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Multiplex PCR detection primers and multiplex PCR detection method for Escherichia coli O<157>

A technology for the detection of Escherichia coli, which is applied in the field of microbial molecular biology detection, can solve the problems of poor specificity, low sensitivity, and long identification time, and achieve the effect of improving sensitivity

Pending Publication Date: 2018-12-07
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] In order to solve the Escherichia coli O mentioned in the above-mentioned background technology 157 The identification of O 157 Multiplex PCR detection primers and detection method

Method used

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  • Multiplex PCR detection primers and multiplex PCR detection method for Escherichia coli O&lt;157&gt;
  • Multiplex PCR detection primers and multiplex PCR detection method for Escherichia coli O&lt;157&gt;
  • Multiplex PCR detection primers and multiplex PCR detection method for Escherichia coli O&lt;157&gt;

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: O 157 Design and synthesis of PCR detection primers

[0030] Primers were found in Genbank for Escherichia coli O 157 The sequences of the stx1, flic, rfbE genes of the target gene were compared and analyzed with CLUSTAL, DNAMAN and other biological software to find the conserved region sequence of each target gene, and the conserved region sequence was used as the PCR amplification region, using Primers Premier, The primers were designed by biological software such as Oligo, the specificity and feasibility of the primers were verified by online BLAST, and the primers were synthesized according to conventional methods in the field.

[0031] The primer sequences are:

[0032] rfbE upstream primer F1: 5'-CGTAAGAGGGACCGTAGA-3' (SEQ ID No.1),

[0033] rfbE downstream primer F2: 5'-TGGCTGGATTATCGTTTG-3' (SEQ ID No.2),

[0034] fliC upstream primer F3: 5'-GCTGTCGAGTTCTATCGAG-3' (SEQ ID No.3),

[0035] FliC downstream primer F4: 5'-GTGACTTTATCGCCATTCC-3' (SEQ I...

Embodiment 2

[0039] Example 2: O 157 PCR detection annealing temperature optimization

[0040] 1. Escherichia coli O 157 DNA Extraction

[0041] Escherichia coli O was lysed by boiling 157 (ATCC35150), using phenol-chloroform-isoamyl alcohol reagent to extract bacterial DNA. The specific steps are: take O 157 Put 1ml of bacterial solution in a 1.5ml centrifuge tube and boil in a water bath for 10 minutes. Centrifuge at 10000r / min for 5min, take the supernatant, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) respectively, then mix slightly, leave at room temperature for 5-10min, then centrifuge at 12000rpm for 10min. Repeat the extraction 2-3 times. Aspirate the supernatant, add an equal volume of absolute ethanol, place it at low temperature for 30 minutes, and then centrifuge at 12000 r / min for 10 minutes to retain the precipitate. Wash the DNA with pre-cooled 70% ethanol to remove excess impurities. Centrifuge at 12000r / min for 10min, pour off the upper laye...

Embodiment 3

[0050] Example 3: Effect of Primer Ratio on Multiplex PCR Detection

[0051] E. coli O 157 The DNA extraction, PCR reaction system and amplification procedures refer to Example 2. In the PCR reaction system, only the ratio of the primer pair is changed. The molar ratio of the upstream and downstream primers in each pair of primers is 1:1, and the molar ratio between the primer pairs of stx1, flic, and rfbE is respectively set to 1:1: 1. 1:2:1, 2:3:1, research primers for Escherichia coli O 157 The effect of multiplex PCR detection.

[0052] The experimental results found that the brightness of the three bands was better when the molar ratio between the primer pairs of stx1, flic, and rfbE was 1:2:1 and 2:3:1 than when the molar ratio was 1:1:1, indicating that this The three pairs of primers have different amplification efficiencies for their respective target genes.

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Abstract

The invention relates to multiplex PCR detection primers and a multiplex PCR detection method for Escherichia coli O<157>. The multiplex PCR detection primers comprise three pairs of primers. The three pairs of primers respectively amplify the rfbE, fliC, and stx1 genes of O<157>, and the sequences of the primers are as shown in a sequence table in the specification. The above primers are used formultiplex PCR detection of Escherichia coli O<157> DNAs; a positive control, a negative control and a blank control are set; and PCR amplification conditions comprise denaturation at 94 DEG C for 5 min, pre-denaturation at 94 DEG C for 30 s, annealing at 48-52 DEG C for 45 s, extension at 72 DEG C for 45 s, 30-35 amplification cycles, and finally, extension at 72 DEG C for 5 min. The detection primers and the detection method for the Escherichia coli O<157> in the invention have the advantages of specificity and sensitivity, and can accurately identify the Escherichia coli O<157>.

Description

technical field [0001] The present invention relates to microbial molecular biology detection technology, particularly relates to Escherichia coli O 157 Multiplex PCR detection primers and detection method. Background technique [0002] Escherichia coli (Escherichia coli) was discovered by Escherich in 1885. For a long period of time, it has been regarded as a component of normal intestinal flora and considered to be non-pathogenic bacteria. It was not until the middle of the 20th century that some bacteria were recognized. Escherichia coli of special serotypes are pathogenic to humans and animals, especially to infants and young livestock and poultry, often causing severe diarrhea and sepsis. [0003] The antigen composition of Escherichia coli is complex, which can be divided into bacterial antigen (O), flagellar antigen (H) and surface antigen (K). According to the different O antigens, Escherichia coli can be divided into more than 150 serotypes, of which 16 serotypes ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12R1/19
CPCC12Q1/686C12Q1/689C12Q2537/143C12Q2565/125
Inventor 颜世敢朱丽萍
Owner QILU UNIV OF TECH
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