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CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method

A detection kit and the technology of Escherichia coli are applied in the field of molecular biology, which can solve the problems of high cost and complicated detection steps, and achieve the effects of low detection cost, simple operation, and good popularization and application value.

Inactive Publication Date: 2015-12-02
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

而此前报道的基于CRISPR的定量PCR特异性检测大肠杆菌O157:H7等相关血清菌株(UseofclusteredregularlyinterspacedshortpalindromicrepeatsequencepolymorphismsforspecificdetectionofenterohemorrhagicEscherichiacolistrainsofserotypesO26:H11,O45:H2,O103:H2,O111:H8,O121:H19,O145:H28,andO157:H7byreal- timePCR) method, it is necessary to design three pairs of primers and probes, the detection steps are complicated, and the cost is high

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  • CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method
  • CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method
  • CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method

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Embodiment 1

[0056] In this embodiment, the CRISPR-based Escherichia coli O157:H7 strain detection kit includes the following primers:

[0057] Upstream primer: 5'-GTTATGCGGATAATGCTACC-3',

[0058] Downstream primer: 5'-CGTATTCCGGTGGATTTGGA-3'.

Embodiment 2

[0060] Basic principle: The detection kit contains various reagent components required for polymerase chain reaction, such as primers, dNTPs, buffer, Taq enzyme, etc., and the detection sample can be added to the PCR reaction mixture to perform PCR amplification. Increased response. PCR amplification products were judged by agarose electrophoresis staining, and positive samples would appear specific bands at fragments of corresponding sizes.

[0061] Product overview: The detection kit adopts polymerase chain reaction (PCR) technology. Since the primers contained can specifically amplify the CRISPR1 locus gene sequence of Escherichia coli O157:H7 strain, it can be used to detect various samples to be tested (such as feces , food, etc.) infection / contamination status.

[0062] In this embodiment, the CRISPR-based Escherichia coli O157:H7 strain detection kit includes: 2×TaqPCR MasterMix (0.1U / μl TaqDNA Polymerase, 2×PCR reaction buffer, 0.4mMdNTP, 4mMMgSO 4 ) 20mL, sterilized...

Embodiment 3

[0084] In this embodiment, the CRISPR-based Escherichia coli O157:H7 bacterial strain detection method comprises the following steps:

[0085] (1) Sample collection and pretreatment

[0086] Add 500 μL of the sample to be tested (feces or food to be tested, etc.) to 10 mL of enrichment solution, enrich the bacteria at 37°C for 5 hours (4-6 hours are acceptable), centrifuge at 12000 rpm / min for 5 minutes in a desktop high-speed centrifuge, discard the supernatant, and precipitate As a test template;

[0087] (2) PCR amplification

[0088] Use the upstream and downstream primers for PCR amplification, and the primers are as follows:

[0089] Upstream primer: 5'-GTTATGCGGATAATGCTACC-3',

[0090] Downstream primer: 5'-CGTATTCCGGTGGATTTGGA-3';

[0091] PCR amplification reaction system: 25 μL in total, 1 μL of 8 μmol / L upstream primer, 1 μL of 8 μmol / L downstream primer, 2×TaqPCR MasterMix (0.1U / μl TaqDNA Polymerase, 2×PCR reaction buffer, 0.4mMdNTP, 4mMMgSO 4 ) 12.5 μL, singl...

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Abstract

The invention discloses a CRISPR-based Escherichia coli O157:H7 strain detection reagent box and a detection method and belongs to the technical field of molecular biology. The detection reagent box comprises primers designed for a CRISPR1 locus gene sequence or a homologous sequence with homology reaching higher than 95% of an Escherichia coli O157:H7 strain and shown as SEQ ID NO.1 and SEQ ID NO.2 in a sequence table. The primers are utilized for PCR amplification, and three unique spacer sequences (shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7) of CRISPR1 are detected to be bacterially positive in an amplification product. The detection method is simple to operate, high in sensitivity and specificity, uniform with serotyping result of Escherichia coli O157:H7 and low in detection expense and has good popularization and application value.

Description

technical field [0001] The invention relates to a CRISPR-based E. coli O157:H7 strain detection kit, and the invention also relates to a CRISPR-based E. coli O157:H7 strain detection method, which belongs to the technical field of molecular biology. Background technique [0002] Escherichia coli O157:H7 is a major pathogenic strain of Escherichia coli, and O157:H7 infection can not only cause serious gastrointestinal complications such as hemorrhagic colitis, appendicitis, esophageal stricture and colon perforation (PaiCH, GordonR, SimsHV, et al. SporadicCasesofHemorrhagicColitisAssociatedwithEscherichiaColiO157:H7.Clinical,Epidemiologic,andBacteriologicFeatures[J].AnnalsofInternalMedicine,1984,101(6):738-742), and can also cause hemolytic urinary tract syndrome (hemolyticuremics) syndrome and thrombosis in children and the elderly population, HUS 性血小板紫癜(thromboticthrombocytopenicpurpura,TTP)等全身性并发症,严重者肾衰竭导致死亡(MoriY,WadaH,TamakiS,etal.OutcomeofThromboticThrombocytopenicPurpur...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/19
CPCC12Q1/689
Inventor 段广才梁文娟张荣光杨海燕张卫东郗园林范清堂
Owner ZHENGZHOU UNIV
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