Method for detecting enteropathogenic bacteria (salmonella) through seven-channel chip and application

A Salmonella and chip detection technology, applied in the field of microbial detection, can solve the problems of high cost and complicated operation, achieve the effects of short reaction time, low reaction temperature, and avoid refrigerated transportation

Pending Publication Date: 2019-11-15
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are not only cumbersome to operate, limited

Method used

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  • Method for detecting enteropathogenic bacteria (salmonella) through seven-channel chip and application
  • Method for detecting enteropathogenic bacteria (salmonella) through seven-channel chip and application
  • Method for detecting enteropathogenic bacteria (salmonella) through seven-channel chip and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] This example is used to illustrate the specificity and sensitivity of the Salmonella RPA method.

[0085] 1. Salmonella primer and probe design

[0086] Primers were designed according to the InvA gene encoding the secreted protein of Salmonella, and the published InvA gene sequence (NC_003198.1) was searched through NCBI. According to the conservative region, after homology analysis, select such as figure 1 Design primers and probes for the indicated fragment (133bp):

[0087] Forward primer: 5’-TACCGGGCATACCATCCAGAGAAAATCGGGCCGC-3’ (34bp)

[0088] Reverse primer: 5’-ATTGGCGATAGCCTGGCGGTGGGTTTTGTTGT-3’ (32bp)

[0089] The sequence information of the probe is as follows:

[0090] 5’-CTCTATTGTCACCGTGGTTCAGTTTATCGTTATTACCAAAGGTTC A-3’

[0091] Among them, the 30th T is labeled with carboxyfluorescein FAM, the 34th T is modified with a quencher group BHQ, the 31-33 TAT is replaced with tetrahydrofuran, and the 3'end is modified with a phosphate group -P. (The modification was done b...

Embodiment 2

[0108] This example is used to illustrate the sensitivity of the Salmonella real-time RPA reaction system.

[0109] According to the instructions of the TwistAmpTM DNA amplification kit, add 2.1 μL of 10 μM forward primer, 10 μM reverse primer 2.1 μL, 10 μM probe 0.6 μL, buffer 29.5 μL, and 12.2 μL distilled water to a 50 μL reaction system. In the EP tube containing the recombinase, polymerase and single-chain polymerase, the last 1 μL of template and 2.5 μL of MgAc at a concentration of 280 mM were added to the EP tube, covered with a lid, and centrifuged, shaking repeatedly 10 times, and centrifuged. Place in a real-time fluorescent PCR machine for isothermal amplification at 39°C, and incubate for 30 minutes.

[0110] Set the template gradient of Salmonella to 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL and 0.1fg / μL. As the negative control, two sterile distilled water was selected.

[0111] From Figure 5 It can be seen that when the template concentration is 0.1fg / μL...

Embodiment 3

[0113] This example is used to illustrate the RPA detection of Salmonella in the seven pipelines.

[0114] The specific dimensions of the microchannel in the seven-channel chip are (width: 500 μm, height: 500 μm, length: 2 cm).

[0115] Real-time RPA system, add 2.1 μL of 10 μM forward primer, 10 μM reverse primer 2.1 μL, 10 μM probe 0.6 μL, buffer 29.5 μL, and 12.2 μL distilled water to a 50 μL reaction system. In the EP tube of polymerase and single-chain polymerase, the last 1 μL of template and 2.5 μL of MgAc at a concentration of 280 mM were added to the EP tube, covered with a lid, and centrifuged, shaking repeatedly 10 times, and centrifuged.

[0116] Set the template gradient of Salmonella as 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL and 0.1fg / μL. As the negative control, two sterile distilled water was selected.

[0117] Use a pipette to pipette 5μL of the above system into the microchannel, seal the inlet and outlet with scotch tape or uncured polydimethylsiloxane to preve...

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Abstract

The invention provides a method for detecting enteropathogenic bacteria (salmonella) through a seven-channel chip and application. The seven-channel chip includes a specific recombinase polymerase primer for detecting the enteropathogenic bacteria (salmonella), a probe and a seven-channel chip body, the sequence of a forward primer is SEQ ID No:1, the sequence of a reverse primer is SEQ ID No:2, the corresponding sequence of the probe is SEQ ID No:3, and the seven-channel chip takes a polymer as a material and is prepared through micro processing. The seven-channel chip includes seven orderedmicro channels which can distinguish multiple signals and prevents cross contamination between multiple systems. The method for detecting the enteropathogenic bacteria (salmonella) based on the micro-fluidic chip is provided to achieve fast, safe, specific and simple on-site detection of the salmonella. The probability of simultaneous detection of multiple samples is provided, and an effective scheme is provided for preventing the occurrence of salmonella epidemic situations and ensuring the food safety.

Description

Technical field [0001] The invention belongs to the technical field of microbial detection, and specifically relates to a method and application for detecting intestinal pathogenic bacteria Salmonella using a seven-channel chip. Background technique [0002] Salmonella is a gram-negative bacterium, which is a kind of intestinal pathogenic bacteria. So far, 1,000 species have been found. Salmonella infection can easily cause typhoid fever, infectious diarrhea, enteric fever, gastroenteritis and other diseases. The World Health Organization (WHO) has listed Salmonella as a serious and moderately harmful food-borne pathogen. Therefore, simple and quick detection of Salmonella can effectively suppress the emergence of public diseases. [0003] At present, the methods commonly used for Salmonella detection include microbial culture methods, immunoassay methods and polymerase chain reaction (PCR). However, these methods are not only cumbersome to operate, restricted by the expertise a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/10B29C69/00C12R1/42
CPCC12Q1/6844C12Q1/689B29C69/001B29L2031/34B29K2083/00
Inventor 蒋兴宇钱伟
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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