The research hotspot of "He". Although gene chip technology has only a few years of development history, it has shown great potential and attractive prospects in many fields of biology. Many people think that gene chip technology can be compared with monoclonal antibody, PCR technology and recombinant DNA technology. Chips used for DNA analysis are also called DNA chips or genes.
Chip, according to the three main steps in the process of nucleic acid analysis, DNA chips can be divided into three categories: DNA chips for nucleic acid sample preparation, DNA chips for nucleic acid fragment amplification reaction and DNA chips for gene detection. The latter is also called microarray, that is, DNA chips. This paper introduces the application of gene chip in the detection of food pathogenic bacteria.
Technical principle of 1 gene chip technology for detecting food pathogenic bacteria
The basic principle of gene chip is to spot various gene oligonucleotides on the surface of the chip. The DNA of microbial samples was amplified by PCR, and fluorescent labeled probes were prepared, and then hybridized with oligonucleotide spots on the chip. Finally, the scanner quantitatively analyzes the fluorescence distribution pattern to determine whether there are some specific microorganisms in the detected sample.
Application of double gene chip technology in detection of food pathogenic bacteria
2. 1 Harm of pathogenic bacteria in food
Food is easily contaminated by pathogenic microorganisms in the process of production, transportation and sales, so it is very important to detect pathogenic microorganisms in food in time and accurately. The existence of these pathogenic microorganisms will bring great harm to the health of consumers.
2.2 Deficiencies of routine detection methods for pathogenic bacteria in food
At present, the detection of food microorganisms at home and abroad includes common pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Shigella, Salmonella and so on. Conventional detection methods mainly include traditional biochemical culture detection, probe hybridization and PCR. The traditional methods are bacterial culture, biochemical identification and serotyping, which are widely used because of their simple operation and economy, but the detection time is long (usually 1 ~ 2 weeks), with low efficiency and low sensitivity. Probe hybridization and PCR are accurate, sensitive and rapid (PCR takes 2 ~ 4 h, and probe hybridization is a little longer), which makes up for the shortcomings of traditional methods. There are many reports on the application of these two technologies in medical microbial detection at home and abroad. However, due to the influence of false positive and high detection cost, the application of PCR method is limited. The probe hybridization operation is complex and the detection time is long, so these two technologies have not been widely used in actual detection. It can be seen that the conventional detection methods can no longer meet the requirements of rapid detection, and it is difficult to adapt to the rapid development of modern food production and circulation. Therefore, in order to ensure the safety of food, it is of great significance to develop a rapid detection method for pathogenic bacteria and accurately detect pathogenic bacteria in food.
2.3. Using gene chip technology to detect common pathogenic bacteria in food.
1996, the world's first commercial DNA chip came out, which marked that the gene chip technology has entered a stage of extensive research and application [2], and it has incomparable advantages over traditional detection methods [3]: ① The gene chip can detect pathogenic bacteria in food.
Qualcomm and parallel detection are realized, all of which can be obtained in one experiment.
Test results; ② The operation is simple and quick, and the whole detection is within a few hours.
The detection result can be obtained; ③ Strong specificity and high sensitivity. along with
The continuous development and perfection of gene chip technology for detecting pathogenic bacteria in food.
Ok, it will be widely used in entry and exit, food safety indicators and emergencies.
Through the detection of pathogenic bacteria such as samples, food safety will be further guaranteed.
Barriers, and will have a far-reaching impact on the entire food field.
Li Guangxing from the College of Animal Medicine of Northeast Agricultural University prepared the land.
Highly sensitive labeled campylobacter jejuni probe, clinical examination in Beijing hospital
Yang Huawei of the center has developed two biotin-labeled conjugates.
DNA probe specific for Mycobacterium tuberculosis [4]; Li Junwen and others [5]
Detection of common pathogenic bacteria in water by gene chip: it can cause
Qualcomm quantity and parallel detection can be carried out on germs, and the whole can be obtained in one experiment.
Partial results; Jin Lianqun et al. [6] used gene chip technology to detect intestine.
Pathogens are in. The results show that the prepared gene chip can detect
Salmonella, Shigella, Staphylococcus, etc. For unknown colonies
The identification of unknown bacteria can be completed within 3 hours by using gene chip.
Settings; Professor Wang Leyan of Nankai University [7] undertakes "intestinal pathogens".
-The research and development project of gene chip for Shigella detection has achieved great success recently.
This project has made great progress, targeting Shigella and
Its probe has applied for patent 12. Detection of food by gene chip
The time of Shigella in products is shortened from 6 days by traditional detection methods.
A few hours later.
The hybrid genome microarray constructed by Borucki et al [8] is accurate.
Identify various Listeria monocytogenes isolates;
Volokhov et al [9] used single-tube multiplex amplification and gene chip.
Technical detection and identification of six Listeria species: telephone calls, etc. [10] passing point.
Analysis of Shiga-like Toxin Ⅰ and Shiga-like Toxin in Escherichia coli O 157: H7
Toxin Ⅱ and hemolysin A, it was found that the gene chip could accurately detect each one.
E. Escherichia coli O 157 :H7 isolate. J ack[ 1 1] is designed.
Universal primers amplify bacterial ribosomes 16S rRNA, which will be amplified.
The product hybridizes with a low-density chip containing a probe, so it is straight.
Then detect and identify microorganisms. Wilson et al. [12] used pathogen diagnosis.
Fragment gene amplification and 20 oligonucleotide phycoerythrin labeled probe, open
A set of multi-pathogen recognition microarray was released, which can accurately identify 18.
Three pathogenic viruses, prokaryotes and eukaryotes.
2.4 Procedures for detecting food pathogenic bacteria by gene chip technology
2.4. Carefully designed 1 PCR amplification primers and gene chip probes.
Interbacterial 16S rRNA is highly conserved, which is more conservative than that in biological evolution.
Other genes evolved slowly and were crowned as "fossils" of bacterial classification.
But the conservation of bacterial 16S rRNA is relative, at 16S.
There are both constant regions and sequences in rRNA gene.
Different variable regions, constant regions and variable regions are staggered.
Therefore, PCR primers can be designed and used in the constant region of 16S rRNA.
A pair of primers can complete the corresponding gene fragments of all pathogenic bacteria.
After amplification, detection probes can be designed in the variable region and bases can be made.
Because of the chip.
2.4.2. Prepare the chip and aminate the dielectric surface of the chip.
Chemical modification, silanization or disulfide bond modification are gene chip technologies.
Important components [13]. Use gene chip spotter to mix primers and primers.
The probe DNA molecules are spot coated on a carrier, such as a modified glass slide.
On, that is, to make a chip. There are several different ways to make DNA chips.
Methods: ① Chip was prepared by off-chip synthesis, such as chemical spraying and bonding.
Contact point coating method [14]; (2) In-situ synthesis of chips, such as high-voltage electricity.
Nozzle synthesis method [15] and light guide in-situ synthesis method [16]. Spend less money
Nucleotides are synthesized first and then immobilized on the carrier. The oligonucleotides used are all synthetic and fixed on the carrier to make chips.
After the chip is prepared, it can be used to detect unknown pathogens in food.
2.4.3. Samples of food pathogens to be detected shall be treated with pathogens to be detected.
After culture, the samples were lysed to extract the template of pathogenic bacteria.
The DNA was amplified by polymerase chain reaction (PCR), and the amplified DNA was analyzed.
The product has a fluorescent label. Then use 2. 0% agarose gel.
Electrophoresis detection showed that the fluorescent labeled products could be used for hybridization detection.
2.4.4. Hybridize the amplified and labeled pathogens to be detected.
The DNA standard liquid drops on the gene chip, which is different from the specificity on the chip.
DNA hybridization. If the detected pathogen exists, it
DNA successfully hybridized with chip DNA, washed and dried, and then put into
Row result analysis.
2.4.5 The results are detected and analyzed by chip scanner, such as fluorescence.
Optical scanner, focusing microscope, etc. , according to the fluorescence display.
The presence or absence of the detected pathogenic bacteria can be determined.
Problems and prospects of 3 gene chip technology
As a new technology, gene chip technology is fast and accurate.
Accurate, sensitive and so on, can detect a large number of samples in parallel at the same time, but
But there are still some key problems to be solved: ① At present,
Generally speaking, gene chips use fluorescent labeling technology to make genes
Chip inspection not only needs expensive chip manufacturing system, but also needs
For the expensive laser focusing scanner, the high price is seriously limited.
This chip technology has been popularized and applied. ② The operation is complicated and expensive.
At the same time, the requirements for the professional quality of operators are relatively high, which is also the limit.
One of the obstacles to its popularization and application; ③ Enlarge the sample target.
In the process of reaction, it is easy to cause sample pollution, which will also affect the inspection.
In order to measure the signal-to-noise ratio, researchers tried to adsorb samples through biosensors.
Products should avoid being combined with semiconductor technology, nanotechnology and biology.
Luminescence technology improves its sensitivity; ④ Micromachining technology of biochip.
How to improve the density of biochip microarray is also a big problem.
It limits the market demand of this technology. I believe that with the development of these technologies,
With further improvement, gene chip technology may become the most important technology in 2 1 century.
One of the dynamic technologies.
Gene chip technology is complete in detecting food pathogenic bacteria.
In the new field, foreign countries have started the research of food pathogenic bacteria detection chip.
But it is still in the early stage of research and development. In China, this field still exists.
In the initial exploration stage. Detection of pathogenicity in food by gene chip technology
It is expected that bacteria will be standardized and commercialized in the near future.
Almost all pathogenic bacteria are detected on one chip, which realizes the real revolution of pathogenic bacteria detection technology.