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Primer, probe, kit and detection method for detecting real-time fluorescence RAA of shiga toxin-producing escherichia coli

A technology for Escherichia coli and Shiga toxin production, applied in the field of molecular biology detection, can solve the problems of expensive instruments, high requirements for operators, and difficulties in popularization and application in grassroots laboratories, and achieve the effect of simple method and low price

Pending Publication Date: 2021-01-01
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the international STEC detection method is mainly based on real-time fluorescent PCR method, which detects two types of Shiga toxin genes (stx1, stx2). However, real-time fluorescent PCR is based on a thermal cycler, which is expensive and requires high requirements for operators. Therefore, it is difficult to popularize and apply in the grassroots laboratories

Method used

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  • Primer, probe, kit and detection method for detecting real-time fluorescence RAA of shiga toxin-producing escherichia coli
  • Primer, probe, kit and detection method for detecting real-time fluorescence RAA of shiga toxin-producing escherichia coli
  • Primer, probe, kit and detection method for detecting real-time fluorescence RAA of shiga toxin-producing escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Primer and probe design

[0044] There are three main variants of Stx1: stx1a, stx1c and stx1d, stx1a is the most common, stx1c and stx1d are relatively rare, so the primer probe of the present invention is mainly designed for stx1a, but due to the sequence differences of the primer probe in the three variants Small, positive for the other two variants can also be amplified, but the signal is weaker than stx1a.

[0045] stx1a (NC_002695.1)

[0046]

[0047] Stx1c (DQ449666.1)

[0048]

[0049] Stx1d (AY170851.1)

[0050]

[0051] There are seven main variants of Stx2: stx2a, stx2c, stx2d, stx2dactivatable, stx2e, stx2f and stx2g, among which stx2a, stx2c and stx2dactivatable are the most common, and the remaining variants are relatively rare. Stx2f is quite different from other variants, and the primer probe of the present invention can amplify other variants except stx2f.

[0052] Stx2a (NC_002695.1)

[0053]

[0054] Stx2c (FR851896.1)

[00...

Embodiment 2

[0071] RAA detection method, comprises the steps:

[0072] (1), extract sample DNA;

[0073] a. Preparation of enrichment solution template DNA

[0074] i) Take 1mL of the bacterial solution and add it to a 1.5mL centrifuge tube, centrifuge at 12000g for 3min, and discard the supernatant;

[0075] ii) Add 1 mL of 0.85% sterile saline to completely dissolve the precipitate, centrifuge at 12000 g for 3 min, and discard the supernatant;

[0076] iii) Add 100 μL of sterile water and mix well, then boil in water bath for 10 minutes, and cool on ice;

[0077] iv) centrifuge at 12000g for 12min, and the supernatant is the template DNA.

[0078] b. Preparation of suspicious colony template DNA

[0079] For the isolated suspicious colonies, you can directly pick the suspicious colonies and follow step iii to prepare template DNA for detection.

[0080] (2) Using the sample DNA as a template, the primers and probes in Example 1 are used to detect RAA.

[0081] (3) Fluorescence sig...

Embodiment 3

[0089] Embodiment 3: specificity test

[0090] The following materials were tested using the RAA detection method in Example 2.

[0091] The strains used in the test were Enterohaemorrhagic Escherichia coli CICC10670 (including stx1 and stx2), CICC24187 (including stx1 and stx2), Enterohaemorrhagic Escherichia coli ATCC03820 (including stx1 and stx2), Shiga toxin-producing Escherichia coli Shigella flexneri CMCC51572 (stx1-, stx2-), Escherichia coli coli ATCC25922, enteroaggregative Escherichia coli EAEC24186, enteropathogenic Escherichia coli EPEC24189, enterotoxigenic Escherichia coli EIEC24188, Listeria monocytogenes ATCC BAA-751, Salmonella ATCC14028, parahaemolyticus Vibrio ATCC33847, Escherichia coli O157 FSCC149015 (without Shiga toxin gene), Bacillus cereus ATCC11778, Proteus mirabilis ATCC12543, Klebsiella pneumoniae ATCC13883, Pseudomonas aeruginosa ATCC27853, Cronobacter ATCC25944, Enterobacter aerogenes ATCC13048. The detection reaction conditions are: 39°C for ...

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Abstract

The invention provides a primer, a probe, a kit and a detection method for detecting real-time fluorescence RAA of shiga toxin-producing escherichia coli. The primer sequences of an stx1 gene are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of an stx1 gene probe is shown as SEQ ID NO.3; the primer sequences of an stx2 gene are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, and the sequence of the stx2 gene probe is shown as SEQ ID NO.6; and the kit comprises the primer, the probe, a buffer solution and purified water. The specificity of the real-time fluorescence RAAdetection method reaches 100%, the sensitivity reaches 1.6*10<3> cfu / mL, a result can be obtained within 15 min, and the real-time fluorescence RAA detection method is faster, more sensitive, simplerand more convenient than a real-time fluorescence PCR method, can be used for disease monitoring and food safety detection, and is suitable for popularization and application in primary laboratories.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a primer, a probe, a kit and a detection method for detecting real-time fluorescent RAA of Shiga toxin-producing Escherichia coli. Background technique [0002] Shiga toxin-producing Escherichia coli (Shiga toxin-producing E.coli, STEC) is a Shiga-like toxin-producing Escherichia coli, also known as verocytotoxigenic E.coli ( VTEC). STEC is the most important emerging pathogen in recent years, which can cause diarrhea, hemorrhagic colitis and even hemolytic uremic syndrome, and is the main cause of acute renal failure in children. Human infection with STEC is mainly through ingestion of contaminated food or water, direct contact with animals or through human-to-human transmission. At present, the international STEC detection method is mainly based on real-time fluorescent PCR method, which detects two types of Shiga toxin genes (stx1, stx2). Howe...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/19
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2522/101C12Q2561/113C12Q2563/107
Inventor 申进玲蒋原赵丽娜杨捷琳郭德华
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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