-04-08
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

[0003] Existing norovirus detection methods mainly contain direct electron microscopy, immunoelectron microscopy, radioimmunoassay, enzyme-linked immunoassay and RT-PCR etc., but the sensitivity of direct electron microscopy is low; the sensitivity of immunoelectron microscopy is high, but the cost is also very high. High; radioimmunoassay takes a long time and requires radioisotope labeling; enzyme-linked immunoassay has a narrow range of applications; RT-PCR is sensitive and accurate, but it also has the problem of high cost
However, the loop-mediated isothermal amplification method can better make up for these shortcomings, and there is no report on the detection of norovirus by the loop-mediated isothermal amplification method.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Prepare the ring-mediated isothermal amplification reaction solution of GII type norovirus according to the following formula:

[0035] (1) LAMP reaction solution:

[0036] Contains 2.5 μL 10× Thermopol reaction buffer, 1.4 μL 25 mmol / L dNTP (a mixture of four kinds of DNA), 4.0 μL 10 μmol / L upstream internal primer (FIP), 4.0 μL 10 μmol / L downstream internal primer (BIP), 0.5 μL 10 μmol / L upstream outer primer (F3), 0.5 μL 10 μmol / L downstream outer primer (B3), 1.5 μL 100 mmol / L MgSO 4 , 5 μL 5M betaine and 1.6 μL ddH 2 O (sterilized double distilled water).

[0037] The upstream internal primers described therein:

[0038]5-CGGGCTCCAGAGCCATAACCT-ACGCCAACCCATCTGATG-3,

[0039] Downstream internal primers:

[0040] 5-GTGGCGGGCCAACAAAACGTA-CACTGTGAACTTCTCCACCAG-3,

[0041] Upstream outer primer: 5-GTGAATGAAGATGGCGTCGA-3,

[0042] Downstream outer primer: 5-CTGGAGCGTTTCTAGGGGA-3

[0043] The mass ratio of the four deoxyribose nucleic acids in the mixture dNTP is ...

Embodiment 2

[0059] Prepare the ring-mediated isothermal amplification reaction solution of GII type norovirus according to the following formula:

[0060] (1) LAMP reaction solution:

[0061] Contains 2.5 μL 10× Thermopol reaction buffer, 1.4 μL 25 mmol / L dNTP (mixture of four kinds of DNA), 4.0 μL 10 μmol / L upstream internal primer (FIP), 4.0 μL 10 μmol / L downstream internal primer (BIP), 0.5 μL 10 μmol / L upstream outer primer (F3), 0.5 μL 10 μmol / L downstream outer primer (B3), 1.5 μL 100 mmol / L MgSO 4 , 5 μL 5M betaine and 1.6 μL ddH 2 O (sterilized double distilled water).

[0062] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above.

[0063] The mass ratio of the four deoxyribose nucleic acids in the above mixture dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0064] (2) UNG enzyme: 1U / μL;

[0065] (3) Bst DNA polymerase: 8U / μL;

[0066] (4) ReverTra Ace: 100U / μL;

[0067] (5) Chromogen: 10% fluoresce...

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Abstract

The invention relates to a rapid detection method of the loop-mediated isothermal amplification of a G II type norovirus. Reagents thereof comprise a reaction solution of the loop-mediated isothermal amplification, Bst DNA polymerase and a color development agent; wherein, the reaction solution comprises a reaction buffer solution, dNTP, bitter salt, an upstream inner primer 5-CGGGCTCCAGAGCCATAACCT-ACGCCAACCCATCTGATG-3, a downstream inner primer 5-GTGGCGGGCCAACAAAACGTA-CACTGTGAACTCTCCACCAG-3, an upstream outer primer 5-GTGAATGAAGATGGCGTCGA-3 and a downstream outer primer 5-CTGGAGCGTTTCTAGGGGA-3, and lycine. The detection method of the G II type norovirus includes the extraction of bacterial RNA, the loop-mediated isothermal amplification of the G II type norovirus, and color development detection. The method has the advantages of fast speed, strong specificity, high sensitivity and low cost.

Description

technical field [0001] The invention relates to a method for rapid detection of bacterial samples using a loop-mediated isothermal amplification (LAMP) technology, in particular to a rapid detection method for GII type norovirus loop-mediated isothermal amplification. Background technique [0002] Norovirus (NV) is a group of virus particles with similar morphology and slightly different antigenicity. Norwalk virus is the prototype representative strain of this genus. It was first isolated from the stool of a patient with acute diarrhea in Norwalk, USA in 1968. Since then, it has been isolated from the stool of patients with gastroenteritis in various parts of the world. A variety of virus-like particles with similar morphology but slightly different antigenicity are named after the place of discovery, such as: Hawaii Virus (HV), Snow Mountain Virus (SMV), Mexico Virus (MxV) Southampton Virus (SOV), etc., in 2002 In August, the Eighth International Virus Nomenclature Commit...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 雷质文贺楠刘云国张健祝素珍姜英辉李正义房保海唐静贾俊涛赵丽青马维兴
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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