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Nano-PCR kit for simultaneous detection of enterohemorrhagic Escherichia coli O45 and O145 and its application

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

Active Publication Date: 2022-01-11
JIANGSU ACAD OF AGRI SCI
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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 kit for simultaneous detection of enterohemorrhagic Escherichia coli O45 and O145 and its application
  • Nano-PCR kit for simultaneous detection of enterohemorrhagic Escherichia coli O45 and O145 and its application
  • Nano-PCR kit for simultaneous detection of enterohemorrhagic Escherichia coli O45 and O145 and its application

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 (wzx) sequence of EHEC O45 (GenBank accession number: AY771223.1) issued by GenBank, use the routine primer design software Primer Premier5 in this field to design 2 pairs of primers; according to the O145 issued by GenBank O antigen flippase gene (wzx) sequence (CP006027.1), design 3 pairs of specific primers. Primer sequences are listed in Table 1.

[0025] Table 1 Primer Sequence

[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., product number NHS20-3) 10 μL, 1 μL DNA positive template, dNTP Mixture (2.5mM, Beijing Tiangen Biochemical Technology Co., Ltd., product number CD117-02) 1 μL, DNA polymerase (Beijing ...

Embodiment 2

[0033] Embodiment 2 A kind of nano-PCR kit that detects 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×NanoPCR buffer (Shanghai Hushi Pharmaceutical Technology Co., Ltd., catalog number NHS20-3), dNTP Mixture (2.5mM,...

Embodiment 3

[0044] Embodiment 3 non-diagnostic purpose detects the method for EHEC O45 and O145 with nano-PCR method

[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 comprises 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 , centrifuge at 12000 rpm for 10 min at 4° C...

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Abstract

The invention provides a nano-PCR kit for simultaneously detecting enterohaemorrhagic Escherichia coli O45 and O145 and an application thereof, and relates to the technical field of detection of hemorrhagic Escherichia coli. The test kit includes a first primer pair and a second primer pair, the first primer pair includes primers with sequences such as SEQ ID No: 3 and SEQ ID No: 4; the second primer pair includes primers with sequences such as SEQ ID No: Primers shown in ID No: 7 and SEQ ID No: 8. The present invention also provides the application of the nano-PCR kit in detecting enterohemorrhagic Escherichia coli O45 and O145 for the purpose of non-diagnosis. The nano-PCR kit of the invention can quickly and specifically detect two serotypes of EHEC in a sample at the same time, and has good application prospects.

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] Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic food-borne pathogen that produces one or two Shiga toxins. It can be divided into O157 EHEC and non-O157 EHEC. EHEC has strong pathogenicity to humans, and the infection dose is low, and the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 ACAD OF AGRI SCI
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