KR20160107632A - Method for identification of causative bacteria using bacteria-derived extracellular vesicles after nuclease treatment - Google Patents
Method for identification of causative bacteria using bacteria-derived extracellular vesicles after nuclease treatment Download PDFInfo
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Abstract
The present invention relates to a method for identifying bacteria by using a nucleic acid of an extracellular endoplasmic reticulum whose external nucleic acid has been removed by treatment with a nuclease.
According to the method of the present invention, it is possible to increase the possibility of using the method of identifying bacteria using the extracellular endoplasmic reticulum by treating the bacterial-derived extracellular endoplasmic reticulum with nuclease to remove the nucleic acid outside the extracellular endoplasmic reticulum. Accuracy can be improved.
Description
The present invention relates to a method for identifying a bacterium by analyzing a nucleic acid of an extracellular endoplasmic reticulum from which an external nucleic acid has been removed by a nuclease treatment.
Gram-negative bacteria and Gram-positive bacteria secrete extracellular vesicles into the extracellular environment for intercellular information exchange. The extracellular endoplasmic reticulum is composed of a spherical phospholipid bilayer with a diameter of 20-200 nm, and is often referred to as nanoseves. Extracellular ERs derived from Gram-negative bacteria have lipopolysaccharide (LPS), toxic proteins, and DNA and RNA, which are nucleic acids of bacteria. The extracellular endoplasmic reticulum from Gram-positive bacteria also contains peptidoglycan and lipoteichoic acid, which are cell wall components, in addition to toxic proteins and nucleic acids. In addition, substances that may be unstable outside the cell, such as nucleic acids, are encapsulated within the extracellular endoplasmic reticulum and can secrete outside the cell by secretion. Thus, the extracellular endoplasmic reticulum represents cells or bacteria from which the constituents are derived. For example, extracellular endoplasmic reticulum derived from S. pneumoniae contains pneumococcal specific proteins and nucleic acids.
On the other hand, ribosomal RNA (rRNA) is a RNA that constitutes a ribosome, which accounts for about 80% of intracellular RNA. The rRNA consists of a variable region which is different from the same base sequence portion for each individual, and the bacterium can be distinguished by analyzing different base sequence portions. For example, the sequence of 16S rRNA that constitutes a small 30S ribosomal unit of bacteria is composed of about 1500 bases, and most of them are highly conserved, while some sequences exhibit high sequence diversity. In other words, 16S rRNA gene is most suitable for evolutionary system analysis and is widely used for bacterial identification because there is little sequence diversity among the bacterial species, and diversity among species is very high.
Since the extracellular endoplasmic reticulum represents cells or bacteria that are derived, it contains nucleic acids such as DNA, rRNA and mRNA of bacteria. Therefore, studies are being carried out to identify bacteria by using nucleic acid present in the extracellular endoplasmic reticulum and to diagnose infection. However, the nucleic acid present in the extracellular endoplasmic reticulum is present both inside and outside the extracellular endoplasmic reticulum, and the exogenous nucleic acid may be derived from dead bacteria of other species, and its origin is unclear. When used for diagnosis, bacteria can not be identified accurately and the accuracy of infection diagnosis can be reduced.
However, there are no cases in which nucleic acids existing inside and outside the extracellular matrix are distinguished from each other and used for diagnosis.
The inventors of the present invention found that, when the nucleic acid outside the extracellular matrix was removed by treating the extracellular endoplasmic reticulum with the nuclease, the present inventors discovered that specific bacteria can be identified only by the nucleic acid present in the extracellular endoplasmic reticulum Thereby completing the present invention.
Accordingly, an object of the present invention is to provide a method for identifying a bacterium using a nucleic acid of a bacterial-derived extracellular endoplasmic reticulum treated with nuclease.
However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.
In order to accomplish the object of the present invention as described above, the present invention provides a method for preparing a sample, comprising: (a) separating extracellular endoplasmic reticulum from a sample; (b) treating the extracellular endoplasmic reticulum with a nuclease; (c) analyzing the nucleic acid using a bacterial species-specific primer for the product treated with the nuclease; And (d) determining the presence or absence of a bacterium in accordance with the presence or absence of the detection of the analytical product. The present invention also provides a method for identifying a bacterium using the bacterium-derived extracellular endoplasmic reticulum treated with a nuclease.
The present invention also relates to a method for the treatment of cancer, comprising: (a) treating a sample with a nuclease; (b) separating the extracellular endoplasmic reticulum from the nuclease treated sample; (c) analyzing the nucleic acid using a bacterial species-specific primer for the separated extracellular endoplasmic reticulum; And (d) determining the presence or absence of a bacterium in accordance with the presence or absence of the detection of the analytical product. The present invention also provides a method for identifying a bacterium using the bacterium-derived extracellular endoplasmic reticulum treated with a nuclease.
In one embodiment of the present invention, the step of separating the extracellular endoplasmic reticulum is selected from the group consisting of continuous centrifugation, ultrafast centrifugation, polymer-based precipitation, and gel filtration .
In another embodiment of the present invention, the method may further include the step of separating and purifying the nucleic acid between steps (b) and (c).
In another embodiment of the present invention, the sample may be a body fluid selected from the group consisting of blood, urine, ascites, synovial fluid, breast milk, sputum, saliva, sweat, tears, runny nose, and feces.
In yet another embodiment of the present invention, the sample may be a surrounding environment selected from the group consisting of dust, tap water, seawater, soil, air, and rainwater.
In another embodiment of the present invention, the sample may be a fermented food selected from the group consisting of beer, miso, soy sauce, kimchi, yogurt, and yoghurt.
In another embodiment of the present invention, the nuclease is a deoxyribonuclease or a ribonuclease.
In another embodiment of the present invention, the ribonuclease is selected from the group consisting of RNase A, RNase H, RNase I, RNase II, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, , RNase V1, Polynucleotide Phosphorylase, RNase PH, RNase R, RNase D, Oligoribonuclease, Exoribonuclease I, and Exoribonuclease II.
In another embodiment of the present invention, the nucleic acid may be 16S rDNA, 16S rRNA, DNA or mRNA.
In another embodiment of the present invention, the primer is a bacterial species-specific 16S rRNA, 16S rDNA; Or may be targeted to DNA or RNA against an antibiotic resistance gene or a pathogenic gene.
In another embodiment of the present invention, the nucleic acid analysis can be performed using a polymerase chain reaction (PCR), a reverse transcription polymerase chain reaction (RT-PCR), real time PCR, real time RT- ≪ / RTI >
According to the method of the present invention, it is possible to increase the possibility of using a method of identifying a bacterium using an extracellular endoplasmic reticulum by treating a bacterium-derived extracellular endoplasmic reticulum with a nuclease to remove nucleic acids outside the extracellular endoplasmic reticulum. Can be improved.
FIG. 1 is a graph showing the results obtained when P. aeruginosa- derived extracellular endoplasmic reticulum was isolated and treated with RNA of S. aureus and treated with or without ribonuclease, The results are shown in Fig.
FIG. 2A shows the results of confirming the presence of P. aeruginosa , S. aureus , and the extracellular endoplasmic reticulum endoplasmic reticulum endoplasmic reticulum (ER) RNA. FIG. 2B shows the results of PCR for P. aeruginosa , , Staphylococcus aureus (S. aureus), these bacteria-derived extracellular vesicles, and mammalian cells (SW480, Colon26) bacteria common to the 16S rRNA, Pseudomonas (Pseudomonas) or staphylococcal species (Staphylococcus) species-specific 16S rRNA, and mammalian The result of confirming common 18S rRNA.
FIG. 3 is a result of confirming RNAs of P. aeruginosa and S. aureus- derived extracellular endoplasmic reticulum with or without ribonuclease treatment. FIG. the result shows that a reduced amount of RNA, Figure 3B, and the result shows the 16S rRNA and the reduction of the 23S rRNA of Pseudomonas aeruginosa by ribonuclease treatment, Figure 3C, Pseudomonas (Pseudomonas) species or Staphylococcus aureus (Staphylococcus ) The result of PCR confirmed the presence of 16S rRNA of P. aeruginosa and Staphylococcus aureus using a species-specific 16S rRNA primer.
Figure 4 shows the amount of mRNA transcripts of bacterial common 16S rRNA and pseudomonas spp. Genes, acpP, aprA, katB, recN, and rpoD in P. aeruginosa- derived extracellular endoplasmic reticulum with or without ribonuclease treatment FIG. 4A is a table showing Ct Value (cycles) as a result of real-time PCR, FIG. 4B is a table showing Ct Value (cycles), and FIG. 16S rRNA and each transcript are located outside and inside the extracellular endoplasmic reticulum.
FIG. 5 is a graph showing the relationship between the amount of mRNA from the common 16S rRNA of S. aureus- derived extracellular endoplasmic reticulum and the amount of mRNA transcript of atlA and clfB, which are specific genes of Staphylococcus aureus, 5A is a table showing Ct Value (cycles) as a result of real-time PCR, FIG. 5B is a table showing Ct Value (cycles), and FIG. 5C is a table showing the 16S rRNA And the positions where the respective transcripts are present outside and inside the extracellular endoplasmic reticulum.
FIG. 6 is a graph showing the results of the administration of P. aeruginosa- derived extracellular endoplasmic reticulum to the mouse, blood and urine of the mouse were obtained and the ribonuclease was treated, and then Pseudomonas species specific to blood and urine The presence of 16S rRNA and 18S rRNA in mouse were confirmed by PCR.
The present invention provides a method for identifying a bacterium using a nucleic acid of a bacterial-derived extracellular endoplasmic reticulum from which an external nucleic acid has been removed by nuclease treatment.
In one embodiment of the present invention, Pseudomonas aeruginosa) Staphylococcus aureus derived from the extracellular vesicles separation (Staphylococcus aureus ) and then treated with ribonuclease (RNase) in the extracellular endoplasmic reticulum derived from P. aeruginosa. After rinsing the endoplasmic reticulum to obtain all the nucleic acids, deoxyribonuclease (DNase) was treated to obtain only the RNA, and PCR was performed. As a result, the RNA derived from Staphylococcus aureus was not decomposed by ribonuclease treatment, Pseudomonas aeruginosa belongs to (Pseudomonas) species-specific 16S rRNA was identified. Therefore, it was confirmed that a specific bacterium can be identified through the ribosomal RNA of the extracellular endoplasmic reticulum in the presence of various bacteria (see Example 1).
In another embodiment of the present invention, it was confirmed that common 16S rRNA and 23S rRNA of bacteria were present from both Pseudomonas aeruginosa and Staphylococcus aureus cells and the extracellular endoplasmic reticulum from the bacterium. In addition, RNA was isolated from human and mouse-derived mammalian cells (SW480, Colon26) and PCR was carried out together with the bacterium-derived extracellular endoplasmic reticulum from the bacterium. As a result, was confirmed in the common 16S rRNA, Pseudomonas (Pseudomonas) or Staphylococcus aureus (Staphylococcus) species when using the specific primers were each identified the Pseudomonas aeruginosa and Staphylococcus aureus. Only mammalian common 18S rRNA was identified in mammalian cells (see Example 2).
In another embodiment of the present invention, it was confirmed that about 96-97% of total RNA was removed when ribonuclease was treated with P. aeruginosa or extracellular endoplasmic reticulum-derived endoplasmic reticulum. However, for 16S rRNA ribonuclease it was the small amount present inside the ER after the treatment, ribonuclease treatment by using the PCR Identification common 16S rRNA, Pseudomonas (Pseudomonas) or Staphylococcus aureus (Staphylococcus) species-specific 16S rRNA of the bacteria , It was confirmed that bacteria could be identified through a small amount of nucleic acid inside the extracellular endoplasmic reticulum (see Example 3).
In another embodiment of the present invention, the extracellular endoplasmic reticulum of Pseudomonas aeruginosa or Staphylococcus aureus is treated with a ribonuclease, the endoplasmic reticulum is ruptured to obtain all the nucleic acids, the deoxyribonuclease is treated to obtain only RNA, The amount of gene transcript specific to bacterial 16S rRNA and Pseudomonas aeruginosa or Staphylococcus aureus was confirmed. In both of the above two bacteria, it was confirmed that a small amount of 16S rRNA common to bacteria remained in the extracellular endoplasmic reticulum, and that transcripts specific for each bacterium were mostly present in the endoplasmic reticulum (see Example 4 ).
In another embodiment of the present invention, the parenteral injection of Pseudomonas aeruginosa-derived extracellular endoplasmic reticulum into the mouse was performed to obtain blood and urine. After the treatment with ribonuclease, the Pseudomonas sp . By identifying 16S rRNA, P. aeruginosa was identified (see Example 5).
The present invention relates to a method for separating extracellular endoplasmic reticulum from a sample; Treating the extracellular endoplasmic reticulum with a nuclease; Analyzing the nucleic acid using a bacterial species-specific primer for the nuclease-treated product; And determining the presence or absence of a bacterium in accordance with the presence or absence of the detection of the analytical product. The present invention also provides a method for identifying a bacterium using the bacterium-derived extracellular endoplasmic reticulum treated with a nuclease.
The present invention also relates to a method for the treatment of cancer, comprising the steps of: treating a sample with a nuclease; Separating the extracellular endoplasmic reticulum from the nuclease treated sample; Analyzing the nucleic acid using a bacterial species-specific primer for the separated extracellular endoplasmic reticulum; And determining the presence or absence of a bacterium in accordance with the presence or absence of the detection of the analytical product. The present invention also provides a method for identifying a bacterium using the bacterium-derived extracellular endoplasmic reticulum treated with a nuclease.
The extracellular endoplasmic reticulum of the present invention is secreted from cells, and is divided into internal and external by a double lipid membrane. The extracellular endoplasmic reticulum has cell membrane lipids, cell membrane proteins, genes, and metabolites of the derived cells. The extracellular endoplasmic reticulum has a spherical shape and may have a diameter of 20 to 1000 nm, but is not limited thereto.
The step of separating the nucleic acid from the extracellular endoplasmic reticulum may further comprise the step of separating and purifying the nucleic acid from the extracellular endoplasmic reticulum, And the method of isolating the gene is not limited thereto.
In the present invention, the sample is derived from the body such as blood, urine, ascites, synovial fluid, breast milk, sputum, saliva, sweat, tears, runny nose and faeces, and is contained in the vicinity of dust, tap water, seawater, soil, air, Environment-derived, or fermented foods such as beer, miso, soy sauce, kimchi, yogurt, and yoghurt.
Methods for separating extracellular endoplasmic reticulum from the sample of the present invention include continuous centrifugation, ultracentrifugation, polymer-based precipitation, and gel filtration. And may be, but is not limited to, a method for separating extracellular endoplasmic reticulum.
As used herein, the term " nuclease " refers to an enzyme that catalyzes hydrolysis of a nucleic acid to a nucleotide, and broadly speaking, is involved in the hydrolysis of a nucleotide or a nucleoside It also includes enzymes. The nuclease of the present invention may be, but is not limited to, deoxyribonuclease (DNase) or ribonuclease (RNase).
In addition, the ribonuclease of the present invention can be used as a ribonuclease in the present invention, including RNase A, RNase H, RNase I, RNase II, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, But are not limited to, phosphorylase, RNase PH, RNase R, RNase D, Oligoribonuclease, Exoribonuclease I, and Exoribonuclease II.
As used herein, the term " nucleic acid " is a genetic material of a living organism composed of a monomer that is a nucleotide consisting of a pentose, a phosphate group, and a base, and typically includes DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) do. More specifically, the deoxyribonucleic acid containing DNA includes DNA, mtDNA, and cDNA, and ribonucleic acid including RNA includes RNA, mRNA, tRNA, rRNA, ncRNA, sgRNA, shRNA, siRNA, snRNA, miRNA, , And other nucleic acid analogues, such as GNA, PNA, TNA, and morpholino. The nucleic acid of the present invention may be, but is not limited to, 16S rDNA, 16S rRNA, DNA, and / or mRNA.
Among the constituents of the extracellular endoplasmic reticulum, nucleic acids used for the analysis include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction time polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) But is not limited thereto. The primers for the polymerase chain reaction were bacterial species-specific 16S rRNA and 16S rDNA; Or may target DNA or RNA to an antibiotic resistance gene or pathogenic gene, but is not limited thereto.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
[ Example ]
Example 1. Bacterial origin in the presence of various bacteria Extracellular Endoplasmic reticulum Ribosome RNA Identification of a specific bacterium through
As a method for identifying a specific bacterium in the presence of various bacterium, a bacterium was identified by using ribosomal RNA (rRNA) existing in the extracellular endoplasmic reticulum.
Specifically, Pseudomonas aeruginosa ) was centrifuged, and the supernatant was obtained. The cells were then filtered through 0.45 ㎛ and 0.22 ㎛ filters in order to remove the cells in the culture. Staphylococcus aureus ). Subsequently, the culture was centrifuged at 150,000 g for 4 hours at 4 ° C for 3 hours to isolate the extracellular endoplasmic reticulum from the culture. Ribonuclease (RNase), an RNA degradation enzyme, is added to the extracellular endoplasmic reticulum-derived endoplasmic reticulum-derived endoplasmic reticulum-derived endoplasmic reticulum-derived extracellular endoplasmic reticulum and cultured at 95 ° C for 10 minutes to remove all the nucleic acids present inside and outside the extracellular endoplasmic reticulum Respectively. Deoxyribonuclease (DNase) is treated to remove DNA from the extracellular ER DNA, DNase activity is inhibited by DNase inhibitor, random primer, AMV reverse transcriptase, dNTP, RNase inhibitor (RNase inhibitor) was added to perform cDNA synthesis. After using the synthesized cDNA as a template was confirmed by PCR for the presence of Pseudomonas (Pseudomonas) species-specific 16S rRNA and Staphylococcus aureus (Staphylococcus) species-specific 16S rRNA.
As a result, it was identified both in Fig., Derived from Pseudomonas aeruginosa out not treated with ribonuclease reticulum cell as shown in Fig. 1 Pseudomonas (Pseudomonas) species-specific 16S rRNA and Staphylococcus aureus (Staphylococcus) are species-specific 16S rRNA. On the other hand, only the Pseudomonas species-specific 16S rRNA was identified in the Pseudomonas aeruginosa-derived extracellular endoplasmic reticulum treated with ribonuclease. Therefore, it was confirmed that the RNA of the staphylococci put into the culture broth was not decomposed by the ribonuclease treatment.
Example 2. Bacterial Origin Extracellular From the endoplasm Ribosome RNA Check presence
In order to confirm the presence of ribosomal RNA in the extracellular endoplasmic reticulum derived from bacteria, Gram-negative bacteria such as Pseudomonas aeruginosa ) and gram-positive bacteria ( Staphylococcus aureus ), and extracellular endoplasmic reticulum isolated from the culture of the two bacteria.
The extracellular endoplasmic reticulum-derived endoplasmic reticulum-derived endoplasmic reticulum was put into a flask containing LB medium and incubated at 37 ° C in an incubator. 600 was measured until the value reached 1.5 and centrifuged at 6,000 g for 20 minutes at 4 ° C to remove P. aeruginosa cells. The supernatant was passed through a 0.45 mu m filter to remove remaining bacteria, and then a cell culture having a size larger than 100 kD was separated and concentrated using a filter having a 100 kD membrane. The concentrated cell culture solution having a size larger than 100 kD was filtered with a 0.22 mu m filter and subjected to ultrafast centrifugation at 150,000 g at 4 DEG C for 3 hours to obtain extracellular endoplasmic reticulum.
Staphylococcus aureus-derived extracellular endoplasmic reticulum was prepared by placing Staphylococcus aureus in a flask containing nutrient broth medium and incubating at 37 ° C in an incubator. 600 was measured until the value reached 1.5, and the cells were removed by centrifugation at 10,000 g for 20 minutes at 4 ° C. The subsequent procedure was carried out in the same manner as in the case of the above-mentioned Pseudomonas aeruginosa to obtain extracellular endoplasmic reticulum derived from Staphylococcus aureus.
The PowerMicrobiome TM RNA isolation kit (MoBio) was used to separate RNA from cells obtained from Pseudomonas aeruginosa or Staphylococcus aureus and the extracellular endoplasmic reticulum. The lysis buffer containing β-mercaptoethanol was applied to each cell and extracellular endoplasmic reticulum, followed by chemical and physical disruption using glass beads. The glass beads were removed by centrifugation, and the supernatant obtained was treated on a column that only bound RNA. The RNA was bound to the column and DNase was treated to isolate only the RNA from which the DNA was finally removed.
RNA was also isolated from mammalian cells for comparison with RNA isolated from bacterial or bacterial extracellular endoplasmic reticulum. RNA extraction was performed using SW480 cells for human-derived cells and Colon26 cells for mouse-derived cells using miRNeasy RNA isolation kit (QIAGEN). Each cell was treated with TRIzol solution and cultured for 5 minutes, followed by treatment with chloroform to obtain an aqueous phase. After mixing the aquenous phase with 100% ethanol, the RNA was bound only to the column. The RNA was bound to the column and DNase was treated to isolate only the RNA from which the DNA was finally removed.
RNA from P. aeruginosa and S. aureus cells and extracellular endoplasmic reticulum were analyzed for total RNA using a Bioanalyzer 2100 instrument. As a result, as shown in Fig. 2A, the pneumococcal cells ( P. aeruginosa cells) and P. aeruginosa EVs, S. aureus cells and S. aureus-derived extracellular endoplasmic reticulum S. aureus EV) were found to be present in both 16S rRNA and 23S rRNA.
In addition to the above results, in the same manner as in Example 1, using the above-obtained Pseudomonas aeruginosa, Staphylococcus aureus cells, extracellular endoplasmic reticulum from each bacterium, and RNA (1 ㎍) obtained from mammalian cells to confirm the presence of ribosomal RNA by PCR Gt; cDNA < / RTI > Using the synthesized cDNA as a template bacteria common 16S rRNA, Pseudomonas (Pseudomonas) species-specific 16S rRNA, Staphylococcus aureus (Staphylococcus) species-specific 16S rRNA, and mammalian individual primers specific for amplifying a common 18S rRNA PCR was performed.
As a result, as shown in FIG. 2B, there was a common 16S rRNA of bacteria in both Pseudomonas aeruginosa and Staphylococcus aureus cells and extracellular endoplasmic reticulum, while there was no bacterial common 16S rRNA in mammalian cells. Was Pseudomonas (Pseudomonas) species-specific 16S rRNA is identified only in Pseudomonas aeruginosa-derived cells and the extracellular vesicles, Staphylococcus aureus (Staphylococcus) species-specific 16S rRNA showed a species-specific characteristic which is identified only in Staphylococcus aureus-derived cells and the extracellular vesicles . On the other hand, mammalian common 18S rRNA was identified in mammalian cells, but not in both bacterial-derived cells and extracellular endoplasmic reticulum.
Example 3. Bacterial origin after ribonuclease treatment Extracellular Endoplasmic reticulum Ribosome RNA Check presence
The cells were treated with 1 / / ml of ribonuclease at 37 캜 for 10 minutes, then treated with ribonuclease inhibitor and incubated at 4 캜 for 150,000 g Ultra high speed centrifugation was performed for 3 hours to obtain a ribonuclease treated extracellular endoplasmic reticulum.
RNA was obtained and quantified in the extracellular endoplasmic reticulum treated with or without the ribonuclease as described in Example 2. As a result, as shown in FIG. 3A, 96-97% of the total RNA amount of the extracellular endoplasmic reticulum Was removed by ribonuclease treatment. As shown in FIG. 3B, RNA was isolated from Pseudomonas aeruginosa-treated extracellular endoplasmic reticulum treated with or without ribonuclease and analyzed with Bioanalyzer 2100 instrument. As a result, most 16S rRNA and 23S rRNA were removed, Of 16S rRNA remains after the ribonuclease treatment.
In addition, cDNA synthesis was carried out in the same manner as in Example 1 using RNA (1 ㎍) obtained from Pseudomonas aeruginosa or an extracellular endoplasmic reticulum derived from Staphylococcus aureus treated or not treated with ribonuclease. Using the synthesized cDNA as a template, the presence of 16S rRNA specific for Pseudomonas species or 16S rRNA for Staphylococcus species was confirmed by PCR.
As a result, as shown in FIG. 3C, it was confirmed that most of 16S rRNA was removed by treatment with ribonuclease, and a small amount of 16S rRNA was present compared with 16S rRNA existing before treatment.
These results suggest that 16S rRNA existing in the extracellular endoplasmic reticulum from P. aeruginosa or Staphylococcus aureus is present mostly in the endoplasmic reticulum and is removed by ribonuclease treatment. However, a small amount of 16S rRNA is still present in the endoplasmic reticulum, It was confirmed that the primers can be amplified by PCR.
Example 4. After ribonuclease treatment Extracellular Present in the endoplasmic reticulum Ribosome RNA and Transcript Confirm
After treatment with the ribonuclease or without treatment of the extracellular endoplasmic reticulum from the Pseudomonas aeruginosa or Staphylococcus aureus by the method of Example 3, each of the above-described extracellular endoplasmic reticulum (10 in in proteins) was incubated at 95 째 C for 10 minutes The extracellular endoplasmic reticulum was exploded to allow all of the nucleic acids present inside and outside the extracellular endoplasmic reticulum. After the DNA was treated with DNase, DNase activity was inhibited by treatment with DNase inhibitor, and cDNA synthesis was carried out in the same manner as in Example 1. Using the synthesized cDNA as a template, real-time PCR was performed on bacterial common 16S rRNA and Pseudomonas aeruginosa or Staphylococcus aureus-specific transcripts to confirm the change of each RNA amount.
In the case of Pseudomonas aeruginosa, an acpP gene encoding an acyl carrier protein (ACP), an aprA gene encoding an alkaline protease, a katB gene encoding a catalase, a DNA repair protein The recN gene encoding the DNA repair protein RecN, and the rpoD gene encoding the RNA polymerase sigma factor RpoD, that is, the amount of mRNA produced through transcription of each gene.
As a result, as shown in FIG. 4A and FIG. 4B, the extracellular endoplasmic reticulum-derived endoplasmic reticulum exhibited a Ct (threshold cycle) value of 15 cycles after the ribonuclease treatment, whereas transcripts (acpP, aprA, katB, recN, and rpoD) did not show any difference, or most of them were 5 cycles later. As shown in FIG. 4C, most of the 16S rRNA was present outside but some were still inside the extracellular endoplasmic reticulum. Since aprA in the transcript was not affected by ribonuclease, , While acpP, katB, recN, and rpoD are mostly present inside the extracellular ER, while others are located outside.
In the case of Staphylococcus aureus, the amount of each transcript, mRNA, of the clfB gene encoding the atlA gene encoding the hydrolyzing enzyme AtlA, which is a specific gene for Staphylococcus aureus, and the clumping factor B (ClfB) was confirmed.
As a result, as shown in Fig. 5A and Fig. 5B, the extracellular endoplasmic reticulum-derived extracellular endoplasmic reticulum exhibited a Ct (threshold cycle) value of 16S rRNA by about 10 cycles after ribonuclease treatment, clfB) showed no significant difference in cycle. As shown in FIG. 5C, most of the bacterium-common 16S rRNA is present outside but some are still in the inside. Since the transcripts atlA and clfB are not affected by ribonuclease, It means that it exists inside.
Example 5. In mouse animal models, the presence of blood and urine Extracellular Escherichia coli-derived ribosome RNA Wow Transcript Confirm
After injecting the extracellular endoplasmic reticulum (ER) derived from Pseudomonas aeruginosa into the mouse, blood and urine of the mouse were taken and the ribonuclease was treated to confirm the identification of P. aeruginosa through identification of the ribosomal RNA and transcript from the extracellular endoplasmic reticulum.
Blood and urine were obtained at 0, 0.5, 2, 6, and 24 hours after intraperitoneal injection of Pseudomonas aeruginosa (10 ㎍ in proteins) into mice (C57BL / 6, male, 6-8 weeks). After treating mouse blood and urine with ribonuclease, RNA was isolated using PowerMicrobiome TM RNA isolation kit (MoBio) without separate bacterial-derived extracellular endoplasmic reticulum. CDNA synthesis was performed according to the method of Example 1 using mouse blood and RNA (1 占 퐂) obtained from the urine. PCR was performed using the synthesized cDNA as a template to confirm the presence of Pseudomonas species-specific 16S rRNA and mouse-specific 18S rRNA.
As a result, as shown in FIG. 6A, the 16S rRNA specific for Pseudomonas species in the mouse blood began to be identified from 0.5 hour, and then decreased toward 24 hours, and the mouse specific 18S rRNA was observed only at 0.5 hour It was identified.
As shown in FIG. 6B, the 16S rRNA specific for Pseudomonas species in the mouse urine started to be identified from 2 hours, and then decreased toward 24 hours, and 18S rRNA was not identified.
The above results indicate that the nucleic acid existing outside the extracellular endoplasmic reticulum derived from the peritoneal injection of the mouse blood and urine, particularly the ribosomal RNA existing inside the extracellular endoplasmic reticulum even if the ribosomal RNA is completely removed by the ribonuclease treatment And that the identification of P. aeruginosa was possible.
It will be understood by those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
Claims (12)
(a) separating the extracellular endoplasmic reticulum from the sample;
(b) treating the extracellular endoplasmic reticulum with a nuclease;
(c) analyzing the nucleic acid using a bacterial species-specific primer for the product treated with the nuclease; And
(d) determining whether or not the bacteria are present according to the presence or absence of the detection of the analytical product.
(a) treating the sample with a nuclease;
(b) separating the extracellular endoplasmic reticulum from the nuclease treated sample;
(c) analyzing the nucleic acid using a bacterial species-specific primer for the separated extracellular endoplasmic reticulum; And
(d) determining whether or not the bacteria are present according to the presence or absence of the detection of the analytical product.
Wherein the step of separating the extracellular endoplasmic reticulum comprises a method selected from the group consisting of continuous centrifugation, ultrafast centrifugation, polymer-based precipitation, and gel filtration. .
Further comprising separating and purifying the nucleic acid between steps (b) and (c).
Wherein the sample is a body fluid selected from the group consisting of blood, urine, ascites, synovial fluid, breast milk, sputum, saliva, sweat, tears, runny nose, and feces.
Wherein the sample is a surrounding environment selected from the group consisting of dust, tap water, seawater, soil, air, and rainwater.
Wherein the sample is a fermented food selected from the group consisting of beer, miso, soy sauce, kimchi, yogurt, and yoghurt.
Wherein the nuclease is a deoxyribonuclease or a ribonuclease. ≪ Desc / Clms Page number 20 >
The ribonuclease may be selected from the group consisting of RNase A, RNase H, RNase I, RNase II, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, RNase V1, Polynucleotide Phosphorylase, RNase PH , RNase R, RNase D, Oligoribonuclease, Exoribonuclease I, and Exoribonuclease II.
Wherein the nucleic acid is 16S rDNA, 16S rRNA, DNA, or mRNA.
The primers include bacterial species-specific 16S rRNA, 16S rDNA; Or a DNA or RNA for an antibiotic resistance gene or a pathogenic gene is targeted.
In step (c), the nucleic acid analysis may be performed by a group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real time PCR, real-time RT-PCR, and fluorescence in situ hybridization Wherein the method is performed in a selected method.
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| KR101704828B1 (en) * | 2011-03-31 | 2017-02-09 | 포항공과대학교 산학협력단 | Method for diagnosing inflammatory diseases through analysis of protein or gene of extracellular vesicle in a body fluid |
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