JPH0740B2 - Methods for the detection and identification of infectious disease-causing bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a diagnostic system for infectious diseases and a method for detecting and identifying microorganisms for use in said system.

[Problems to be solved by the prior art and the invention]

Infection, pathologically, refers to the invasion of a pathogenic microorganism (hereafter referred to as "bacteria") into the body and establishment of a foothold for proliferation. The onset of disease due to infection depends on the interaction between the host organism's resistance and the virulence of the bacteria.

Among infectious diseases, improving the treatment of bacteremia is considered an important issue as follows:

In other words, bacteremia is not a disease caused by a specific bacterium, but begins when various bacteria appear and live in the blood.
Clinically, the disease is suspected when a fever of nearly 40 degrees Celsius continues for more than two days.

Symptoms include high fever, and if left untreated, it can last for several days or
It is a serious and urgent disease that can lead to death within one to two days, particularly in children and patients with terminal cancer whose immune systems are weakened.

In infectious diseases, neutrophils are the primary pathogens in living tissues.
Phagocytic cells of the monocyte and macrophage system play a role in this defense.

The appearance of bacteria in the blood in bacteremia is thought to be due to bacteria becoming dominant in the battle with phagocytes and leaking out of the tissues into the blood.

Bacteremia refers to the state in which such bacteria appear, and treatment involves administering large amounts of antibiotics to which the causative bacteria are sensitive.

However, since antibiotics generally impair the function of the body (organs) such as the liver, it is necessary to avoid as much as possible giving ineffective antibiotics to patients in critical condition.

Generally, bacteremia is defined as a condition in which the phagocytic ability of cells is not strong enough to counter the virulence of bacteria, and the bacteria spread throughout the bloodstream. If bacteremia is caused by the action of toxins produced by bacteria, and severe symptoms are present, sepsis is the condition. To prove sepsis (i.e., to establish a diagnosis), the following are required: (1) clinical symptoms,
(2) Culture, (3) Gram staining, and (4) confirmation of shock are important items, and only after these items are met can a treatment plan be decided.

Therefore, rapid and reliable identification of the bacteria is essential for treatment.

According to the current method for identifying this bacterium, the routine detection and identification of bacteria in laboratory specimens suspected of bacteremia is performed using culture bottles (hereafter referred to as "C/B").
The general procedure is to identify the causative bacteria using selective media only for positive samples. However, in reality, the success rate of culturing bacteria from these blood samples is extremely low. Furthermore, if a patient is given large amounts of antibiotics when bacteremia is suspected, even if bacteria are present in the blood, they often cannot be multiplied or grow, and therefore the rate of C and B positive results is extremely low.

As a subroutine method, instrumental analysis of bacterial components and metabolic products of bacteria (Yoshimi Benno, Rapid identification of bacteria by gas chromatography, Clinical Testing, vol. No. 12,
November 1985, Igaku Shoin], a method using specific antibodies [Japanese Patent Laid-Open No. 60-224068], and a method using hybridization utilizing the specificity of DNA [Japanese Patent Laid-Open No. 2003-2004068].
61-502376], highly accurate methods based on the specificity of the method have been developed, but all of them require isolation of the bacteria and enrichment culture.

On the other hand, there are studies that focus on the function of phagocytes in infectious diseases, such as
There is a method for microscopically examining a smear-stained sample of buffy coat, which is the region where white blood cells are concentrated in a blood sample.

Generally, the frequency of bacteria being detected in buffy coat specimens in adult bacteremia is only 30%, similar to that in earlobe blood.
In newborns, there are reports of bacteria being detected in 7 out of 10 cases (70%), and information on the presence or absence of bacteria in peripheral blood by microscopic examination of smear specimens is an important guide for treatment.

Thus, although rapidity and accuracy are essential in the field of diagnosis, conventional methods have hardly contributed to treatment.

In the pretreatment step of the prior art, it takes at least 1 to 2 days for selective isolation from the specimen, 1 day for bacterial enrichment, and 1 day for fixation.
It takes at least 10 days, a total of 3-4 days, and in reality, the culture is continued until the bacteria grow, so even if the result is C and B positive, it often takes more than a week. This is the reason for the high mortality rate of C and B positive cases using the conventional method. For example, according to a 1984 report in the Japanese Journal of Infectious Diseases, Vol. 58, No. 2, p. 122, even though the blood culture positivity rate was 28.6% (163/569 cases), the mortality rate among these cases was reported to be 84.6% (138/163 cases).

Furthermore, culture may not be able to distinguish between the presence of normal bacteria.

For example, Staphylococcus epidermidis (Epidermides), a type of staphylococcus that is considered one of the most likely causes of bacteremia, is also found on the skin of normal people, so there is a risk that it may be absorbed when the needle is inserted into the skin and become mixed into the sample.

And what is important is that, due to the aforementioned circumstances, many of the bacteria in the specimen to be cultured are taken up by the phagocytes and are dead or in a quiescent state due to the administration of antibiotics, so even under culture conditions, the number of bacteria that can grow is small.
In fact, the detection rate of bacteria by culture using clinical specimens is 10%.
It is very low both front and back.

In other words, even if blood from patients who are clinically suspected of having bacteremia is cultured and tested for over a day and night, the presence of bacteria is not even detected in 90% of cases. Therefore, as mentioned above, treatment is now started as soon as sepsis is clinically suspected, without waiting for the detection results. In other words, the trial method is to administer an antibiotic that is effective against the widest range of bacteria, observe the condition for a day or two, and switch to a different antibiotic if no effect is observed.

Furthermore, in the staining method described above, biological components are stained in the same way as bacteria, so rapid identification of bacteria from the image based solely on their morphology requires skill and can be difficult.

Generally, cells can be stained and visualized. The cells then attempt to digest the proteins that coat the outside of the bacteria that have entered. When bacteria first enter, the outer proteins remain intact and are stained, but gradually the outer proteins are broken down, and it is assumed that the DNA or RNA is then destroyed, but it is not clear to what extent these proteins, DNA, or RNA are broken down.

In any case, since it is assumed that DNA or RNA is maintained longer, it would be more efficient and accurate to identify bacteria within cells by focusing on DNA or RNA, which is less likely to be denatured and more highly preserved, rather than on proteins, which are more likely to be digested intracellularly. It goes without saying that bacteria may be ingested and remain resistant to the DNA or RNA, even if they are ingested. For example, bacteria that are not the main causative agents of sepsis but can survive within phagocytes, such as Mycobacterium and Tuberculosis, may be identified.
sis), Listeria, Salmonella
These include Brucella, Legionella pneumophila, and others, and are applicable to the diagnostic method of the present invention.

Experiments have also shown that after cells ingest bacteria, the bacteria's DNA
Alternatively, it has been demonstrated that RNA specificity is maintained.

[Means for solving the problem]

In the diagnosis of infectious diseases for treatment, the ultimate goal of testing is to identify the bacteria in the specimen.

In the present invention, in order to efficiently carry out the work,
The object of the present invention is to provide the following method (1) and the following methods (2) and (3) that can be applied to the method (1).

(1) Identify the bacteria in specimens in which it has been confirmed that they have been taken up by phagocytes.

(2) High detection rate is achieved by detecting bacteria that have been ingested by phagocytes.

(3) Bacteria can be rapidly and accurately identified by directly detecting bacteria taken up by phagocytes without enrichment.

That is, the gist of the present invention is: [1] a method for identifying infectious disease causative bacteria, comprising the steps of: (a) fixing a specimen containing a large amount of phagocytes prepared from a biological component selected from the group consisting of blood, ascites, dialysate, urine, and pus; (b) hybridizing the specimen with a bacterial DNA probe containing a gene fragment of the infectious disease causative bacteria; and (c) detecting the bacterial DNA probe hybridized to the phagocyte fraction by the hybridization; [2] a method for detecting infectious disease causative bacteria, comprising: (a) fixing a specimen containing a large amount of phagocytes prepared from a biological component selected from the group consisting of blood, ascites, dialysate, urine, and pus; (b) contacting the specimen with an adipic acid-like protein; (c) contacting the specimen contacted with the avidin-like protein with biotin bound to a labeling molecule selected from the group consisting of enzymes, antibodies, dyes, and gold, and (d) detecting the labeling molecule bound to the biotin bound to the specimen via the avidin-like protein; and [3] a method for detecting infectious disease-causing bacteria, comprising the steps of: (e) detecting the labeling molecule in the step (d);
(f) isolating a phagocyte fraction containing the infectious disease-causing bacteria; and (g) hybridizing the phagocyte fraction with a bacterial DNA probe containing a gene fragment of the infectious disease-causing bacteria; and (g) detecting the bacterial DNA probe that has hybridized to the phagocyte fraction during the hybridization.

[Effect]

The present inventors have found that streptavidin or avidin conjugated to a labeling molecule can quantitatively detect all types of bacterial components, including yeast, and that these molecules have no affinity for phagocytes.

This is thought to be because avidin has an affinity for biotin that is several hundred times stronger than its binding strength with other substances, and the outer membrane of the bacterial cell components may uniformly incorporate biotin, making it possible to visualize the presence of bacteria within the cell using a labeling molecule with extremely high sensitivity via avidin.

Furthermore, the binding speed of avidin-biotin to living organisms is much faster than that of antibodies, which have been used as detection reagents in the past, and is 2 to 10 times faster than that of antibodies.
It also has the advantage of being detectable within three hours.

For example, bacteria can be detected in a sample as a specific color by binding an enzyme to streptavidin, which binds to bacteria, via biotin, to cause a color reaction.

Furthermore, as mentioned above, by focusing on the fact that phagocytes ingest and collect bacteria, the DNA still retained in the ingested and destroyed bacteria can be detected by performing in situ hybridization on the sample without enrichment, without further processing. Therefore, since no enrichment is required, detection is rapid and reliable.

Although this in situ hybridization method is used in the field of histopathology to detect infectious viruses, its application to bacterial infections has not been established for the following reasons.

In other words, viruses multiply within cells, and DNA or RNA that can be detected by hybridization is present in a free state. However, bacteria do not multiply in this manner, and the state in which the preserved DNA (RNA) is taken up into the cell is complex and there are various possibilities, such as whether the preserved DNA (RNA) is protected by the bacterial body or whether it is in close contact with destroyed proteins, and no attempt has been made to do this yet.

To further summarize the above, (1) The state of existence of bacterial DNA or RNA is essentially different from the state of existence of viral DNA or RNA, but it is most concentrated in phagocytes.

(2) The DNA (RNA) is less damaged than bacterial proteins and, in some cases, is likely to be well preserved.

As a result of focusing on the above two points, the present invention has established the first hybridization detection method for bacteria.

If the probe used in hybridization is non-radioactive, for example, biotinylated, it can be detected using an optical microscope even in a general laboratory that does not have facilities using radioisotopes, making the process quick and simple.

The method for diagnosing infectious diseases according to the present invention is different from conventional detection methods, such as culture methods, in that even in the case of a positive result, it takes more than 24 hours to simply confirm the presence or absence of bacteria, and then 24 to 48 hours or more to identify the bacteria (determine the genus and species), which takes 3 to 48 hours in total.
In comparison with the conventional method requiring 4 days for the detection of bacterial components, by using either of the two methods having the above-mentioned effects alone or in combination, the presence or absence of bacteria can be detected quickly and clearly in just 2-3 hours, or at most 1-2 days. Furthermore, the detected specimens can also be rapidly and accurately identified by the in situ hybridization method having the above-mentioned effects, so that overall, accurate detection can be achieved in at most 1-2 days, which is a groundbreaking effect. Furthermore, even if bacterial components are almost completely digested and cannot be detected, their native DNA (RNA) can be detected.
can be hybridized and detected.

[Embodiment]

A. Sample Preparation Method Samples prepared according to the following method (1) were fixed on slide glasses by the following method (2) to provide samples for use in each example of the present invention.

(1) Preparation of various clinical specimens for the smear method Sample preparation from blood (heparinized whole blood from humans and animals) Sample preparation from blood was carried out by one of the following two methods.

-1: Mono-poly solution (M-PRM: Flow Laboratories, In
c) Method: Place 3 ml of M-PRM in a 13 x 100 mm test tube, carefully layer 3.5 ml of heparinized whole blood (collected within 2 hours) as the sample on top, and centrifuge at 300 g using a swinging bucket rotor for 30 minutes at room temperature to collect only the band of polymorphonuclear leukocytes, which is then washed and centrifuged with PBS (isotonic phosphate buffer). The resulting cell pellet is suspended in an appropriate amount (approximately 1 ml) of PBS and used as the sample.

-2:6% dextran method: Add 1/3 to 1/4 volume of 6% dextran to 1 volume (1 ml) of heparinized whole blood as a sample, mix, and leave to stand at 37°C for 1 hour. Collect the upper layer, dilute it with PBS, and then centrifuge. The resulting cell precipitate is suspended in an appropriate amount of PBS and used as the sample.

Urine sample preparation: Urine samples are smeared directly.

Preparation of ascites samples: Ascites fluid was taken into a test tube and heated at 800 rpm or 3,000 rpm for 15-18 minutes.
Centrifuge at room temperature for 30 minutes, and add the resulting cell pellet to an appropriate amount of PBS.
Suspend the mixture in the following manner and use it as a specimen.

Sample preparation from pus: Smear the specimen directly from the pus.

(2) Slide Glass Fixation Sample Smear Method: Place an appropriate amount of the specimen obtained by the procedure in (1) above on a slide glass coated with 2% gelatin (preferably an "HT Coating Slide" manufactured by Boxy Brown), spread it with the belly of a pipette, etc., and air-dry until completely dry. Next, immerse the slide glass in Carnoy's fixative (ethanol:chloroform:acetic acid = 6:3:1) for 10 minutes to fix the cells.

The slides are then washed briefly in 70% ethanol and air-dried.

Example 1: Bacteria detection method using avidin-like protein (hereinafter referred to as "streptavidin method") In this example, streptavidin was used as the avidin-like protein.

Each fixed sample on a slide glass prepared by the method A above was rehydrated in PBS solution at room temperature for 10 minutes or more, and an appropriate amount of 5 μg/ml streptavidin (Amersham), 1% bovine serum albumin (Sigma Fraction V), and 0.1% Tween 20 (Tween 20) was applied to the fixed sample.
PBS containing 20 (Sigma) was placed on the plate and the plate was placed in a humidified box at 37°C.
After reacting for 60 minutes, an appropriate amount of 0.1% Tween 2
Wash three times in PBS containing 0 for 10 minutes at room temperature with gentle shaking.

Next, 5 μg/ml biotinylated alkaline phosphatase (EY LABS. Inc.), 1% bovine serum albumin, and 0.1%
PBS containing Tween 20 was placed on the sample fixed on the slide glass, and the sample was reacted in a humidified box at 37°C for 60 minutes.
Wash once in PBS containing 0.05% Tween 20 for 10 minutes at room temperature with gentle shaking, then in PBS containing 0.05% Triton
X-100 (Sigma) (hereafter referred to as TX) containing AP7.5 (100 mM
The cells were washed twice for 10 minutes at room temperature with gentle shaking in AP9.5 (100 mM Tris.HCl pH 7.5, 100 mM CaCl).
The membrane is washed three times for 10 minutes at room temperature with gentle shaking in 100 mM Tris·HCl pH 9.5, 100 mM NaCl, 10 mM MgCl ₂ .

Next, 0.3 mg/ml NBT (nitroblue tetrazolium, Be
Thesda Research Laboratories) and 0.17 mg/ml BCIP
AP9.5 containing (5-bromo-4-chloro-3-indolyl phosphate, Bethesda Research Laboratories)
The reaction is stopped at 10°C in a dark place at 37°C for an appropriate time.
This is done by immersing in 2 mM EDTA for several minutes and then air drying.

Finally, naphthol blue black (Sigma) of appropriate concentration was used.
Counterstain using a), wash under running water, air dry until completely dry, and observe the presence or absence of bacteria (signal) under a microscope using oil immersion.

Bacteria can be observed as purple signals, while cells such as neutrophils can be observed as light blue to blue.

The results of the experiments carried out on each plate are explained below according to the lines attached to the micrographs of each plate.

The optical microscope used had a magnification of 1000 times.

Plate 1: Bacteria (gonococci) in urine (clinical specimen); gonococci in human cells appear as blue-stained particulate signals (Figure 1).

Plate 2: Bacteria (unidentified) detected from human blood (clinical specimen); blood was prepared by the dextran method.

A signal indicating bacteria engulfed in human leukocytes is visible (Figure 2).

Plate 3: Staphylococcus detected from human blood (in vitro); blood was prepared using the mono-polysolvent method, incubating blood with staphylococcus for 20 minutes.

A signal indicating staphylococci taken up by human leukocytes is visible (Fig. 3).

Plate 4: Staphylococcus detected in human blood (in vivo); Staphylococcus was intravenously injected into mice, and 4 hours later,
Blood samples were taken according to routine methods.

A signal indicating staphylococci taken up by mouse leukocytes is visible (Fig. 4).

Plate 5: Yeast detected in the dialysate after peritoneal lavage;
Obtained from a dialysis patient.

A signal indicating yeast from the dialysate of a dialysis patient is visible (Figure 5).

The preparation of plates 2, 3 and 4 can be carried out by either the dextran method or the mono-polysolvent method.

Example 2: Detection and identification of bacteria (infectious bacteria) inside and outside of cells by in situ hybridization using biotinylated probes (hereinafter referred to as the "probe method") Each slide glass fixed sample prepared according to the method A above was immersed in PBS solution at room temperature for 10 minutes or more to rehydrate, and then the sample was transferred to a PBS solution containing 0.25% (w/v) each of saponin and TX-100.
The cells were treated with PBS solution for 10 minutes at room temperature with gentle shaking.

Next, lysozyme (Sigma) and lysostaphin (trade name, S
Approximately 100 μl of a PBS-saponin (0.05%) solution containing 5 mg/ml of N-acetyl muramidis SG (trade name, manufactured by Seikagaku Corporation) and 0.5 mg/ml of N-acetyl muramidis SG was dropped into each well.
Leave in a humidified box at room temperature or 37°C for 20 minutes.
The sections were washed with 2N saline containing the acid salt for 20 minutes, and then immersed in a solution of 0.5% acetic anhydride in 0.1 M triethanolamine-HCl buffer (pH 8.0) for 20 minutes.

Then, each slide was washed with 70% and then 95% ethanol, thoroughly air-dried, and then immersed in a saline solution containing 70 mM NaOH for 3 minutes, followed by 70% and then 95%.
The sections are washed with ethanol and then thoroughly air-dried to serve as samples for in situ hybridization.

Next, an appropriate amount of hybridization solution having the following composition is placed on the sample fixed on the slide glass, and the reaction is carried out overnight at 37°C in a humidified box.

Composition: 45% formamide, 2x SSC, 1x Denhardt's solution (0.02% polyvinylpyrrolidone (Si
sma), 0.02% Ficoll (Pharmacia Fine Chemicals), 0.02% bovine serum albumin), 250 μg/ml salmon sperm DNA (previously heated to 100°C for 5 minutes, quickly cooled on ice, and denatured (Pharmacia Fine Chemicals)
ls), 10% dextran sulfate (Pharmacis Fine Che
(micals), and an appropriate amount of biotinylated probe DNA (previously denatured). The probe DNA is biotinylated by the nick translation method. For example, a nick translation kit (Batheda Research Laboratories) is used.

Next, the membrane is washed in 2×SSC containing 50% formamide at 37°C for 30 minutes, and then in 1×SSC containing 50% formamide at a temperature of 37°C or higher for 30 minutes. After washing three times in 1×SSC with gentle shaking at room temperature for 20 minutes, the membrane is then placed in PRS at room temperature for 1 minute.
Soak for 0 minutes.

Next, 10% normal rabbit serum (Vector Laboratories) was applied to the fixed sample slide for blocking.
The cells are placed on a PRS containing PRS (Plastic Resistance Testing Co., Ltd.) and incubated in a humidified box at 37°C for 60 minutes, and then immersed in PRS at room temperature for several minutes.

Next, PRS containing 2 μg/ml streptavidin, 1% bovine serum albumin, and 0.1% Tween 20 was placed on the fixed sample on the slide glass, and the sample was incubated at 37°C in a humidified chamber for 60 minutes. The sample was then washed three times at room temperature for 10 minutes with gentle shaking in PBS containing 0.1% Tween 20.

Next, PBS containing 2 μg/ml biotinylated alkaline phosphatase, 1% BSA, and 0.1% Tween 20 was placed on the slide-mounted sample, which was then incubated at 37°C for 60 minutes in a humidified chamber. After washing with gentle shaking in PBS containing 0.1% Tween 20 for 10 minutes at room temperature, the slide was then washed with AP7.5 containing 0.05% Triton X-100.
Wash twice for 10 minutes at room temperature with gentle shaking in
Wash three times in AP9.5 for 10 minutes at room temperature with gentle shaking.

Next, the color is developed in AP9.5 containing 330 μg/ml NBT and 170 μg/ml BCIP at 37°C in the dark for an appropriate period of time.
This is done by immersing in EDTA for a few minutes and then air drying.

Finally, counterstain with an appropriate concentration of naphthol blue black, wash with running water, air dry until completely dry, and then use oil immersion to determine the presence or absence of bacteria from the color and specific morphology under a microscope.

The results of each experiment are explained below according to the lines attached to the micrographs of each plate. The optical microscope used had a magnification of 1000x.

Experimental Example 1: Preparation of DNA probe derived from Staphylococcus aureus The properties of the DNA probe derived from Staphylococcus aureus used in the following Experimental Examples 2 and 3 are as follows.

The preparation method is described in textbooks, e.g., Guide to Molecular Cloning Techniques.
(Methods in Enzymology, vol.
152) Academic Press, 1987. Using established cloning techniques, staphylococcal chromosomal DNA is inserted into a vector (e.g., PBR).

From each of the obtained clones, those carrying DNA fragments specific to Staphylococcus were selected and used as probes.

These probes do not cross-react with other bacteria (eg, Escherichia coli, Klebsiella, Pseudomonas, Enterobacter, etc., or the related genus Staphylococcus epidermidis).

The restriction enzyme maps of these probes are shown in FIG.

In addition, each probe and clinical strain (Staphylococcus aureus and Staphylococcus epidermidis (these two species are the most common cause of infectious diseases,
The reactivity with the related bacteria was examined as follows.

[Preparation method]

The preparation method involves culturing a clinical strain and extracting its DNA according to the above-mentioned text.

A certain amount (for example, 5 μl) of this is spotted onto a nylon filter, and after alkali denaturation, it is used as a sample for hybridization.

[Dot blot hybridization]

According to the same text, 50% formamide, 5xSSC, 42
Hybridization is carried out overnight at 37°C with the above ³² P-labeled probe.

Thereafter, the sections were washed twice with 0.1×SSC, 0.1% SDS at 55° C. for 20 minutes, irradiated overnight at −70° C., and then developed.

The reactivity of each of these probes with clinical strains (S. aureus and S. epidermidis) is shown in plates 10, 11, 12 and 13.

In both cases, the areas where Staphylococcus aureus was spotted (10th,
Signals were observed in the spots in the top two rows of Figures 11, 12, and 13.
They are not visible where Staphylococcus epidermidis was spotted (the spots in the bottom two rows of each figure). In other words, these probes clearly detect Staphylococcus aureus, but do not cross-react with Staphylococcus epidermidis, a related bacterium of Staphylococcus aureus.

Experimental Example 2: Detection of Staphylococcus in Mouse Ascites After Staphylococcus was injected into the abdominal cavity of a mouse, the abdomen was opened 4 to 6 hours later, and the ascites fluid was collected. The centrifuged precipitate obtained according to the preparation method in A(1) above was suspended in 100 μl of PBS, and 20 to 30 ml of the suspension was collected.
50 μl of the solution was placed on a well of a slide glass to prepare a fixed sample, which was hybridized with the following probes according to the method described in Example 2 above.

The experimental results are shown below for each experimental plate.

Plate 6: Hybridization of staphylococci taken up in the ascites with a staphylococcal DNA probe; hybrid signals are visible in dispersed leukocytes (Figure 6).

Plate 7: E. coli and Klebsiella
Hybridization using a DNA probe derived from the nucleotide sequence: no hybrid signal was detected (Figure 7).

Experimental Example 3: Detection of Staphylococcus aureus in human subphrenic abscess. Human subphrenic abscess was suspended in saline, and 20 μl of the suspension was
was smeared and spread in each well, and then air-dried (40 minutes). Thereafter, the plates were pretreated in the same manner as in the case of plates 6 and 7, and in situ hybridization was carried out according to the method of Example 2.

The experimental results are shown below for each experimental plate.

Plate 8: Hybridization with the DNA probe used in plates 6 and 7; hybrid signals are visible (Figure 8).

Plate 9: Hybridization using the same probes as those on plates 6 and 7; when DNA probes derived from E. coli and Klebsiella were used, no hybrid signals were detected.

Example 3: Comparative experiment between in situ hybridization method and blood culture method. From a patient in whom bacteria were not detected by the streptavidin method of Example 1, 6% of the above A(1)-2 was obtained.
Human blood samples were prepared by preparing fixed samples according to the dextran method and the slide glass fixed sample smear method described in A(2).

This was hybridized with the Staphylococcus-derived DNA probe prepared in Experimental Example 1 according to the method described in Example 2 (see plates 16 to 19 in Table I below and Figures 16 to 19).

A DNA probe derived from Pseudomonas aeruginosa was prepared in the same manner as in Experimental Example 1. Specifically, Pseudomonas aeruginosa
The chromosomal DNA of E. nosa is fragmented with the restriction enzyme HindIII, and then amplified in an appropriate vector (e.g., pBR322). This DNA is then used to clone other bacterial DNA (e.g., E. coli, Klebsiella, Enterobacter, Staphylococcus, Staphylococcus aureus, etc.).
From these, probes of appropriate length (for example, 10 kb, 5 kb, 4.5 kb, etc.) are used as probes.

This was hybridized with a human blood sample prepared as described above according to the method described in Example 2 above (see Plate 15, Figure 15 of Table I below).

In addition, 10 ml of blood was collected aseptically from the vein of the same patient from which the above-mentioned human blood samples were prepared, using a Blood Collecting Unit (Roche) and placed in a Recondo BC Bottle (Roche) (manufactured by Nippon Roche Co., Ltd.).
These were incubated at room temperature and observed over time. As a result, as shown in Table I below, no bacteria were detected in bottles 15 to 19 (microscope photographs not shown).

The results are shown in Table I below.

As is clear from the results in Table I above, even in cases where bacteria were not detected by the conventional blood culture method or the direct streptavidin method, the probe method was able to detect bacteria, demonstrating its excellent detection ability.

[Effects of the Invention]

First, for specimens that were not positive in blood culture, the in situ hybridization method focusing on phagocytes enabled rapid and reliable identification of bacteria.
The establishment of this diagnostic method will provide a revolutionary guideline for the treatment of bacteremia, which is expected to reduce the mortality rate.

Furthermore, in conventional blood culture methods, specimen preparation requires the collection of approximately 5 to 10 ml of blood sample from the patient every day until bacteria are detected, which causes physical and mental distress to the patient. In contrast, in the diagnostic method of the present invention, a blood sample can be collected from the patient only once, does not require strict aseptic procedures, and the amount collected is extremely small, for example, approximately 1 ml in the dextran method, thereby reducing the physical and mental distress to the patient.

Furthermore, in the diagnostic method for infectious diseases according to the present invention using only the probe method, bacteria can be detected even in samples in which bacteria were not detected by the streptavidin method in the above diagnostic method.
It is possible to identify them, and its detection and identification capabilities are significantly superior.

Furthermore, the detection method of the present invention, which uses only the streptavidin method, rapidly gives very clear detection results. In other words, this invention itself is a major advancement in that it can confirm bacteremia in situations where antibiotic treatment had to be attempted based on the previous clinically suspected bacteremia.

Furthermore, the detection and identification method of the present invention, which uses only the probe method, also has the following effect in relation to the identification of the causative bacteria of bacteremia: Namely, with this hybridization method, it is possible to identify the type of bacteria with a single sample, and the time required, which would have taken 3-4 days with the conventional method (and the detection rate was low), can be dramatically reduced to 1-2 days with this method, and the detection rate is also significantly higher.

In particular, when a non-radioactive label, particularly a biotinylated probe, is used, the assay can be carried out easily and at any location.

However, in this DNA hybridization method, the challenge is to find probes that are as specific as possible.

For example, if a DNA specific to Staphylococcus aureus is developed and used, and if the sample does not contain Staphylococcus aureus, the detection result will be negative.Even if the sample is tested using different types of DNA and all of the DNA samples tested are negative, the presence or absence of the bacteria itself will still be unknown.

Therefore, the diagnostic method for infectious diseases according to the present invention, which combines the streptavidin method and the probe method, quickly and accurately fixes bacteria in samples in which the presence or absence of bacteria has been rapidly detected, thereby achieving the overall effect of being rapid and accurate.

That is, confirming the presence or absence of bacteria using the streptavidin method is extremely useful in preventing the wastefulness of repeating hybridization on a specimen that does not contain bacteria.

Furthermore, unlike conventional staining methods, it does not stain biological components.
Since it specifically stains bacteria in smear-stained specimens, the infection status can be determined quickly and accurately without requiring expertise.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing the morphology of gonococci (particulate signals) detected in human cells on plate 1.

FIG. 2 is a photograph showing the morphology of bacteria (unidentified: clinical examination) detected in plate 2 and incorporated into human leukocytes.

FIG. 3 is a photograph showing the morphology of Staphylococcus aureus (in vitro) incorporated into human leukocytes detected on plate 3.

FIG. 4 is a photograph showing the morphology of Staphylococcus aureus (in vivo) incorporated into mouse leukocytes detected on plate 4.

FIG. 5 is a photograph showing the morphology of yeast detected in the dialysate of a dialysis patient on plate 5.

FIG. 6 is a photograph showing hybrids with DNA derived from Staphylococcus aureus that had been incorporated into mouse ascites fluid (hybrid signals with a probe derived from Staphylococcus aureus) detected on plate 6.

FIG. 7 is a photograph showing the results of Plate 7 using other probes in the method of Plate 6.

FIG. 8 is a photograph showing hybrids with staphylococcal DNA detected in human subphrenic abscesses (hybrid signals with the staphylococcal probe) on plate 8.

FIG. 9 is a photograph showing the results of Plate 9 using other probes in the method of Plate 8.

FIG. 10 is a photograph showing the results of dot hybridization using probe 24 on plate 10.

FIG. 11 is a photograph showing the results of dot hybridization using probe 77 on plate 11.

FIG. 12 is a photograph showing the results of dot hybridization using probe 7 on plate 12.

FIG. 13 is a photograph showing the results of dot hybridization using probe 36 on plate 13.

FIG. 14 shows a restriction enzyme map of the Staphylococcus-derived probe used in the examples of the present invention.

FIG. 15 is a photograph (magnification: 1000 times) showing the hybrids detected on plate 15 between a human blood sample and a DNA probe derived from Periplaneta aeruginosa.

FIG. 16 is a photograph (magnification 1000x) showing hybrids between a human blood sample and a DNA probe derived from Staphylococcus aureus on plate 16.

FIG. 17 is a photograph (magnification 1000x) showing hybrids between a human blood sample and a DNA probe derived from Staphylococcus aureus on plate 17.

FIG. 18 is a photograph (magnification 400x) showing hybrids between a human blood sample and a DNA probe derived from Staphylococcus aureus on plate 18.

FIG. 19 is a photograph (magnification 1000x) showing hybrids between a human blood sample and a DNA probe derived from Staphylococcus aureus on plate 19.

FIG. 20 is a photograph (magnification 400 times of FIG. 17) showing hybrids between a human blood sample and a DNA probe derived from Staphylococcus aureus on plate 20.

Claims (7)

[Claims] [Claim 1] A method for identifying infectious disease-causing bacteria, comprising the following steps: (a) fixing a specimen containing a large amount of phagocytes prepared from a biological component selected from the group consisting of blood, ascites, dialysis fluid, urine, and pus; (b) hybridizing the specimen with a bacterial DNA probe containing a gene fragment of the infectious disease-causing bacteria; and (c) detecting the bacterial DNA probe that has hybridized to the phagocyte fraction during the hybridization. 2. The method of claim 1, wherein the bacterial DNA probe is a biotinylated DNA probe. [Claim 3] A method for detecting infectious disease-causing bacteria, comprising the following steps: (a) immobilizing a specimen containing a large amount of phagocytes prepared from a biological component selected from the group consisting of blood, ascites, dialysis fluid, urine, and pus; (b) contacting the specimen with an avidin-like protein; (c) contacting the specimen contacted with the avidin-like protein with biotin bound to a labeling molecule selected from the group consisting of enzymes, antibodies, dyes, and gold; and (d) detecting the labeling molecule bound to the biotin bound to the specimen via the avidin-like protein. 4. The detection method according to claim 1, wherein the detection method comprises the following steps: (e) detecting the labeled molecule in the step (d);
The method of claim 3, further comprising the steps of: (f) isolating a phagocyte fraction containing the infectious disease-causing bacterium; (g) hybridizing the phagocyte fraction with a bacterial DNA probe containing a gene fragment of the infectious disease-causing bacterium; and (g) detecting the bacterial DNA probe that has hybridized to the phagocyte fraction during the hybridization. 5. The method according to claim 4, wherein the bacterial DNA probe is a biotinylated DNA probe. 6. The method according to any one of claims 3 to 5, wherein the infectious disease-causing bacterium is a bacterium or yeast having biotin in the outer membrane of a bacterial cell component. 7. The method according to any one of claims 3 to 6, wherein the avidin-like protein is streptavidin.