Rapid diagnostic kit based on loop-mediated isothermal amplification technique for hepatitis A virus genes and detection method thereof

A hepatitis A virus, ring-mediated isothermal technology, applied in the directions of microorganism-based methods, resistance to vector-borne diseases, and microbial assay/examination, which can solve problems such as genetic rapid diagnostic kits for Mycobacterium tuberculosis , to achieve the effect of significant color difference, high specificity, and rapid amplification

Active Publication Date: 2011-08-31
GUANGZHOU HUAFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, isothermal amplification (Isothermal Amplification) nucleic acid rapid detection technology is a great progress in pathogenic nucleic acid detection technology. The established loop-mediated isothermal amplification technology (LAMP) has many advantages, and there is no useful loop at present. Gene rapid diagnostic kit for detection of Mycobacterium tuberculosis by mediated isothermal amplification technique

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 kit

[0039] (1) The DNA synthesizer synthesizes the following oligodeoxynucleic acid primers:

[0040] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0041] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0042] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0043]The nucleotide sequence of the internal primer BIP is shown in SEQ ID NO:4.

[0044] (2) Purchasing DNA polymerase: Bst DNA polymerase is placed in the container.

[0045] (3) Preparation of reaction solution: the reaction solution contains 2mmol / LdNTP, 25mmol / L Tris-Cl, 12.5mmol / L potassium chloride, 12.5mmol / L ammonium sulfate, 10mmol / L magnesium sulfate, 0.125% by volume TritonX-100, 1mol / L betaine, 2 mol / L each of the inner primers FIP / BIP and 0.25 mol / L each of the outer primers F3 / B3 were placed in the container.

[0046] (4) Purchasing reverse transcriptase: AMV reverse ...

Embodiment 2

[0062] The preparation of embodiment 2 kit

[0063] The formula of the reaction solution is: the reaction solution contains 1.6mmol / LdNTP, 20mmol / L Tris-Cl, 10mmol / L potassium chloride, 10mmol / L ammonium sulfate, 8mmol / L magnesium sulfate, 0.1vol%TritonX-100, 0.8mol / L betaine, inner primer FIP / BIP each 1.6mol / L and outer primer F3 / B3 each 0.2mol / L.

[0064] The chromogenic solution is EvaGreen.

[0065] Others are the same as embodiment 1.

Embodiment 3

[0066] Example 3 Application of Hepatitis A Virus Gene Rapid Diagnosis Kit

[0067] Collect samples according to the method in 2.1 of GB / T 18936-2003, add 175mL glycine buffer solution (pH 7.5, glycine 0.5mol / L, NaCL 0.3mol / L) to 25g shellfish sample, mix well with agitator, 37℃ 30min. 4°C 1000r / m20min, take the supernatant. Use 8% PEG8000 (with a final NaCl concentration of 0.75 mol / L) to settle the virus overnight at 4°C. 10000r / m15min, discard the supernatant, resuspend the pellet with 10mL PBS buffer (pH7.4), add an equal volume of 3-chloro-3-fluoro-ethane, mix well, 10000r / m15min at 4°C, carefully absorb the supernatant, The virus was sedimented overnight at 4° C. with 8% PEG8000 (with a final NaCl concentration of 0.75 mol / L). 14000r / m15min at 4°C, discard the supernatant, and resuspend the pellet in an appropriate amount of PBS buffer. Take 1 / 4 volume of the suspension, extract it once with an equal volume of chloroform, and carefully collect the supernatant for RNA...

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Abstract

The invention discloses a rapid diagnostic kit based on a loop-mediated isothermal amplification technique for hepatitis A virus genes and a detection method thereof. The kit comprises two pairs of primers, Bst DNA polymerase, revertase, an RNase inhibitor, a stabilizing solution, a reaction solution, a chromogenic solution and a positive contrast solution, wherein the nucleotide sequences of thetwo pairs of primers are shown in SEQ ID NO: 1-4; and the eight solutions are respectively contained in containers. The kit and the detection method can detect the hepatitis A virus with high efficiency and high specificity, are based on the loop-mediated isothermal amplification technique, apply six segments, four primers and one constant temperature to complete an amplification reaction within less than one hour, and have the advantages of low detection cost, short time consumption, high yield, high specificity, significant chromogenic difference between a positive result and a negative result, high authentication rate, distinctness and reliability.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a rapid diagnosis kit of hepatitis A virus gene based on a loop-mediated isothermal amplification technology and a detection method thereof. Background technique [0002] At present, there are many detection methods for hepatitis A virus, from the national standard (GB / T 18936-2003) based on the isolation and identification of pathogenic microorganisms, morphological identification and serological identification, to the immunological detection technology of specific proteins, nucleic acid Molecular biological detection methods such as probes and polymerase chain reaction (PCR) technology (GB / T 22287-2008). Among them, the detection of pathogenic nucleic acids has greatly improved in terms of rapidity, safety, accuracy and sensitivity. These new technologies try to break through the traditional microbiological detection modes such as morphology and biochemical reactions, and do no...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/93C12Q1/70
CPCY02A50/30
Inventor 曹以诚黄逸男李志勇杜正平陈洵谭惠媚王志强
Owner GUANGZHOU HUAFENG BIOTECH
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