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RPA-LFD primer pair and probe for detecting pathogenic escherichia coli O157:H7 and application thereof

A technology of Escherichia coli and O157, applied in the field of food safety testing, can solve the problems of unfavorable on-site rapid diagnosis and accurate traceability, long testing period, expensive instruments, etc. Effect

Pending Publication Date: 2018-09-14
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these technical means have many limitations such as cumbersome sample processing, long detection cycle, expensive instruments, high reagent costs, low sensitivity, prone to false detection and missed detection, and inability to detect pathogenic bacteria that are difficult to cultivate artificially. Therefore, It is not conducive to on-site rapid diagnosis and accurate traceability during outbreaks, and does not meet the requirements of rapid, specific and sensitive principles for foodborne pathogen detection

Method used

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  • RPA-LFD primer pair and probe for detecting pathogenic escherichia coli O157:H7 and application thereof
  • RPA-LFD primer pair and probe for detecting pathogenic escherichia coli O157:H7 and application thereof
  • RPA-LFD primer pair and probe for detecting pathogenic escherichia coli O157:H7 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, primer screening and labeling

[0038] 1. RPA primer design

[0039] According to the Escherichia coli O157:H7rfbE gene (Genbank no: S83460.1), the conserved region was analyzed by Blast, RPA primers were designed, and 4 upstream and downstream primers were designed respectively, and PCR and RPA double screening were performed on 7 different upstream and downstream primer combinations. The primer sets and their sequence information are as follows:

[0040] Group 1 (EO1)

[0041] Upstream primer EOF1:

[0042] 5'-GCCCAGTTAGAACAAGCTGATGATTTTATATCACG-3' (SEQ ID NO.1)

[0043] Downstream primer EOR1:

[0044] 5'-CCTTGTTTCGATGAGTTTTATCTGCAAGGTGATTCC-3' (SEQ ID NO.2)

[0045] Product length: 207bp

[0046] Group 2 (EO2)

[0047] Upstream primer EOF2:

[0048] 5'-CATCCATGTGATATGGAACAAATTGTAGAACTGGC-3' (SEQ ID NO.3)

[0049] Downstream primer EOR2:

[0050] 5'-CCCACATATTTACCTTTATATTTAGAACCAAAGGC-3' (SEQ ID NO.4)

[0051] Product length: 110bp

[0052] ...

Embodiment 2

[0096] Embodiment 2, RPA-LFD establishment

[0097] 1. Probe design

[0098]Design probe EOProb for optimal primer pair EOF4 / EOR3: 5'-FAM-TGTCTGTTAGTGACATAGAACAAAAAATCACT-THF-ATAAAACTAAAGCTATT-C3-Spacer-3'(SEQ ID NO.9)( figure 2 ).

[0099] Probe length: 49bp

[0100] Corresponding RPA product length: 233bp

[0101] 2. RPA reaction system (10μL)

[0102]

[0103]

[0104] 3. RPA reaction system amplification

[0105] Add the above reagents except magnesium acetate into a sterile centrifuge tube and mix well. Next, add the magnesium acetate solution to the inside of the cap of the sterile centrifuge tube, cap tightly and then centrifuge instantly. After centrifuging, mix well and quickly put it into a PCR instrument with the hot cap closed or incubate in a constant temperature water bath at 40°C for 10 minutes.

[0106] 4. LFD detection of RPA products

[0107] Add 2 μL of RPA product into a 96-well microplate containing 100 μL Tris-Cl Buffer (pH 8.0) and mix well...

Embodiment 3

[0108] Embodiment 3, RPA reaction condition optimization

[0109] The optimum reaction volume, optimum reaction time and optimum reaction temperature of Escherichia coli O157:H7RPA were explored. The RPA reaction volume sets 10 gradients: 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL and 50 μL. The content of the RPA reaction components corresponding to each reaction volume is in equal proportion to its volume. The RPA reaction was carried out in a constant temperature electric hot water bath, and the RPA products corresponding to different reaction volumes were purified and analyzed by 2% AGE and LFD. The result is as image 3 As shown, the amount of RPA amplification product was the lowest when the RPA reaction volume was 5 μL, and remained basically unchanged in the 9 gradients from 10 μL to 30 μL. The RPA reaction time sets 6 gradients: 5min, 10min, 15min, 20min, 25min and 30min. The RPA reaction system is the same except for the time. The RPA reaction wa...

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Abstract

The invention discloses an RPA-LFD primer pair and probe for detecting pathogenic escherichia coli O157:H7 and application thereof. As for the sequences of the primer pair and the probe, the nucleotide sequence of an upstream primer EOF4 is SEQ ID NO.6; the nucleotide sequence of a downstream primer EOR3 is SEQ ID NO.7; the nucleotide sequence of the probe EOProb is SEQ ID NO.9. By means of a method, the defects of existing detection technologies are overcome through safe, specific, rapid, sensitive and simple on-site detection. It is proved through experiments that the minimum reaction volumeof an RPA-LFD technology is 10 microliters, the optimum reaction temperature is 40 DEG C, the shortest detection time is 15 min, the reaction cost is greatly saved, and the time required for detection is shortened. The optimum primers screened by the RPA reaction are high in specificity and sensitivity, and the sensitivity of RPA-LFD is 100 times that of PCR.

Description

technical field [0001] The invention relates to an RPA-LFD primer pair and probe for detecting pathogenic Escherichia coli O157:H7 and an application thereof, belonging to the technical field of food safety detection. Background technique [0002] In recent years, food safety incidents at home and abroad have emerged one after another. Foodborne pathogenic bacteria are widely distributed all over the world and cause thousands of human diseases every year. It is the number one food safety problem in the world. According to the latest report released by the World Health Organization (WHO), there are billions of food-borne diseases every year in the world, and even in developed countries, at least one-third of people suffer from food-borne diseases. For example, in the United States, it is estimated that there are about 48 million patients with food-borne diseases every year, of which 128,000 are hospitalized and 3,000 die. In my country, the biggest threat to food safety is a...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6804C12Q1/6844C12Q1/10C12N15/11C12R1/19
CPCC12Q1/6804C12Q1/6844C12Q1/689C12Q2521/507C12Q2565/625C12Q2563/131
Inventor 胡金强王一赵卫东魏向珂黄润娜孙新城王章存景建洲耿尧高辉姜春鹏董彩文
Owner ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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