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Primers, probes, method and kit for detecting vomit-type Bacillus cereus

A Bacillus cereus, detection method technology, applied in the field of microbial detection, can solve problems such as missed detection, and achieve the effect of strong specificity, high conservation and specificity, high specificity

Inactive Publication Date: 2013-12-18
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

Since Bacillus cereus has a variety of genes with different characteristics, only a single test for one gene can easily lead to missed detection

Method used

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  • Primers, probes, method and kit for detecting vomit-type Bacillus cereus
  • Primers, probes, method and kit for detecting vomit-type Bacillus cereus
  • Primers, probes, method and kit for detecting vomit-type Bacillus cereus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Design and synthesis of primer probes for Bacillus cereus gyrB gene and vomitoxin synthase cesB gene

[0080] Find the full-length sequences of the Bacillus cereus gyrB gene and the vomitoxin synthase cesB gene in Genbank, import all the full-length sequences of the gyrB gene and cesB gene into the Mega4 software, and use the Alignment function to compare and analyze the conservation of the gyrB gene and the cesB gene Sequence, to obtain the conserved sequence segment.

[0081] The base sequence of the gyrB gene is shown in SEQ ID No.1, and the base sequence of the cesB gene is shown in SEQ ID No.2.

[0082] Input the conserved sequences of gyrB gene and cesB gene into Primer Express3.0 software, and automatically analyze and obtain various primer probe design schemes. On this basis, manually adjust the primer probe position and sequence length to avoid hairpin structures, primer dimers and false priming. BLAST analysis was performed on the primer and probe ...

Embodiment 2

[0086] Embodiment 2 gyrB gene and cesB gene primer probe screening test

[0087] The alternatives of primer probes for gyrB gene and cesB gene in Table 2 were screened, and the specificity and sensitivity of the primer probes were mainly evaluated.

[0088] Specificity evaluation: Bacillus thuringiensis, Bacillus mycoides, Bacillus wei, Bacillus pseudomycota, Bacillus anthracis, Bacillus megaterium, Bacillus subtilis, non-pathogenic Escherichia coli ATCC25922, Sakazaki Enterobacter, Shigella, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Listeria monocytogenes, Vibrio cholerae, Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica Bacteria and other food-borne pathogenic bacteria are used as strains for specificity evaluation. DNA templates were obtained from the above-mentioned bacteria using a bacterial genome extraction kit. Take 10 μL of the extract from each bacterium, mix it into a comprehensive DNA template, and use it as a template for speci...

Embodiment 3

[0101] Embodiment 3 Formation of Bacillus cereus real-time fluorescent PCR detection kit

[0102] The kit consists of reaction system buffer, DNA polymerase, dNTP, MgCl 2 , primer-probe mixture, positive control, and ultrapure water, the specific components of which are as follows:

[0103] Reaction system buffer: DNA polymerase: 5U / μL; 10×Taq Buffer with (NH 4 ) 2 SO 4 (100mM Tris-HCl (pH8.8), 750mM (NH 4 ) 2 SO 4 ) 2.5 μL; 10 mM dNTPs; 25 mM MgCl 2 ; Primer-probe mixture (12.5 μM for each primer, 7.5 μM for each probe), positive control (10 5 CFU / mL Bacillus cereus template).

[0104] The reaction system detected by the kit is 25 μL, and its configuration is as follows:

[0105]

[0106] Set up sample detection system, negative control, positive control and blank control. The total DNA to be tested was added to the sample detection system, the total DNA of non-target bacteria was added to the negative control, the positive control in the kit was added to the pos...

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Abstract

The invention provides real-time fluorescence quantitative PCR (polymerase chain reaction) primers and probes for detecting vomit-type Bacillus cereus, and a method for detecting the vomit-type Bacillus cereus by using the primers and probes. The nucleotide sequences of the primers and probes are respectively disclosed as SEQ ID NO.3 through SEQ ID NO.8. The invention also provides a kit for detecting the vomit-type Bacillus cereus. The detection method and kit thereof have the advantages of high detection accuracy, high sensitivity and specificity, are simple, convenient and quick, and have favorable clinical specimen detection capability.

Description

technical field [0001] The invention belongs to the field of microbial detection, and relates to a primer, a probe, a method and a kit for detecting Bacillus cereus Background technique [0002] Bacillus cereus, an aerobic, mesophilic, spore-forming bacillus, is a common contaminating bacterium in food and cosmetics. The bacterium is widely distributed in nature, and almost all kinds of food have been reported to be related to food poisoning caused by Bacillus cereus. Bacillus cereus food poisoning can be clinically divided into two types: vomiting type and diarrhea type. In Asia, vomiting type is the main type, while in Europe and North America, diarrhea type is more common. Some non-gastrointestinal infections. Because the symptoms of food poisoning caused by Bacillus cereus are usually mild and do not exceed 24 hours, a large number of sporadic Bacillus cereus food poisoning incidents have not been reported, and the number of Bacillus cereus infections has been greatly ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 王雷莫莎王晓艳张志强
Owner 北京卓诚惠生生物科技股份有限公司
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