KR102070914B1 - Method for improving efficiency of detecting microorganisms by FISH - Google Patents

Abstract

The present invention relates to a method for improving microbial detection efficacy through FISH. Since the method according to one aspect may improve the efficiency of immobilization and permeation of microorganisms in a sample, most microorganisms including Gram-positive bacteria, which are difficult to analyze by FISH, may be efficiently analyzed.

Description

Method for improving efficiency of detecting microorganisms by FISH}

The present invention relates to a method for improving microbial detection efficacy through FISH.

Pathogenic microorganisms worldwide cause infections in water and humans, causing many diseases. Representative methods for detecting conventional microorganisms are polymerase chain reaction (PCR), a cell culture method and a gene detection method. However, since cell culture is a method for separating and identifying pathogenic and non-pathogenic viruses, it is generally judged based on whether a cytopathic effect (CPE) occurs or not. It takes a long time. Therefore, the cell culture method is less effective in determining the presence of harmful microorganisms to prepare a countermeasure. In addition, the polymerase chain reaction method, which is one of the gene diagnosis methods, is a method of amplifying a small amount of DNA or RNA, and thus the sensitivity, specificity, and rapidity are superior to the cell culture method. Since it is necessary to amplify the information of the microorganism to be detected. Thus, there are limitations to detecting unknown microorganisms.

Fluorescene in situ hybridization (FISH) method is used to solve this problem. FISH is a technology that can detect microorganisms through fluorescence microscopy by attaching fluorescent material to nucleic acid probes. In order to bind nucleic acid probes to nucleotide sequences, cell fixation and permeabilization processes are required. . In general, ethanol or paraformaldehyde is used for immobilization and permeation of cells. In the conventional monolithic treatment method, FISH efficiency is greatly changed because the efficiency of entering a probe into a cell varies depending on the type and characteristics of the cell. In particular, a stereo Philo Rhodococcus (Staphylcoccus) or a disadvantage that when the Gram-positive bacteria comprising the Streptococcus (Streptococcus), the cell wall than in other types of microorganisms is more thick to decrease the FISH efficiency by a common protocol that causes the main disease.

Therefore, there is a demand for development of a method capable of detecting various kinds of microorganisms by improving FISH efficiency.

One aspect provides a method for improving FISH assay efficiency by enhancing the efficiency of immobilization and / or permeation of microorganisms in the FISH process.

One aspect includes treating 20-25% (v / v) ethanol to a sample comprising microorganisms; And treating 95 to 100% (v / v) methanol to the sample.

In the present specification, the "sample" may include a biological sample, an environmental sample, or a food sample.

The biological sample may refer to a sample obtained from a biological subject including a sample of biological tissue or bodily fluid origin obtained in vivo or in vitro. The biological sample may be, but is not limited to, body fluids (eg, blood, plasma, serum, saliva, sputum or urine), organs, tissues, fractions, and cells isolated from a mammal, including a human. It may also include extracts from biological samples, such as antibodies, proteins, and the like, from biological fluids (eg, blood or urine).

The environmental sample may include a water sample or a soil sample.

The sample may include microorganisms such as Gram-positive bacteria such as B. subtilis , S. aureus , S. epidermidis, and Gram-negative bacteria such as E. coli , P. aeruginosa , and S. typhimurium .

The microorganism may include bacteria, viruses, fungi, fungi, or combinations thereof, and specifically may include gram-positive bacteria. In the detection of microorganisms through conventional FISH analysis, the detection efficiency of Gram-positive bacteria was not good because the Gram-positive bacteria immobilization and permeation by the known method were not sufficient. However, according to the method according to one aspect is excellent in the immobilization, permeation effect on all microorganisms including Gram-positive bacteria.

In this method, it may be to increase the efficiency of the FISH assay by increasing the degree of immobilization and permeation of the microorganism.

In the present specification, the "fixation of microorganisms" may refer to fixing the microorganism internal environment in order to bind the probes to the microorganisms, and the "permeabilization" of the microorganisms may refer to the microorganisms so that the probes are introduced into the microorganisms. It may refer to making the permeable state. Generally, in FISH analysis, the immobilization and / or permeation of microorganisms is performed by 4% paraformaldehyde, 50% cold ethanol or 70% ethanol, but not enough to immobilize and permeate some gram-negative bacteria or most gram-positive bacteria. not. However, according to the method according to one aspect, FISH analysis can be effectively immobilized and permeable to most microorganisms including Gram-positive bacteria without using 4% paraformaldehyde, 50% cold ethanol or 70% ethanol as in the conventional method The efficiency can be increased.

The method may comprise treating about 20-25% (v / v) ethanol to a sample comprising the microorganism. The ethanol may be at a concentration of about 20 to 25% (v / v), for example about 20 to 24.5% (v / v), or about 20 to 24% (v / v).

In the method, ethanol is used for about 1 minute to 60 minutes, for example about 2 to 50 minutes, about 3 to 40 minutes, about 4 to 30 minutes, or about 5 to 20 minutes, or about 10 May be for minutes. If ethanol is treated for too short a time, it may not be sufficient for immobilization and permeation of the microorganism, and if ethanol is treated for too long, the microbe may be damaged and difficult to analyze.

In the above method, ethanol may be treated at room temperature, but is not limited thereto.

The method may include treating about 95-100% (v / v) methanol to the sample. The methanol may be about 95-100% (v / v), about 96-100% (v / v), about 97-100% (v / v), about 98-100% (v / v), or about 99 To 100% (v / v) concentration.

In the process, methanol is added for about 1 minute to 60 minutes, for example about 2 to 50 minutes, about 3 to 40 minutes, about 4 to 30 minutes, or about 5 to 20 minutes, or about May be for 10 minutes. If ethanol is treated for too short a time, it may not be sufficient for immobilization and permeation of the microorganism, and if ethanol is treated for too long, the microbe may be damaged and difficult to analyze.

In the above method, methanol may be treated at room temperature, but is not limited thereto.

In the above method, when the step of treating methanol after the step of treating ethanol, the efficiency of immobilization and / or permeation of the microorganism may be improved to improve the FISH analysis efficiency.

In the above method, the method may further include treating 1 × phosphate buffered saline (PBS), saline (saline), or 1 × tris buffered saline (TBS). The PBS, saline, or TBS is not limited to the type as long as it is a solution for removing the methanol treated in the previous step. The treatment of the PBS, saline, or TBS may be to be performed once, or repeatedly two or more times.

In the method, the method may further include hybridizing the probe after immobilization and permeation of the microorganism. Hybridizing the probe may be performed by methods known in the art, for example, by treating formamide, saline sodium citrate buffer, hybridization solution.

In one embodiment, the hybridization solution is 15% (v / v) Ethylene Carbonate, 20% (v / v) Dextran sulfate, 600 mM NaCl, and 10 mM Citrate. It may include, but it is obvious that those skilled in the art can select the concentration of each component in an appropriate range. The hybridization solution may be pH 6.0 to pH 6.5, pH 6.1 to pH 6.3, or pH 6.2, but is not limited thereto.

The step of treating the formamide or SSC buffer may be performed at room temperature, but is not limited thereto.

The step of treating the hybridization solution may be performed at about 40 to 50 ℃, about 42 to 48 ℃, about 43 to 47 ℃, about 44 to 46 ℃, or about 45 ℃.

The "probe" is a part of the microorganism, for example, a polysaccharide expressed on the surface of the microorganism, genetic material (DNA, RNA, etc.) in the microorganism, antibodies, aptamers, DNA, RNA, PNA (Peptide) that binds to the protein of the microorganism Nucleic Acid), oligosaccharides, peptides or proteins.

The probe may be labeled with a detectable label. The labeling material may be a fluorescent material, a coloring material, or a chemiluminescent material, and may be labeled with the labeling material to visualize (or image) the microorganism and detect the microorganism.

The fluorescent material is selected from the group consisting of FAM, VIC, HEX, Cy5, Cy3, SYBR Green, DAPI, TAMRA, FITC, RITC, Rhodamine, Fluoredite, FluorePrime, and ROX It may include one or more fluorescent materials, but is not limited thereto. Using a probe labeled with such a fluorescent substance, hybridization and dissociation can be easily confirmed only by simple imaging due to variations in respective fluorescent signals.

The coloring material may include horse radish peroxidase (HRP), alkaline phosphatase (ALP), β-D-galactosidase (β-Gal) However, the present invention is not limited thereto.

The chemiluminescent material may include GFP, luciferin, but is not limited thereto.

In this method, detecting the signal emitted from the hybridized probe may be performed by methods known in the art, for example, histology, immunohistochemistry, immunofluorescence, in situ hybridization, FISH and the like.

The presence or absence of the microorganism and the type of the microorganism can be determined by comparing the signal of the portion where there is a signal emitted from the hybridized microbe with the signal of the other portion. In addition, the concentration of the microorganisms contained in the sample can be obtained by comparison using a standard sample containing the microorganisms at a known concentration.

Since the method according to one aspect may improve the efficiency of immobilization and permeation of microorganisms in a sample, most microorganisms including Gram-positive bacteria, which are difficult to analyze by FISH, may be efficiently analyzed.

Figure 1 is a FISH analysis of S. aureus by the conventional method.
Figure 2 is a FISH analysis of E. coli by the conventional method.
Figure 3 is a FISH analysis of S. aureus by the method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples, which are merely illustrative and are not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below may be modified without departing from the essential gist of the invention.

Comparative example  1: through FISH analysis S. aureus of  detection

Comparative Example 1 is a method using a conventional FISH protocol for detecting microorganisms.

1.1 Cleaning of the detection device

As a microbial detection apparatus, a microfluidic channel, which is a rectangular PDMS (polydimethylsiloxane) channel of 20 x 40 x 0.2 mm, was used. To clean the microfluidic channel, use a syringe pump or pneumatic pump to inject 99.9% (v / v) ethanol at a rate of 10 μl / min for 10 minutes and distilled water at 10 μl / min. The device was washed by flowing 10 × PBS (Phosphate Buffered Saline) at a rate of 10 μl / min for 10 minutes at a rate of 10 minutes.

1.2 Immobilization of Microorganisms and Permeation

The body fluid containing the sample, S. aureus , was flowed into the washed microfluidic channel to capture pathogenic microorganisms.

For immobilization and permeation of the microorganisms in the captured sample, 50% (v / v) cold ethanol was added at 10 μl / min for 10 minutes and 3.7% (v / v) form for immobilization of the microorganisms. The aldehyde solution was treated at 10 μl / min for 4 minutes and 1 × PBS at 10 μl / min for 10 minutes, and 70% (v / v) ethanol was added at 10 μl / min for permeation. The treatment was carried out for 60 minutes at a rate of 1 μl / min.

1.3 Probe Hybridization

In order to hybridize the probe to the immobilized and permeable microorganisms as described above, Cy3 and Cy5 labeled probes were hybridized with 40% (v / v) formamide for 10 minutes at a rate of 10 μl / min for 2X SSC. The solution was flowed in order for 2 hours at a rate of 5 μl / min to hybridize the probe to the microorganism. Wherein the hybridization solution comprises 15% (v / v) Ethylene Carbonate, 20% (v / v) Dextran sulfate, 600 mM NaCl, 10 mM Citrate, pH 6.2 .

1.4 Dyeing and Imaging

When the probe hybridizes as described above, 2X SSC containing 40% (v / v) formamide and the fluorescent substance DAPI was used at a rate of 10 μl / min for 15 minutes, and 2X SSC at 10 μl / min. Flowed in order for minutes. All processes except hybridization solution treatment were performed at room temperature. Treatment of the hybridization solution was carried out at 45 ° C. conditions. The microorganisms treated as above were imaged with a fluorescence microscope to detect the microorganisms. DAPI is a fluorescent material for staining unspecified DNA located between double-stranded DNA of all bacteria, Cy3 is a fluorescent material for staining probes specific to the sequence common to bacteria, Cy5 specific for S. aureus It is a fluorescent material for dyeing with. Microbe detection results are shown in FIG.

The FISH efficiency for S. aureus was evaluated by measuring the number of fluorescent signals obtained from Cy5 stained with S. aureus compared to the number of fluorescent signals obtained from DAPI staining all bacteria included in the sample.

As shown in FIG. 1, the FISH efficiency for S. aureus was found to be 36%.

Comparative example  2: through FISH analysis E. coli of  detection

In order to evaluate the efficiency of detecting E. coli through the conventional FISH analysis method, FISH analysis was performed in the same manner as in Comparative Example 1 using the microbial sample as E. coli .

However, Cy5 is a fluorescent material for specifically staining E. coli . Microbe detection results are shown in FIG.

FISH efficiency for E. coli by measuring the number of fluorescent signals obtained from Cy5 stained with E. coli stained compared to the number of fluorescent signals obtained from DAPI stained with all bacteria contained in the sample. Was evaluated.

As shown in Figure 2, the FISH efficiency for E. coli was confirmed to be 95% or more.

Through Comparative Examples 1 and 2, the efficiency of detecting Gram-negative bacteria was excellent in the conventional FISH analysis method, but the efficiency of detecting Gram-positive bacteria was not very good.

Example  1: through FISH analysis of the present invention S. aureus of  Detection efficiency evaluation

Since the efficiency of detecting Gram-positive bacteria S. aureus by the conventional FISH method was confirmed from the results of Comparative Examples 1 and 2, The microbial immobilization and permeation methods were optimized to increase the detection efficacy of S. aureus .

1.1 Cleaning of the detection device

It carried out similarly to Comparative Example 1.

1.2 Immobilization of Microorganisms and Permeation

For immobilization and permeation of the microorganisms in the captured sample, 1X PBS was added at 10 μl / min for 10 minutes, 24% (v / v) ethanol at 10 μl / min for 10 minutes, and 1X PBS For 10 minutes at a rate of 10 μl / min, 99.9% (v / v) methanol was flowed at a rate of 10 μl / min for 10 minutes, and then 1 × PBS was flowed in sequence at 10 μl / min for 10 minutes. Treated.

1.3 Probe Hybridization

It carried out similarly to Comparative Example 1.

1.4 Dyeing and Imaging

When the probe hybridizes as described above, the 2X SSC containing the fluorescent substance DAPI with 40% (v / v) formamide at a rate of 10 μl / min for 15 minutes, and the 2X SSC at 10 μl / min Flowed in order for 10 minutes. All processes except hybridization solution treatment were performed at room temperature. Treatment of the hybridization solution was carried out at 45 ° C. conditions. The microorganisms treated as above were imaged with a fluorescence microscope to detect the microorganisms. DAPI is a fluorescent material for staining unspecified DNA located between the double helix DNA of all bacteria, Cy5 is a fluorescent material for specifically staining S. aureus . Microbe detection results are shown in FIG.

The FISH efficiency for S. aureus was evaluated by measuring the number of fluorescent signals obtained from Cy5 stained with S. aureus compared to the number of fluorescent signals obtained from DAPI staining all bacteria included in the sample.

As shown in Figure 3, the FISH efficiency for S. aureus was confirmed to be 96% or more.

Therefore, when FISH analysis was performed by the method according to an embodiment, it was confirmed that S. aureus , which was not well detected by the conventional FISH method, could be detected with high efficiency.

Claims (7)

In the microbial immobilization and permeation step of the FISH assay,
Treating 20 to 25% (v / v) ethanol without treating formaldehyde with a sample containing microorganisms; And
Treating 95 to 100% (v / v) methanol to the sample. The method of claim 1, wherein the sample is a biological sample, an environmental sample, or a food sample. The method of claim 1, wherein the ethanol is treated for 1 to 60 minutes. The method of claim 1, wherein the methanol is treated for 1 to 60 minutes. The method of claim 1, wherein the microorganism comprises Gram-positive bacteria. The method of claim 1, further comprising treating 1 × PBS, saline, or 1 × TBS. The method of claim 1, wherein the efficiency of the FISH assay is increased by increasing the immobilization and permeation of the microorganism.

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