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Fluorescent quantitative PCR primer probe and kit and method for detecting three kinds of bacillus

A bacillus, primer probe technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of manpower consumption, reagents and time, complicated process, and insufficient detection sensitivity , to save time and increase sensitivity

Active Publication Date: 2019-05-21
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the fluorescent quantitative PCR method, isothermal amplification PCR method, and liquid chip technology for detecting three kinds of bacteria are only detection and identification technologies for a single target. During operation, detection needs to be carried out one by one, which consumes manpower, reagents and time. Do a good job of quality control in one test to ensure the uniformity of the three test results
The rapid detection technology represented by immunochromatography technology has shortcomings such as insufficient detection sensitivity, complex process, and inability to detect multiple targets in the same reaction system.
There is no report on the simultaneous identification of the species of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis using multiplex fluorescent quantitative PCR

Method used

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  • Fluorescent quantitative PCR primer probe and kit and method for detecting three kinds of bacillus
  • Fluorescent quantitative PCR primer probe and kit and method for detecting three kinds of bacillus
  • Fluorescent quantitative PCR primer probe and kit and method for detecting three kinds of bacillus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 Formation of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis triple fluorescent quantitative PCR detection kit:

[0062] The kit consists of 5× reaction system buffer, Hotstar DNA polymerase, 10× primer-probe mixture, positive control, and ultrapure water. The specific components are as follows: 5×PCR buffer (Tris-HCl 100Mm (pH 8.3), KCl 100mM, Tween-20 0.2%, pET28a 0.0003ng, 5mM dNTPs, 20mM MgCl 2 ); 25× Hotstar DNA polymerase (2U / μL); 10× primer-probe mixture (as shown in Table 1, the concentration of each primer including the positive internal control (pET28a plasmid template) is 3 μM, each probe The concentration of the needle is 2μM); Positive control (mixed template of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, each is 10 6 CFU / ml).

[0063] The kit detection reaction system is 25 μL, and its configuration is as follows: 5×PCR Buffer 5 μL; 25×DNA polymerase 1 μL; 10× primer mixture 2.5 μL; template 2 μL, ultrapure...

Embodiment 2

[0064] Operation and result judgment of embodiment 2 kit

[0065] 1. Genome extraction

[0066] Take 1 mL of enrichment solution from food samples, centrifuge at 13,000 rpm for 5 minutes, discard the supernatant, add proteinase K to digest at 55°C for 1 hour, and use the boiling method or a commercial kit to extract the DNA of the target bacteria to be tested as a test sample.

[0067] 2. Preparation of reaction system

[0068] Take a 200 μL PCR tube and configure a 25 μL reaction system, which is configured as follows: 5 μL of 5×PCR Buffer; 1 μL of 25× DNA polymerase; 2.5 μL of 10× primer mix; 2 μL of template, and 14.5 μL of ultrapure water.

[0069] 3. PCR reaction

[0070] Put the PCR tube into the CFX96 fluorescent quantitative PCR instrument, and perform the PCR reaction according to the following procedure: 95°C for 10 min; 95°C for 15 s, 55°C for 30 s, cycle 10 reactions; 95°C 15 s, 60°C 30 s, cycle 30 reactions, Amplification results such as figure 1 shown.

[00...

Embodiment 3

[0077] The shelf life test of embodiment 3 kits

[0078] to 10 5 CFU / mL The mixed template of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis is the test sample for evaluation. On day 0, it is divided into 9 parts and stored in a -70°C refrigerator. The completed kits were stored at -20°C, and kits of 0, 10, 15, 30, 60, 90, 120, 150, 180, and 360 days were used for storage test. The test results of the shelf life are shown in Table 3:

[0079] Table 3 shelf life test results

[0080] shelf life

[0081] It can be seen from Table 3 that the kit is stored in a -20°C refrigerator, and it is effective in detecting the three target bacteria at different storage periods. The experimental results show that the storage period of the kit is at least 6 months.

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Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) primer probe set, a kit and a method for detecting three bacilli. The primer probe set comprises a primer and a probe, wherein the primer has a nucleotide sequence shown as SEQ ID NO. 1-6, and the probe has a nucleotide sequence shown as SEQ ID NO. 7-9. The kit is used for detecting the three bacilli by fluorescent quantitative PCR and comprises the fluorescent quantitative PCR primer probe set and Hotstar DNA (deoxyribonucleic acid). By the aid of the technical scheme, sensitivity, specificity, simplicity and convenience of simultaneously detecting the three bacilli are remarkably improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fluorescent quantitative PCR primer probe set, a kit and a method for detecting three kinds of bacillus. Background technique [0002] Bacillus is an important class of food-borne pathogens, which can easily cause food and cosmetic contamination, and then infect humans to cause disease. The Bacillus cereus group in the Bacillus genus is of great significance to the prevention and control of food safety pollution. Among them, Bacillus anthracis is the most important pathogenic bacteria, which can cause severe pulmonary anthrax, intestinal anthrax, skin anthrax, etc., and patients It is highly contagious. In addition to contact infection, the disease can also be caused by eating meat contaminated with this bacteria. Bacillus cereus is the second pathogenic bacterium in this group, and the food poisoning caused by it is universal, mainly because Bacillus cereus widely exists in soil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 王雷林笑冬张志强
Owner 北京卓诚惠生生物科技股份有限公司
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