KR101247975B1 - PNA microarray for differential diagnosis of aerobic gram-positive food poisoning bacteria and the method for differential diagnosis using the same - Google Patents

Abstract

The present invention relates to a PNA microarray for differential diagnosis of aerobic Gram-positive food poisoning bacteria and a method for differential diagnosis of the bacteria using the same, more specifically, PNA (Peptide nucleic acid) probes of SEQ ID NOs: 1 and 2 and SEQ ID NOs: 3 to 6 Staphylococcus aureus , an aerobic Gram-positive food poisoning bacterium And The present invention relates to a PNA microarray capable of distinguishing Listeria monocytogenes from other bacteria accurately and quickly, and a method for differentially detecting the bacteria using the same.

Description

PNA microarray for differential diagnosis of aerobic Gram-positive food poisoning bacteria and a method for differential diagnosis using the same {PNA microarray for differential diagnosis of aerobic gram-positive food poisoning bacteria and the method for differential diagnosis using the same}

The present invention relates to a PNA microarray for differential diagnosis of aerobic Gram-positive food poisoning bacteria and a method for differential diagnosis of the bacteria using the same, more specifically, PNA (Peptide nucleic acid) probes of SEQ ID NOs: 1 and 2 and SEQ ID NOs: 3 to 6 Staphylococcus aureus , an aerobic Gram-positive food poisoning bacterium And The present invention relates to a PNA microarray capable of distinguishing Listeria monocytogenes from other bacteria accurately and quickly, and a method for differentially detecting the bacteria using the same.

According to the regulations of the Food Code published by the Food and Drug Administration meat (manufacturing and processing a raw material is excluded), disinfection or sterilization process hayeotgeona In further processing, processed food intake without a cooking Staphylococcus aureus according to the characteristic (Staphylococcus aureu s) and Listeria monocytogenes ( Listeria) monocytogenes ) Gram-positive food poisoning bacteria, etc., are not to be detected. However, since there is a high possibility that various bacteria are mixed in the specimen to be tested, it is necessary to quickly and accurately discriminate gram-positive food poisoning bacteria from them.

DNA microarray is a high density arrangement of DNA molecules with known sequence on a small substrate. DNA microarrays, also known as DNA chips, are collections of genes planted on a small piece of glass, nylon, silicon, or silica. When a DNA labeled with fluorescent material is extracted from a tissue such as blood and reacted with a chip, fluorescence is generated when there is DNA of a tissue that matches the DNA on the chip. Using the program of the computer, the gene located at the point is identified, and the function is identified to analyze the meaning of the pattern given by the fluorescent points.

The advantage of such DNA microarrays is that they can collectively explore the state of gene expression. DNA microarray technology is a fusion of various fields such as chemistry, molecular biology, mechanical engineering, and electronic engineering, and contributes greatly to genome-level research related to life phenomena. Because DNA microarray technology can put tens of thousands of fragments into a single microarray, information about the entire gene can be obtained in a single experiment. Unlike the existing technology using Southern or Northern method, which only targets a few genes, it has a characteristic that can get quite a lot of information.

DNA microarrays are used to find genes that are expressed differently in different tissues or conditions and to discover new genes based on known gene expression patterns. It is also widely used to study how one gene interacts with other genes.

However, the DNA microarray method is widely used in the human body, which can be tested even at a high cost because the test cost is high, but in the veterinary field, a minimum test cost is required and it is necessary to analyze the results of the microarray. Since it is impossible to equip all the expensive equipment such as a reader, it is difficult to introduce the microarray method.

There are several types of microarrays, such as DNA chips, protein chips, cell chips, and neuronal chips, depending on the type of material currently loaded on the chip. Microarrays have a wide range of applications such as gene discovery, cancer classification, disease risk assessment, disease prognosis, disease diagnosis, new drug development, toxicology research, food stability assessment and genetic disease research. Among them, the PNA microarray chip is much more sensitive than the existing DNA microarray chip and can be diagnosed using SNP, so that it can be distinguished by only one or two nucleotide sequences. It is expected to be introduced in.

Accordingly, the present inventors are able to discriminate between aerobic Gram-positive food poisoning bacteria Staphylococcus aureus and Listeria monocytogenes, while searching for a method for rapidly and accurately discriminating aerobic Gram-positive food poisoning bacteria from a test specimen containing various bacteria. The present invention finds that the aerobic Gram-positive food poisoning bacteria can be quickly and accurately diagnosed using two PNA probes and two pairs of primers for amplifying a target DNA, and using the PNA microarray chip. It was completed.

Accordingly, an object of the present invention is to provide a PNA microarray capable of rapidly and accurately discriminating aerobic Gram-positive food poisoning bacteria and a method for differentially detecting the bacteria using the same.

In order to achieve the above object, the present invention provides a PNA microarray for aerobic Gram-positive food poisoning bacteria differential diagnosis comprising a PNA (Peptide nucleic acid) probe of SEQ ID NO: 1, 2 and the primers of SEQ ID NO: 3 to 6.

The present invention also provides a method for differential diagnosis of aerobic Gram-positive food poisoning bacteria using the PNA microarray.

The present invention can quickly and accurately discriminate aerobic Gram-positive food poisoning bacteria using the PNA chip technique to secure the safety of the people and establish food safety. In addition, the differentiation method according to the present invention is able to discriminate against serotypes and genotypes of aerobic Gram-positive food poisoning bacteria, which is used for food poisoning test for pre-food ingredients of large group food service schools such as school meals and military meals. It is expected to contribute.

1 is a schematic diagram of the reaction of PNA microarray chip and target DNA for differential diagnosis of aerobic Gram-positive food poisoning bacteria.
2 is a schematic diagram of the chemical reaction of the PNA microarray.
Figure 3 is a general schematic diagram of the PNA microarray experimental procedure.
Figure 4a is a PNA microarray read manual, Figure 4b shows the results of differential diagnosis of aerobic Gram-positive food poisoning bacteria using the PNA microarray.

The present invention relates to a method for rapid and accurate differential diagnosis of aerobic Gram-positive food poisoning bacteria from common bacteria and other pathogenic bacteria. In order to differentially diagnose the aerobic Gram-positive food poisoning bacteria, single nucleotide polymorphim (SNP) is required, but in the case of general DNA microarray method, the sensitivity and specificity are very low, so that differential diagnosis is difficult. Therefore, the present invention differentially diagnoses aerobic Gram-positive food poisoning bacteria using a PNA (Peptide nucleic acid) microarray capable of SNP.

To this end, in the present invention, a PNA microarray for differential diagnosis of aerobic Gram-positive food poisoning bacteria comprising PNA (Peptide nucleic acid) probes of SEQ ID NOs: 1 and 2 and primers of SEQ ID NOs: 3 to 6 was prepared. To provide a method for differential diagnosis of Gram-positive food poisoning bacteria.

The aerobic Gram-positive food poisoning bacterium is Staphylococcus aureus ) And Listeria monocytogenes ( Listeria monocytogenes ) may be one or more selected from the group consisting of, by using the PNA microarray according to the present invention, the aerobic Gram-positive food poisoning bacteria alone or two can be differentially diagnosed at the same time.

1 is a schematic diagram showing the reaction of a PNA microarray chip and a target DNA for differential diagnosis of aerobic Gram-positive food poisoning bacteria. The PCR product amplified by biotin-labeled amplified PNA probes of SEQ ID Nos. Shows hybridization.

Figure 2 is a schematic diagram of the chemical reaction of the PNA microarray PNA probes of SEQ ID NO: 1 and 2 and the target DNA is a hybrid binding form and biotin, streptavidin (streptavidin) and the process of color development by alkaline phosphatase Shows.

Figure 3 is a general schematic diagram of the PNA microarray experimental procedure, showing a series of processes, such as the first total DNA extraction, the target DNA amplification and hybridization by PCR, and the scanning and reading by the equipment.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1

Aerobic gram-positive sikjungdokgyun of Staphylococcus aureus (Staphylococcus aureus ) And PNA probes were synthesized using cyclic bezothiazole-2-sufonyl (Bts) to prepare a PNA microarray for differential diagnosis of Listeria monocytogenes .

The PNA probe synthesis site was selected from the ITS gene of Gram-positive food poisoning bacteria, and the sites that can be distinguished from other food poisoning bacteria were synthesized, respectively, and PNA probes of the following base sequences 1,2 were synthesized, and the cut off signal was 3,000. It was set as. As a result of the differentiation experiment with other food poisoning bacteria, no food poisoning bacteria with a cutoff signal of more than 3,000 were found in other food poisoning bacteria.

The PNA probe was synthesized using Bts (benzothiazole-2-sulfonyl) at 17-21 mer, purity was measured using an Agilent 1100 liquid chromatography instrument, and MALDI-TOF was used for qualitative testing. In addition, the concentration was measured using a NanoDrop (ND-1000) instrument, the PNA probe was attached to the surface of the slide glass by 50 μM. The attachment device used a Qarray mini microarrayer with aQu solid pins, and the humidity was maintained at 75 to 85%.

Two PNA probes prepared for differential diagnosis of aerobic Gram-positive food poisoning bacteria through the above process are shown in Table 1 below. In Table 1, Linker (Panagene) was used as a linker.

division PNA sequence (N → C) SEQ ID NO: St . aureus NH ₂ -Linker-GAACACTCACAAGATTA One  L. monocytogenes NH ₂ -Linker-GAAATACAAATAATCATATCC 2

Staphylococcus aureus And DNA of Listeria monocytogenes was extracted and used for PCR reaction. Target genes for both species were subjected to nested PCR to increase the sensitivity of the reaction to the ITS site. The primers used in the PCR reaction were used by synthesizing the primer pairs of SEQ ID NOs 3 to 6 shown in Table 2 below. In this case, biotin-labeled primers were used for 2nd PCR (nested PCR), and the genomic DNA extracted was used as a template for the first PCR reaction, and 1 μl of 25 μl of the first PCR product was used as a template for the second PCR reaction. . In addition, PCR reaction conditions were performed for 15 seconds in the first PCR reaction and 40 times in the second Nestest PCR reaction for denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 30 seconds, and expansion at 72 ° C. for 1 minute.

designation Primer sequence SEQ ID NO: 1st PCR forward primer 5'-GCCTTGTACACACCGCCC-3 ' 3 1st PCR Reverse Primer 5'-RGTACTTAGATGTTTCARTTCYC-3 ' 4 2nd PCR Forward Primer 5'-ACACC GCCCGTCACACCA-3 ' 5 2nd PCR Reverse Primer 5'-TACTTAGATGTTTCARTTCYC-3 ' 6

In Table 2, R in the primer sequence is A or G, Y is C or T.

10 μl of the PCR product and 10 μl of the biotin-labeled target DNA and 70 μl of Cy5-streptavidin in the PNA hybridization buffer were mixed and hybridized by reacting the PNA microarray chip at 40 ° C. for 2 hours. PNA array was washed twice with 5 minutes after the reaction using a washing buffer (buffing buffer). After drying it was scanned with a non-confocal fluorescent scanner GenePix 4000B. The fluorescence signal was calculated by hybridization signal of probe-target duplexes to determine the gene type.

FIG. 4A is a PNA microarray reading manual, and in the results of FIG. 4B, Staphylococcus aureus , a Gram-positive food poisoning bacterium on the PNA microarray reading manual. And Listeria monocytogenes ( Listeria monocytogenes ) was confirmed that the reaction appeared at the probe position. At this time, the intensity of the signal was based on 3,000 or more.

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries.National Veterinary Research and Quarantine Service (NVRQS)) <120> PNA microarray for differential diagnosis of aerobic          gram-positive food poisoning bacteria and the method for          differential diagnosis using the same <130> YPD / 201012-0007 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PNA probe of St. aureus <400> 1 gaacactcac aagatta 17 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PNA probe of L. monocytogenes <400> 2 gaaatacaaa taatcatatc c 21 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 1st PCR forward primer <400> 3 gccttgtaca caccgccc 18 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 1st PCR reverse primer <400> 4 rgtacttaga tgtttcartt cyc 23 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 2nd PCR forward primer <400> 5 acaccgcccg tcacacca 18 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> 2nd PCR reverse primer <400> 6 tacttagatg tttcarttcy c 21

Claims (3)

A PNA microarray for differential diagnosis of aerobic Gram-positive food poisoning bacteria comprising a PNA (Peptide nucleic acid) probe of SEQ ID NOs: 1 and 2 and a primer of SEQ ID NOs: 3 to 6. A method for differentially diagnosing aerobic Gram-positive food poisoning bacteria using the PNA microarray according to claim 1. According to claim 2, wherein the aerobic Gram-positive food poisoning bacteria Staphylococcus aureus ) And Listeria monocytogenes ( Listeria monocytogenes ) A method for differentially diagnosing aerobic Gram-positive food poisoning bacteria characterized in that at least one member selected from the group consisting of.

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