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Method and kit for detecting clostridium tetani by using loop-mediated isothermal amplification method

A loop-mediated constant temperature, tetanus bacillus technology, applied in the field of molecular biology, can solve the problems of low specificity and sensitivity, short amplification time, complicated operation, etc., and achieve the effect of high specificity and high sensitivity

Inactive Publication Date: 2012-11-21
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct smear method is fast and economical, but it is easy to be confused with other fungi, and has low specificity and sensitivity; the anaerobic culture method is currently the gold standard for the identification of Tetani bacilli, but the culture conditions are high and time-consuming (about 18-72h ), often misses the best treatment time, causing serious consequences; the identification of the automatic bacterial culture identification instrument also requires anaerobic culture of the test sample, which results in the shortcoming of long detection time; nucleic acid analysis is mainly based on fluorescent quantitative PCR technology, However, there are many defects in the current PCR technology: (1) the conditions are high, and the processes of reagent preparation, sample pretreatment, gene amplification and detection are required to be completed in three different operating intervals; (2) the operation is complicated and requires Perform multiple steps such as specimen denaturation, DNA extraction, PCR amplification, and fluorescence detection
Although the loop-mediated constant temperature amplification method has the advantages of high specificity, high sensitivity and short amplification time, it is necessary to design primers when using it, and the accuracy of primer design is related to the specificity and sensitivity of detection.
In addition, the loop-mediated constant temperature amplification method does not have a matching rapid DNA extraction method, and the pretreatment of the specimen is still complicated and time-consuming

Method used

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  • Method and kit for detecting clostridium tetani by using loop-mediated isothermal amplification method
  • Method and kit for detecting clostridium tetani by using loop-mediated isothermal amplification method
  • Method and kit for detecting clostridium tetani by using loop-mediated isothermal amplification method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: kit of the present invention

[0042] 1. Kit

[0043] The test kit for detecting bacillus tetani described in the present invention comprises:

[0044] Upstream inner primer: has the nucleotide sequence shown in SEQ ID NO: 1;

[0045] Downstream inner primer: has the nucleotide sequence shown in SEQ ID NO: 2;

[0046] Upstream outer primer: having the nucleotide sequence shown in SEQ ID NO: 3;

[0047] Downstream outer primer: has the nucleotide sequence shown in SEQ ID NO:4.

[0048] 2. Detection method

[0049] Add the reaction solution containing 2 μL sample DNA to the reaction tube, and then add the upstream internal primer, downstream internal primer, upstream external primer and downstream external primer. The concentrations of the four primers are 8 μmol / L, 8 μmol / L, 1 μmol / L and 1 μmol / L, respectively. L, mixed evenly, placed on the reaction well of the LA320C detection instrument, and amplified at a constant temperature of 65°C for 60min. Ac...

Embodiment 2

[0053] Embodiment 2: kit of the present invention

[0054] 1. Kit

[0055] The test kit for detecting bacillus tetani described in the present invention comprises:

[0056] Upstream inner primer: has the nucleotide sequence shown in SEQ ID NO: 1;

[0057] Downstream inner primer: has the nucleotide sequence shown in SEQ ID NO: 2;

[0058] Upstream outer primer: having the nucleotide sequence shown in SEQ ID NO: 3;

[0059] Downstream outer primer: having the nucleotide sequence shown in SEQ ID NO: 4;

[0060] DNA rapid extraction solution: 0.1mol / L NaOH and 1% Triton X-100.

[0061] 2. Detection method

[0062] Step 1: Place the sample to be tested in a sterile throat swab, add 1.0mL DNA rapid extraction solution into the throat swab tube, mix well, discard the cotton swab, heat the throat swab tube with an alcohol lamp until it boils, and keep it for 1 minute;

[0063] Step 2: Centrifuge at 4000 rpm / centrifuge for 5 minutes, take the supernatant into a 1.5mL EP tube, wh...

Embodiment 3

[0068] Embodiment 3: kit of the present invention

[0069] 1. Kit

[0070] The test kit for detecting bacillus tetani described in the present invention comprises:

[0071] Upstream inner primer: 10 μmol / L, having the nucleotide sequence shown in SEQ ID NO: 1;

[0072] Downstream inner primer: 10 μmol / L, having the nucleotide sequence shown in SEQ ID NO: 2;

[0073] Upstream outer primer: 1.5 μmol / L, having the nucleotide sequence shown in SEQ ID NO: 3;

[0074] Downstream outer primer: 1.5 μmol / L, having the nucleotide sequence shown in SEQ ID NO: 4;

[0075] DNA rapid extraction solution: 0.3mol / L NaOH and 2% Triton X-100.

[0076]Constant temperature amplification solution: 20mmol / L Tris-HCl of pH8.8, 10mmol / L of KCl, 10mmol / L of (NH 4 ) 2 SO 4 , 2mmol / L of MgS0 4 , 0.1% Triton X-100, 40mmol / L dNTP, 320U / mL Bst DNA polymerase.

[0077] 2. Detection method

[0078] Step 1: Place the sample to be tested in a sterile throat swab, add 1.0mL DNA rapid extraction soluti...

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Abstract

The invention relates to the field of molecular biology, and particularly discloses a method and a kit thereof for detecting clostridium tetani by using a loop-mediated isothermal amplification method. The method comprises the following steps of: amplifying clostridium tetani spasm toxin genes in a sample DNA by using loop-mediated isothermal amplification technology and using an upstream inner primer, a downstream inner primer, an upstream outer primer and a downstream outer primer, wherein the nucleotide sequences of the primers are expressed as SEQ ID No: 1-4; and detecting the amplification results. The kit comprises the upstream inner primer, the downstream inner primer, the upstream outer primer, the downstream outer primer, DNA quick extraction liquid and isothermal amplification liquid. The method and the kit thereof for detecting the clostridium tetani by using the loop-mediated isothermal amplification method have the advantages of high clostridium tetani specificity, high sensitivity and simple and quick detection process.

Description

technical field [0001] The present invention relates to the field of molecular biology, and more specifically relates to a method for detecting tetani bacillus by using a loop-mediated constant temperature amplification method, and also relates to a kit for detecting tetani bacillus by using a loop-mediated constant temperature amplification method . Background technique [0002] Tetanus is an infectious disease caused by Bacillus tetani. It is an acute specific infection caused by Bacillus tetani invading human wounds and multiplying and producing exotoxins in the wound. Clinically, the patient's systemic or local muscle persistent Convulsions and paroxysmal convulsions are the main ones. [0003] Bacillus tetani is an obligate anaerobic Clostridium bacterium, Gram staining is positive, and it is an opportunistic pathogenic bacteria. The bacteria can only be infected under anaerobic conditions or deep wounds accompanied by aerobic bacteria infection Easy to grow and repro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 蒲晓允蒋栋能项贵明王左
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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