Recombinant standard plasmid for detecting anthrax bacillus, kit and construction method for the plasmid

A technology of Bacillus anthracis and standard plasmid, applied in the field of bioengineering, can solve the problems of difficulty in general use, difficulty in detection sensitivity of kit detection performance, and difficulty in selecting a kit that meets your own needs, and achieves high specificity and high sensitivity. Effect

Inactive Publication Date: 2011-11-16
中国人民解放军南京军区军事医学研究所
View PDF1 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This makes it difficult for the reference plasmids of each kit to be commonly used in other kits, and it is difficult to objectively compare the detection performance (such as detection sensitivity, etc.) of each kit, making it difficult for people to choose a kit that meets their needs.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant standard plasmid for detecting anthrax bacillus, kit and construction method for the plasmid
  • Recombinant standard plasmid for detecting anthrax bacillus, kit and construction method for the plasmid
  • Recombinant standard plasmid for detecting anthrax bacillus, kit and construction method for the plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Recombination of standard plasmid PGEM-T-easy-RCP

[0051] The recombinant standard plasmid for detecting Bacillus anthracis of this embodiment is as figure 1 As shown, it consists of the PGEM-T easy vector and the PA gene, capA gene, and rpoB gene in series, denoted as PGEM-T-easy-RCP; wherein the PA gene sequence is shown in SEQ.ID.No.1, capA The gene sequence is shown in SEQ.ID.No.2, and the rpoB gene sequence is shown in SEQ.ID.No.3.

[0052] The 5'end of the PA gene is connected to the PGEM-T easy vector via the Sal I restriction enzyme cutting site, and the 3'end of the PA gene is connected to the 5'end of the capA gene via the HindIII restriction enzyme cutting site; the 3 of the capA gene The'end is connected to the 5'end of the rpoB gene through the Nhe I restriction enzyme cut site; the 3'end of the rpoB gene is connected to the PGEM-T easy vector through the Sac I restriction enzyme cut site; among them, HindIII restriction enzyme cuts The site sequenc...

Embodiment 2

[0054] Example 2: Construction of recombinant standard plasmid PGEM-T-easy-RCP

[0055] In this example, a method for constructing a recombinant standard plasmid for detecting Bacillus anthracis was used to construct a recombinant standard plasmid PGEM-T-easy-RCP, and the method includes the following steps:

[0056] (1) Design primer pairs: design primer pairs pa-F and pa-R for PA gene sequence; design primer pairs capa-F and capa-R for capA gene sequence; design primer pairs rpob-F and rpob- for rpoB gene sequence R;

[0057] The PA gene sequence is shown in SEQ.ID.No.1, the capA gene sequence is shown in SEQ.ID.No.2, the rpoB gene sequence is shown in SEQ.ID.No.3; the sequence of pa-F is shown in SEQ. ID.No.9, the sequence of pa-R is shown in SEQ.ID.No.10, the sequence of capa-F is shown in SEQ.ID.No.11, and the sequence of capa-R is shown in SEQ.ID. No. 12, the sequence of rpob-F is shown in SEQ. ID. No. 13, and the sequence of rpob-R is shown in SEQ. ID. No. 14;

[0058] (2) Am...

Embodiment 3

[0093] Example 3: Kit for routine PCR detection of Bacillus anthracis

[0094] The kit of this example includes the recombinant standard plasmid PGEM-T-easy-RCP of the present invention (including but not limited to the recombinant standard plasmid of Example 1).

[0095] The kit of this embodiment also includes a DNA extraction solution containing NaOH at a concentration of 20-30 mM (preferably 25 mM), Tris-HCl at a concentration of 5-15 mM (preferably 10 mM), and a volume percentage of 0.5-1.5% ( Preferably 1%) Triton X-100, 0.5-1.5% by volume (preferably 1%) NP-40, EDTA with a concentration of 0.05-0.15mM (preferably 0.1mM), 1-3% by weight (preferably 2%) Chelex-100. It should be noted that if other existing commercial DNA extraction kits are used to extract DNA, the kit of this embodiment may not include the DNA extraction solution.

[0096] The kit of this embodiment also includes 2×Premix Taq V2.0 (premix containing reagents required for PCR, and dNTPs composed of dATP, dTTP,...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to recombinant standard plasmids for detecting anthrax bacillus. The plasmid comprises a PGEM-T easy carrier and cascaded PA genes, capA genes and rpoB genes and is recorded as PGEM-T-easy-PCR. The invention further relates to a kit containing the recombinant standard plasmids. The invention also relates to a construction method for the recombinant standard plasmids, and themethod comprises the steps of designing primer pairs, amplifying sequences, constructing first intermediate plasmids, constructing second intermediate plasmids and constructing the recombinant standard plasmids. The recombinant standard plasmid PGEM-T-easy-PCR provided in the invention can be used as a substitute of a positive standard sample of anthrax bacillus (ie. nucleic acid of a virulent strain of anthrax bacillus).

Description

Technical field [0001] The invention relates to a recombinant standard plasmid, a kit for detecting pathogenic microorganisms, and a method for constructing the plasmid, in particular to a recombinant standard plasmid, a kit for detecting Bacillus anthracis, and a method for constructing the plasmid, belonging to bioengineering Technical field. Background technique [0002] Bacillus anthracis can cause anthracnose in sheep, cattle, horses and other animals and humans. Among them, pulmonary anthracnose has been listed as a national legal infectious disease (the highest level) in China. Bacillus anthracis can be spread through air droplets and can form aerosols, which can easily cause public health emergencies and seriously threaten public property, health and life safety. [0003] Fast and effective pathogen detection technology plays an extremely critical role in the prevention, control and emergency response decision-making of anthrax, and is of great significance to the protecti...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/70C12R1/19C12R1/645
Inventor 张锦海王忠灿王长军
Owner 中国人民解放军南京军区军事医学研究所
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap