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Nano PCR (Polymerase Chain Reaction) kit for simultaneously detecting enterohemorrhagic escherichia coli O45 and O145 and application thereof

An Escherichia coli and enterohemorrhagic technology, applied in the detection field of hemorrhagic Escherichia coli, can solve the problem of lack of detection kits for EHECO45 and O145, and achieve the effect of good application prospects

Active Publication Date: 2018-12-07
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, there is a lack of kits capable of rapid and specific detection of EHEC O45 and O145 at the same time

Method used

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  • Nano PCR (Polymerase Chain Reaction) kit for simultaneously detecting enterohemorrhagic escherichia coli O45 and O145 and application thereof
  • Nano PCR (Polymerase Chain Reaction) kit for simultaneously detecting enterohemorrhagic escherichia coli O45 and O145 and application thereof
  • Nano PCR (Polymerase Chain Reaction) kit for simultaneously detecting enterohemorrhagic escherichia coli O45 and O145 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Optimal primer pair screening

[0024] 1. Primer design According to the O antigen flippase gene ( wxya ) sequence, two pairs of primers were designed using Primer Premier 5, a routine primer design software in the field; according to the O antigen flippase gene of O145 released by GenBank ( wxya ) sequence (CP006027.1), and three pairs of specific primers were designed. Primer sequences are listed in Table 1.

[0025] Table 1 Primer sequences

[0026]

[0027] 2. PCR amplification

[0028] Firstly, single-plex PCR was performed to detect the amplification efficiency and the optimal annealing temperature of primer pairs 1-5, respectively.

[0029] PCR reaction system: 2×NanoPCR buffer (Shanghai Hushi Pharmaceutical Technology Co., Ltd., catalog number NHS20-3) 10 μL, 1 μL DNA positive template, dNTP Mixture (2.5mM, Beijing Tiangen Biochemical Technology Co., Ltd., catalog number CD117-02) 1 μL , DNA polymerase (Beijing Tiangen Biochemical Technology ...

Embodiment 2

[0033] Example 2 A nano-PCR kit for detecting EHEC O45 and O145

[0034] Test kit of the present invention comprises:

[0035] (1) Primer solution

[0036] The O45 primers include an upstream primer whose sequence is shown in SEQ ID No:3 and a downstream primer whose sequence is shown in SEQ ID No:4. The O145 primers include an upstream primer whose sequence is shown in SEQ ID No: 7 and a downstream primer whose sequence is shown in SEQ ID No: 8.

[0037] O45 primer solution: a solution containing 20 pM O45 upstream primer (SEQ ID No: 3) and 20 pM O45 downstream primer (SEQ ID No: 4), and the solvent is double distilled water.

[0038] O145 primer solution: a solution containing 20 pM O145 upstream primer (SEQ ID No: 7) and 20 pM O145 downstream primer (SEQ ID No: 8), solvent double distilled water.

[0039] (2) 2×NanoPCR Mix

[0040] 2×NanoPCR Mix: 2×NanoPCRbuffer (Shanghai Hushi Pharmaceutical Technology Co., Ltd., catalog number NHS20-3), dNTP Mixture (2.5mM, Beijing Ti...

Embodiment 3

[0044] Example 3 The method for detecting EHEC O45 and O145 by nano-PCR method for non-diagnostic purposes

[0045] In this example, the kit in Example 2 is used to detect whether samples 1 and 2 contain EHEC O45 and EHECO145.

[0046] Sample 1 was feces and sample 2 was meat.

[0047] The sample detection method includes the following steps: (1) sample template preparation; (2) PCR detection.

[0048] (1) Sample template preparation

[0049] Sample template preparation, including two methods, choose one of them.

[0050] method 1

[0051] Bacterial DNA is extracted by conventional boiling methods, including the following steps:

[0052] Take 0.3mL-0.5mL of feces samples and meat tissue grinds, put them in a 1.5mL centrifuge tube, centrifuge at 12000rpm for 10min, discard the supernatant, take the precipitate and resuspend it in sterile distilled water with 1 / 5 of the original volume, and put it in a boiling water bath for 10min , 4°C, 12000rpm centrifugation for 10min, a...

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Abstract

The invention provides a nano PCR (Polymerase Chain Reaction) kit for simultaneously detecting enterohemorrhagic escherichia coli (EHEC) O45 and O145 and application thereof and relates to the technical field of detection of the enterohemorrhagic escherichia coli. The kit comprises a first primer pair and a second primer pair; the first primer pair comprises primers as shown by SEQ ID No: 3 and SEQ ID No: 4; and the second first primer pair comprises primers as shown by SEQ ID No: 7 and SEQ ID No: 8. The invention further provides the application of the kit to detection of the enterohemorrhagic escherichia coli O45 and O145 in a manner of not taking diagnosis as a target. The nano PCR kit provided by the invention can be used for rapidly and specifically simultaneously detecting two bloodserum types of EHEC in a sample and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of detection of hemorrhagic escherichia coli, in particular to a nano-PCR kit for simultaneously detecting enterohaemorrhagic escherichia coli O45 and O145 and an application thereof. Background technique [0002] Foodborne pathogenic microorganisms are the main cause of increasing food safety and public health emergencies in the world today. According to WHO data, within one year of 2011, 3.7 million people died of food-borne diseases mainly caused by infectious diarrhea in the world. and drinking water. Among foodborne pathogenic microorganisms, Salmonella, Listeria, enterohemorrhagic Escherichia coli and Campylobacter play an important role. [0003] Enterohaemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli , EHEC) is a class of highly pathogenic foodborne pathogens that produce one or two types of Shiga toxins, which can be divided into O157 EHEC and non-O157 EHEC. EHEC has strong patho...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/10C12Q1/686C12N15/11C12R1/19
CPCC12Q1/686C12Q1/689
Inventor 张雪寒葛东红张碧成王警郭芸芸俞正玉倪艳秀温立斌李彬汪伟胡屹屹周俊明祝昊丹肖琦范宝超朱雪娇
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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