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A CRISPR detection method and kit for Cronobacter in food

A technology of Cronobacter and detection method, which is applied in the field of CRISPR detection method of Cronobacter in food and its kit, can solve problems such as not causing academia, improve international discourse power and save glue making time , the effect of short operation time

Active Publication Date: 2022-03-01
SHANGHAI INST OF QUALITY INSPECTION & TECHN RES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The discovery of the CRISPR / Cas system can be traced back to 1987, when Nakata et al first discovered the tandem spaced repeat sequence in the genome of Escherichia coli, but it did not attract the attention of the academic community at that time

Method used

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  • A CRISPR detection method and kit for Cronobacter in food
  • A CRISPR detection method and kit for Cronobacter in food
  • A CRISPR detection method and kit for Cronobacter in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Design of primers and probes

[0037] In this example, through genome comparison and candidate target selection, the 16S rRNA gene conservative sequence, Cronobacter strain-specific key gene sequence, coding pathogenicity-related gene sequence, and other literature reports can distinguish strain-specific sequence candidate regions , design 2-3 pairs of amplification primers and guide RNA targeting sequences for each region to conduct bioinformatics tests, select the combination with the highest efficiency and best specificity, and perform sequence analysis and alignment through NCBI online tools, using Prime Express Software V3.0 (ABI, Foster City, CA, USA) designed 4 pairs of primers and guide RNA combinations, after specificity and sensitivity tests, the results are shown in Figure 1-Figure 4. Finally, a pair of primer3 and crRNA-pcr3-1 was screened out, and the sequences are shown in Table 1. The reporter probe of the CRISPR reaction is a single-stranded DNA s...

Embodiment 2

[0060] Embodiment 2 Specificity, inclusiveness and exclusive detection

[0061] (1) specificity

[0062] First, PCR was used to detect the genomic DNA of Cronobacter genus (ATCC25944), and all primers (primer3-F / R, primer4-F / R, primer6-F / R and primer7-F / R) and probes in Table 1 were found. The needles can effectively amplify the target gene. In order to ensure the specificity of the amplification reaction, the standard strains of Cronobacter genus listed in Table 5 and Table 6, as well as the DNA of other common Vibrio and food-borne pathogenic bacteria were detected. It was found that only the primer3-F / R pair of primers showed positive amplification signals for all Cronobacter species, while other common Vibrio and food-borne pathogenic bacteria had no obvious amplification signals (see Figure 1-Figure 5 ).

[0063] The results showed that: primer3-F / R primer-probe set had good specificity. And the fluorescence intensity also increases with the increase of the number of...

Embodiment 3

[0073] Embodiment 3 pure bacteria liquid detection limit

[0074] 10-fold gradient dilution of Cronobacter genus (ATCC 25944) pure bacterial liquid, respectively pipetting 2mL to extract DNA for detection, the results found that 10 3 ( Figure 6 , Table 7).

[0075] Table 7 Detection sensitivity of CRISPR method against targets of Cronobacter genus

[0076] Bacterial concentration repeat 1 repeat 2 repeat 3 Repeat 4 Repeat 5 10 7

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Abstract

The invention discloses a CRISPR detection method of Cronobacter in food, comprising: step 1, performing PCR amplification with the DNA of a sample to be tested as a template to obtain a PCR amplification product; a reaction system for PCR amplification Containing specific primer pairs for amplifying components of the genus Cronobacter; step 2, adding the PCR amplification product to the CRISPR reaction system for CRISPR reaction, the CRISPR reaction system includes Cas12b protein, Cas12b-tracrRNA, crRNA and probes; step 3 1. Detect the change of the fluorescence intensity of the CRISPR reaction, and judge whether there is Cronobacter nucleic acid in the sample to be detected according to the curve. The invention also discloses a CRISPR kit for detecting Cronobacter in food and its application. By adopting the CRISPR detection method of the present invention, the detection specificity of Cronobacter can be improved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology technology CRISPR, and specifically relates to a CRISPR detection method and a kit thereof for Cronobacter in food. Background technique [0002] Food safety is a major strategic issue related to people's life and health and social harmony. Infant formula food is the only food source for some infants and young children, which has an important impact on the health and life safety of infants and young children. Cronobacter is a Gram-negative bacterium and an opportunistic pathogenic bacterium ubiquitous in nature. Once infected with the bacteria, it will cause serious harm to the health of infants and young children, and can cause diseases such as necrotizing enterocolitis, bacteremia, and neonatal meningitis, and may even leave serious neurological sequelae, with a mortality rate of up to 50%. Therefore, the control of Cronobacter in the production process is a key link in the safety co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12R1/01
CPCC12Q1/689C12Q1/686C12Q2521/327C12Q2525/161C12Q2563/107
Inventor 张清平张懿翔刘洋王亮张奕南曲勤凤杨捷琳赵磊诸佩菊王越赵国屏
Owner SHANGHAI INST OF QUALITY INSPECTION & TECHN RES
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