Method for preparing detection chip and method for detecting pathogen by using the same

A technology for detecting chips and pathogens, which is applied in the field of nucleic acid detection of pathogenic microorganisms, and can solve the problems of difficult preservation of primers, high price, and fluorescence quenching.

Inactive Publication Date: 2008-08-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are already some chips used for food testing. However, these chips also have the following shortcomings and deficiencies: 1. Although high-throughput detection of pathogens can be achieved, generally one chip can only detect all pathogens in one sample; 2. At present, there are generally two types of fluorescent labels. One is to add Cy-dCTP while amplifying. However, since Cy is a large molecule and has a large steric hindrance, it is not easy to combine during the chain extension process; the other The first method is to synthesize primers with fluor

Method used

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  • Method for preparing detection chip and method for detecting pathogen by using the same
  • Method for preparing detection chip and method for detecting pathogen by using the same
  • Method for preparing detection chip and method for detecting pathogen by using the same

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1, a kind of preparation method of detection chip, carries out following steps successively:

[0058] 1), design bacterial oligonucleotide probes:

[0059] The genome sequences of Escherichia coli, lactic acid bacteria, Salmonella, Shigella, Staphylococcus aureus, mold and yeast were obtained from the NCBI database, and then designed using Arraydesigner4.2 software. The resulting probe sequences are specifically shown in the table 1.

[0060] All the probes shown in Table 1 were used for each bacterium, namely, the four probes shown in SEQ ID NO: 1 to SEQ ID NO: 4 were used for Escherichia coli, and so on for other bacteria.

[0061] 2), chip preparation:

[0062] Select 28 probes shown in Table 1 for use, and 1 negative (sequence is:

[0063] 5-CTCAATCCTTTGGGTGTATGGGTCGTAGCGAACTGAGAAGGGCCGAGGTATTGTGGCA-3), 1 positive probe (GAPDH:

[0064] 5-GTCCAGTTAATTTCTGACCTTTTACTCCTGCCCTTTGAGTTTGATGATGCTGAGTGTAC-3), a total of 30 probes. Each probe was spotted in d...

Embodiment 2

[0066] Embodiment 2, a method for detecting pathogens in multiple food samples, select the detection chip obtained in embodiment 1, and perform the following steps in sequence:

[0067] 1), design primers:

[0068] Get the genome sequences of Escherichia coli, lactic acid bacteria, Salmonella, Shigella, Staphylococcus aureus, mold and yeast from the NCBI database, and then use the primer5.5 software to design the primers corresponding to each of the above bacteria The sequence is specifically shown in Table 2.

[0069] 2), the extraction of DNA in the sample to be tested, with 12 kinds of emulsion or liquid dairy products as the sample to be tested, each sample to be tested is followed in turn by the following steps:

[0070] ①. Take a 50ml sample, centrifuge at 1000-2000rpm at 4°C for 20min, and discard the supernatant;

[0071] ② Add 10ml of PBS, shake gently to resuspend the pellet, centrifuge at 1000-2000rpm at 4°C for 20min, discard the supernatant;

[0072] ③, repeat ...

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Abstract

The invention discloses a preparation of a detection chip, comprising the steps of: 1) designing oligonucleotide probe of bacteria; 2) preparing the chip. The invention further discloses a method for detecting pathogene in multiple food samples with the detection chip, comprising the following steps: 1) designing a primer; 2) extracting DNA from samples to be detected, and getting extracting solution; 3) multi-PCR amplifying of target gene; 4) hybridization detecting of chip; and 5) detecting and analyzing of hybridization result. The detection step of the invention is simple, easy to operate, and produces high correct rate result.

Description

technical field [0001] The invention belongs to microorganism detection technology, in particular to the nucleic acid detection technology of pathogenic microorganisms in milk and dairy products; in particular, it relates to a method capable of simultaneously detecting pathogens in 12 food samples and a chip used therefor. Background technique [0002] Foodborne pathogenic bacteria often cause mass damage, causing certain harm to people's health, life and property. According to the incomplete statistical report of the World Health Organization (WHO), 2.1 million people died of diarrhea in the world in 2000, most of which were attributed to the contamination of pathogenic bacteria in food and drinking water. According to statistics from the Ministry of Health, in 2000 there were 150 major food poisoning reports, resulting in 6237 poisonings and 135 deaths; in 2001 there were 185 major food poisoning incidents, resulting in 15715 poisonings and 146 deaths. [0003] The basis ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 洪旭涛杨华陈伟光袁谦邵晖项春生
Owner ZHEJIANG UNIV
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