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Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof

A technology of Bacillus cereus and virulence genes, applied in the biological field, can solve the problems of reducing workload, low detection sensitivity, and impossibility, and achieve the effects of time saving, high conservation and specificity

Active Publication Date: 2017-02-22
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] Ordinary single-plex PCR method can realize multi-gene detection by distinguishing the size of the amplification product, but the detection sensitivity is not high; real-time fluorescent PCR method and isothermal amplification PCR method have high detection sensitivity, but the simultaneous detection of multiple target virulence genes The quantity is limited, and the purpose of reducing the workload cannot be achieved

Method used

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  • Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof
  • Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof
  • Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Formation of Bacillus cereus virulence gene multiplex PCR detection kit

[0084] The kit consists of 2× reaction system buffer, DNA polymerase, 10× primer mixture, positive control, and ultrapure water. The specific components are as follows:

[0085] According to the sequences shown in Table 2, primers of SEQ ID NO.1 and 3-26 were synthesized. Configure multiplex PCR reaction system: 2×PCR Buffer (Tris HCl 40mM (pH8.3), KCl 100mM,

[0086]tween-20 0.08%, 0.0006ng / μL pet28a, 1.5mM dNTP, 8mM MgCl 2 ); 25×DNA polymerase (2U / μL); 10×primer mixture (concentration of each long primer pair including IAC is 2μM,

[0087] Homologous universal primer SEQ ID NO 1 concentration is 8μM)); positive control (Bacillus cereus containing 9 enterotoxins (purchased from China Medical Culture Collection Center, No. 63303). The gene contained is hemolysin BL gene A, hemolysin B L gene C, hemolysin B L gene D, non-hemolytic enterotoxin Nhe gene A, non-hemolytic enterotoxin Nhe g...

Embodiment 2

[0089] Operation and result judgment of embodiment 2 kit

[0090] 1. Genome extraction

[0091] Take the enrichment solution or streak-cultured colonies (cultured colonies from the two strains of the above-mentioned positive control) and dissolve them in water, and adjust the concentration of the bacterial suspension to 10 with a turbidimeter. 8 CFU / ml, take 1ml of bacterial suspension, centrifuge at 13000rpm for 3min, resuspend the bacteria in extraction buffer (1.7% SDS, 200mM Tris, pH 8, 20mM EDTA, 200mM NaCl), add proteinase K, and incubate at 55°C for 1h. After 10 minutes in boiling water bath, ice bath, centrifuge at 13000rpm for 10 minutes, and take the supernatant as a template for kit detection.

[0092] 2. Preparation of reaction system

[0093] Take a 200 μL PCR tube and configure a 25 μL reaction system, which is configured as follows: 2×PCR Buffer 12.5 μL; DNA polymerase 1 μL; 10× primer mix 2.5 μL; template 5 μL, ultrapure water 4 μL.

[0094] 3. PCR reaction ...

Embodiment 3

[0104] The shelf life test of embodiment 3 kit

[0105] to 10 5 The CFU / mL template mixture of Bacillus cereus containing 9 enterotoxin genes and Bacillus cereus containing cesB vomitoxin gene is the test sample for evaluation. On day 0, it is divided into 9 parts and stored at -70℃ in the refrigerator. The completed kits were stored at -20°C, and the 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 4:

[0106] Table 4 shelf life test results

[0107]

[0108]

[0109] It can be seen from Table 4 that the kit was stored in a refrigerator at -20°C, and the 12 target genes of Bacillus cereus were all positive during different storage periods. The experimental results showed that the storage period of the kit was at least 6 months.

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Abstract

The invention provides a primer set for detecting bacillus cereus virulence genes by means of a multiplex-PCR. The primer set comprises primers shown in SEQ ID NO.1 and 3-24. The invention further provides a detection method and kit for detecting bacillus cereus virulence genes by means of the multiplex-PCR. According to the technical scheme, the perfect technological means is provided for rapid and accurate screening of the bacillus cereus virulence genes, rapid screening and identification of pathogenicity bacillus cereus after a food poisoning event and outbreak of an epidemic situation can be achieved, and the reliable basis is provided for reasonable and appropriate epidemic treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set and a kit for multiplex PCR detection of Bacillus cereus virulence genes and a detection method thereof. Background technique [0002] Bacillus cereus is a species in Group I of the genus Bacillus, and its biochemical characteristics are very similar to Bacillus thuringiensis, Bacillus mycoides, Bacillus anthracis and Bacillus megaterium. Salt reduction test, casein decomposition test, lysozyme tolerance test, V-P test, glucose utilization (anaerobic) test, root growth test, hemolysis test, protein toxin crystallization test and other biochemical tests can distinguish Bacillus cereus from the same genus. other kinds of distinctions. However, traditional biochemical methods cannot distinguish strains containing pathogenic factors. [0003] The application of molecular biology methods can not only identify the species of Bacillus cereus, but also obtain the information o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/085
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
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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