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Kit for rapidly detecting francisella tularensis

A technology of tularemia and kits, applied in the field of molecular biology, can solve the problems of long detection period and difficulty in popularization and application, and achieve the effects of low cost, reducing the difficulty of experimental operation, and eliminating pollution

Pending Publication Date: 2019-12-17
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of tularemia depends on the specificity of the experiment, and its detection cycle is long, so it is difficult to popularize and apply in the actual work that requires rapid detection

Method used

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  • Kit for rapidly detecting francisella tularensis
  • Kit for rapidly detecting francisella tularensis
  • Kit for rapidly detecting francisella tularensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Kit preparation

[0060] In this embodiment, Chelex-100, betaine, Sigma company; Manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, ammonium sulfate, Sinopharm Chemical Reagent Co., Ltd.; Tris-HCl, Shanghai Shengke Biotechnology Co., Ltd.; TritonX-100, Beijing Milebo Medical Technology Co., Ltd.; dNTP, Pharmacia Corporation.

[0061] According to GeneBank No.: KF607098.1 disclosed tularensis outer membrane protein gene fopA, a specific primer pair for rapid detection of tularensis is designed, and the nucleotide sequence of the primer pair is as follows:

[0062] FP: 5'-CCTTTTGCAAATACTTATAGCGCTTGCCAGTTTCTATCTTGA GGA-3'; SEQ ID NO.1;

[0063] BP: 5'-CCTTTTGCAAATACTTATAGCTTATCGATACGTCAGCAA ACAC-3'; SEQ ID NO.2;

[0064] The kit in this example includes a reaction solution with a pH of 8.8, dNTPs, BstDNA polymerase, FP and BP primers, and an indicator or fluorescent dye.

[0065] In the final reaction system: dNTPs 1.4mM, BstDNA polymeras...

Embodiment 2

[0073] In this example, the kit in Example 1 is used to detect environmental samples. Among them, soak the sterile sampling cotton swab with sterile physiological saline or Chelex lysate, wipe the surface of the contaminated object, and put it into the processing tube containing 2mL Chelex lysate, break the cotton swab rod, cover the processing tube tightly and mix well, Place in an intelligent constant temperature detector at 95°C and heat for 10 minutes to obtain a sample lysate, then take it out and wait for inspection.

[0074] Open the test tube of the experimental group, tear off the covering film, take 14 μL of the sample lysate as the DNA template, divide it into 2 drops, add it dropwise to the test tube cap of the test tube of the experimental group, fasten the cap of the test tube, and invert for about 10 seconds. Set up positive and negative controls at the same time. Pick up the test tubes, positive control tubes, and negative control tubes of the experimental gro...

Embodiment 3

[0077] Take the plasmid clone containing the T. tularensis gene as the detection object, calculate the copy number and perform 10-fold dilution, so that the final nucleic acid concentration is 10 9 copies / μl, 10 8 copies / μl, 10 7 copies / μl, 10 6 copies / μl, 10 5 copies / μl, 10 4 copies / μl, 10 3 copies / μl, 10 2 copies / μl, 10 1 copies / μl, 10 0 copies / μl, and double distilled water was used as a negative control. Utilize the kit in embodiment 1 to detect then; Adopt chromogenic method (adding pH indicator) and fluorescence method respectively (setting absorption wavelength is about 497nm, emission wavelength is about 520nm maximum, is green fluorescence; Reactor is placed In the fluorescence quantitative instrument) to detect, the results are as follows figure 2 , 3 shown.

[0078] Such as figure 2 , the concentration of nucleic acid from left to right is 10 9 copies / μl, 10 8 copies / μl, 10 7 copies / μl, 10 6 copies / μl, 10 5 copies / μl, 10 4 copies / μl, 10 3 copies...

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Abstract

The invention discloses a kit for rapidly detecting francisella tularensis. The kit comprises reaction liquid with the pH being 8.8, dNTPs, BstDNA polymerase, FP and BP primers, an indicator and a sealing agent. The kit for rapidly detecting the francisella tularensis utilizes the two primers to specifically identify an independent region of a target fragment, has high specificity and high sensitivity of nucleic acid detection, meanwhile has the characteristics of being quick in reaction and easy to operate, not depending on large-scale instruments and the like, and is suitable for rapid detection in a laboratory and more suitable for being used on site, and the site quarantine inspection and detection demands under the large-human-traffic and rapid custom clearance background can be met.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for rapid detection of tularemia. Background technique [0002] Francisella tularensis (Francisella tularensis), also known as Francisella tularensis, is a Gram-negative polymorphic small coccus (0.2-0.7μm×0.2μm), generally 0.3-0.5μm in size, and not Spore-forming, aerobic. Tularensis include tularensis (type A), holarctica (type B), novicida and mediasiatica four subspecies. Most tularaemias in humans are caused by types A and B of tularemia, and the reservoir hosts of tularemia are mainly rabbits and hares (type A) and rodents (type B). Type A is mainly transmitted by ticks and blood-sucking insects, while surface water contaminated by rodents is an important source of infection for Type B. [0003] Tularemia, also known as tularemia, is caused by Francisella tularensis, an acute, infectious zoonotic disease that occurs in most countries in the northern hemisphere, and...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 王静张乔刘丽娟慈颖刘威张晓龙杨燕孙筱霞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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