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Kit for detecting primer of bacillus anthracis and bacillus anthracis by double LAMP (Loop-Mediated Isothermal Amplification) and quick visual detection method

A Bacillus anthracis and kit technology, applied in the biological field, can solve the problems of inconvenience, increased use cost, false detection of Bacillus cereus, etc., and achieve the effect of low cost and reliable results

Inactive Publication Date: 2014-01-29
中国人民解放军南京军区军事医学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current detection method faces two main problems: one is how to quickly determine whether there is anthrax bacillus at the scene of the incident in relatively poor conditions; The lives of patients, and at the same time determine the mandatory isolation and epidemic prevention measures for the target population, etc.
[0009] However, almost all the LAMP methods currently used in pathogen detection have a key problem: the detection method of LAMP amplification products is mainly to open the cover after the amplification and add a chromogenic solution such as fluorescent dye SYBR Green I, but in the process, Subsequent false positive results due to nucleic acid aerosol contamination are difficult to avoid
However, the kit can only detect whether the nucleic acid source contains the Bacillus anthracis plasmid PX01, that is to say, it is possible to falsely detect Bacillus cereus containing the toxic plasmid PX01
In addition, in order to avoid aerosol pollution in this patent, special reaction tubes must be used, but in this way, users must purchase additional reaction tubes, which increases the cost of use, which is very inconvenient

Method used

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  • Kit for detecting primer of bacillus anthracis and bacillus anthracis by double LAMP (Loop-Mediated Isothermal Amplification) and quick visual detection method
  • Kit for detecting primer of bacillus anthracis and bacillus anthracis by double LAMP (Loop-Mediated Isothermal Amplification) and quick visual detection method
  • Kit for detecting primer of bacillus anthracis and bacillus anthracis by double LAMP (Loop-Mediated Isothermal Amplification) and quick visual detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: lef, gyrB primer sequence set

[0045] First, the lef gene sequence of the Bacillus anthracis plasmid PX01 and the gyrB gene sequence of the Bacillus anthracis chromosome were searched in the NCBI (National Center for Biotechnology Information) GenBank database, and the sequences were compared by ClustalX software to obtain the specificity of the two genes Conserved sequences; then, use LAMP primer design software (Primer Explorer software, version 4.0) to design LAMP primers for the above-mentioned specific conserved sequences, and obtain lef and gyrB primer sequence sets that can be applied to the same reaction system.

[0046] The lef primer sequence set includes the outer primer pair LF-F3 and LF-B3, and the inner primer pair LF-FIP and LF-BIP. The sequence of LF-F3 is shown in SEQ ID NO:1; the sequence of LF-B3 is shown in SEQ ID NO:2; the sequence of LF-FIP is shown in SEQ ID NO:3; the sequence of LF-BIP is shown in SEQ ID NO:4 shown.

[0047] The ...

Embodiment 2

[0048] Embodiment 2: the kit that detects Bacillus anthracis with LAMP method

[0049] The kit of this example includes the lef primer sequence set and the gyrB primer sequence set of Example 1. Wherein, the primer mixture composed of the lef primer sequence set is placed in the I tube, and the molar ratio of the outer primer pair LF-F3 and LF-B3 to the inner primer pair LF-FIP and LF-BIP is 1:(5-10) , preferably 1:8; the primer mixture composed of the gyrB primer sequence set is placed in tube II, the outer primer pair gy-F3 and gy-B3, the inner primer pair gy-FIP and gy-BIP, and the loop primer pair gy- The molar ratio of LF and gy-LB is 1:(5-10):(2-6), preferably 1:8:4.

[0050] For example: in tube I, each 2.5 μl primer mixture contains 4 pmol LF-F3, 4 pmol LF-B3, 32 pmol LF-FIP, and 32 pmol LF-BIP; in tube II, each 2.5 μl primer mixture contains 4 pmol gy-F3, 4 pmol gy-B3, 32 pmol gy-FIP, 32 pmol gy-BIP, 16 pmol gy-LB, and 16 pmol gy-LF.

[0051] The test kit of this e...

Embodiment 3

[0057] Example 3: A rapid visual detection method for detecting Bacillus anthracis with the LAMP method

[0058] The detection method of this embodiment uses the kit of Embodiment 2 for detection.

[0059] The detection method of this embodiment comprises the following steps:

[0060] Step 1: Add the same volume of 2×reaction buffer of the kit, the primer mixture of tube I or tube II, BstDNA polymerase, chromogenic solution, and ultrapure water into the transparent reaction tubes A and B respectively, and then mix them. Obtain reaction solution A and B with the same volume; add the primer mixture of tube I to reaction tube A, and add the primer mixture of tube II to reaction tube B; kit 2×reaction buffer, primers of tube I or tube II The volume ratio of the mixed solution, BstDNA polymerase, chromogenic solution and ultrapure water is (8-12): (1.5-3.5): (0.5-1.5): (0.3-0.9): (3.1-4.7);

[0061] For example, reaction solution A can be obtained as follows:

[0062] R...

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Abstract

The invention relates to a kit for detecting a primer of bacillus anthracis by an LAMP (Loop-Mediated Isothermal Amplification) method and detecting the bacillus anthracis by double LAMP and a quick visual detection method for detecting the bacillus anthracis by double LAMP as well as a check method for the kit. The primer comprises a lef primer sequence set and a gyrB primer sequence set and can be used for detecting a bacillus anthracis plasmid pX01 and a bacillus anthracis chromosome. The kit comprises the primer and can obtain an accurate detection result. The detection method is carried out by adopting the kit and can realize the visual detection with opening a cover; and the false positive is avoided and no expensive real-time turbidity meter is needed. According to the check method, a reliable result can be obtained by using a normal kit. The kit and the detection method disclosed by the invention are reliable in result and relatively-low in cost and are especially suitable for health emergency places with rough conditions.

Description

technical field [0001] The invention relates to a primer for detecting Bacillus anthracis by a LAMP method, a kit containing the primer, a detection method using the kit, and a testing method for the kit, belonging to the field of biotechnology. Background technique [0002] Bacillus anthracis is a zoonotic pathogen that can infect humans and animals through skin contact, respiratory tract and digestive tract. Bacillus anthracis can spread through air droplets and can form aerosols, which can easily cause public health emergencies; while Bacillus anthracis spores can survive for more than ten years, which can constitute a public health hazard. Bacillus anthracis is a serious threat to human health and life safety, and its detection and prevention have always been paid attention to. [0003] However, the current detection method faces two main problems: one is how to quickly determine whether there is anthrax bacillus at the scene of the incident in relatively poor condition...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 张锦海王长军谭维国吕恒曹勇平
Owner 中国人民解放军南京军区军事医学研究所