JP2009232690A - Method for examining allergic disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for examining an allergic disease, which enables to judge the presence or absence or seriousness risk of the allergic disease without accompanying pains, differently from a series of conventional allergy-diagnosing methods based on the production of antibodies accompanied by the activation of systemic immunities. <P>SOLUTION: The method for examining the allergic disease is characterized by examining the presence or absence or seriousness of the allergic disease on the basis of the number of Bacteroides eggerthii and/or Bacteroides fragilis in an excrement sampled from a testee, as an index. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for examining the presence or severity of an allergic disease using feces, an allergic disease testing kit suitable for the method, and a screening method for an allergic disease treatment composition.

The prevalence of allergic diseases in Japan is 38%, which is recognized as the most common lifestyle related disease.
One of the causes of an increase in allergic diseases mainly in developed countries is the hygiene hypothesis proposed by Dr. Strachan in the UK in 1989. This is the theory that children who have grown up in excessive hygiene are not fully stimulated by microorganisms and thus develop an allergy without the normal development of the immune system. Recently, the “intestinal flora hypothesis” that the intestinal bacterial flora is disturbed with the use of antibiotics and changes in diet and normal immune tolerance is no longer performed has been proposed. (For example, refer nonpatent literature 1).

  For example, in the two-year cedar pollen season, the present inventors analyzed the intestinal microflora of cedar pollinosis patients and healthy individuals, and the Bacteroides fragilis group increases only in hay fever patients after hay fever disperses. (For example, refer nonpatent literature 2). In addition to cedar pollinosis, there are reports at the genus level regarding enteric bacteria suspected of being involved in other allergic diseases such as atopy (see Non-Patent Document 3, for example).

  In addition, it is considered that allergic diseases can be prevented or treated by suppressing IgE production. Based on the above intestinal microbiota hypothesis, various types of intestinal bacteria can be used for suppressing IgE production. A method is disclosed. For example, an insurance product for preventing atopic dermatitis in which a bifidobacteria of a specific bacterial species is used as an active ingredient (see, for example, Patent Document 1), bifidobacteria and acidophilus bacteria are mixed in infant formula. For example, refer to Patent Document 2), and allergy prevention and / or treatment agents (for example, refer to Patent Document 3) containing bifidobacteria of a specific bacterial species as an active ingredient are disclosed. In addition, an allergy-suppressing composition using an IgA antibody production-increasing action by 1-ketose, an IgE antibody production-inhibiting action, and an intestinal bifidobacteria proliferative activity action is also disclosed (see Patent Document 4). Moreover, the composition for immunity promotion which uses Lactobacillus genus bacteria of a specific microbial species, etc., and yeast as an active ingredient is also disclosed (refer patent document 5). However, as described in Non-Patent Document 3 above, even when the number of bacteria such as bifidobacteria capable of suppressing IgE production is measured, it indicates the presence or absence of allergic diseases, allergic symptoms, etc. It may not be a sufficient indicator.

  At present, in the diagnosis of allergic diseases, in general, an inquiry, a family history, and confirmation of the person's past medical condition are important factors. In addition, in order to diagnose allergies based on more objective information, a method of observing a patient's immunological response to an allergen and a test method using a biological sample such as blood have been implemented.

As an example of the former, there is a method in which an immune response observed when a patient is actually contacted with an allergen is used for diagnosis of allergy. Such tests include prick tests, scratch tests, patch tests, intradermal reactions, or provocation tests. While these tests can directly diagnose a patient's allergic reaction, they are highly invasive tests that actually expose a subject to an allergen.
On the other hand, examples of the latter include allergen-specific IgE measurement, leukocyte histamine release test, or lymphocyte blastogenesis test. The presence of allergen-specific IgE is evidence of an allergic reaction to the allergen, but in some patients, allergen-specific IgE may not always be detected. In addition, the leukocyte histamine release test and the lymphocyte blastogenesis test are methods for observing the response of the immune system to allergens in vitro, but these methods are complicated to operate.

In addition, regardless of the allergen, as a test method for demonstrating the involvement of allergic reaction, measurement of serum IgE value can be mentioned. Here, since the serum IgE value is information corresponding to the total amount of allergen-specific IgE, it can be said to be an index containing all the allergies involving IgE. Examples of allergies involving IgE include food allergies, bronchial asthma, urticaria, rhinitis, hay fever, anaphylactic shock and the like. For example, if the serum IgE value is high, it can be estimated that the patient has an allergic reaction.
In addition to a test method using such an antibody reaction, a method that enables a test for an allergic disease by measuring the expression of an indicator gene in blood, for example, has also been disclosed (see, for example, Patent Document 6).
M.M. C. MC Noverr, 1 other, Clinical and Experimental Allergy, Vol. 35, 1511-1520, 2005 Toshitaka Odamaki, 10 others, Journal of Medical Microbiology, Volume 56, 1301-1308, 2007 P. P. Songjinda, 7 others, Bioscience, Biotechnology, and Biochemistry, 35, 1511-1520, 2005 JP 2007-91704 A Japanese Patent No. 4010062 JP 2006-273852 A JP 2006-321786 A JP 2005-68092 A International Publication No. 2004/003198 Pamphlet

In consideration of the patient's QOL, it is very useful if information effective for allergy treatment can be obtained without causing pain to the patient. However, these conventional diagnostic methods have a problem that treatments that are painful for the human body, such as blood collection and intradermal injection, are not preferable from the viewpoint of QOL of patients.
On the other hand, it is considered that intestinal bacteria that specifically increase or decrease in allergic disease patients can be used as an allergic disease marker, so that allergic diseases can be diagnosed non-invasively using feces. As described above, intestinal bacteria suspected to be associated with allergic diseases have been reported at the genus level, but in many cases the results differ depending on the country or research group surveyed. The species of the internal bacteria has not been identified. For example, even in Non-Patent Document 2, a specific bacterial species related to cedar pollinosis is not clarified in the Bacteroides fragilis group. One reason for this is that hundreds of types and hundreds of trillions of bacteria live in the intestines, and many new species are proposed every year.
However, in order to obtain a good disease marker, it is preferable to specify the intestinal bacteria related to the disease not at the genus level but at the bacterial species level. This is because the analysis at the genus level uses a complex system of various bacterial species as a detection target, and thus has a low correlation with allergic diseases and is insufficient as an index of allergic diseases. In fact, Non-Patent Document 2 describes that the total number of bacteria belonging to the Bacteroides fragilis group has a low correlation with cedar pollinosis in feces collected before the cedar pollen is scattered. The Bacteroides fragilis group as a whole had a low correlation with cedar pollinosis and was insufficient as an indicator of cedar pollinosis.

  Unlike a series of allergy diagnosis methods based on antibody production accompanying activation of systemic immunity, the present invention can determine the presence or absence of allergic diseases and the risk of aggravation without pain. The purpose is to provide an inspection method.

  As a result of diligent research to solve the above-mentioned problems, the present inventors analyze and measure subjective symptoms of allergic diseases and blood markers from detailed gut microbiota analysis up to the bacterial species level. Thus, a new bacterial species showing a positive or negative correlation with the total amount of IgE in the blood is found, and the number of bacterial species in the stool is used as an index to determine whether or not allergic diseases are present and the risk of aggravation The present invention has been completed.

That is, the present invention uses the number of Bacteroides eggerii and / or Bacteroides fragilis in feces collected from a subject as an index, and the presence or severity of allergic diseases. It is intended to provide a method for testing allergic diseases, characterized by testing the above.
The present invention also relates to the allergic disease test described above, wherein the test subject determines that the subject is suffering from an allergic disease when the number of Bacteroides aggaeae is less than 1 million per gram of stool A method is provided.
The present invention also relates to the allergic disease test described above, wherein the subject is judged to have an allergic disease when the number of Bacteroides fragilis is 1 million or more per gram of feces A method is provided.
The present invention also determines that the subject is suffering from an allergic disease when the number of Bacteroides aegyce per 1 g of feces is equal to or less than the number of Bacteroides fragilis. When the number is greater than the number of bacteria, it is determined that the subject is not suffering from an allergic disease, and the allergic disease testing method described above is provided.
Further, the present invention determines that the severity of the allergic disease has increased when the number of Bacteroides aegasi in the feces has decreased, and has determined that the severity of the allergic disease has decreased when the number has increased. The above-described allergic disease testing method is provided.
Further, the present invention determines that the severity of the allergic disease has increased when the number of Bacteroides fragilis in the stool increases, and determines that the severity of the allergic disease has decreased when the number has decreased. The above-described allergic disease testing method is provided.
The present invention is characterized in that, in the following first group to fourth group, the severity of allergic disease is determined in the order of the first group, the second group, the third group, and the fourth group. The allergic disease test method described is provided.
First group: The number of bacteria per gram of stool is less than 1 million for Bacteroides aegasi and 1 million or more for Bacteroides fragilis.
Group 2: The number of bacteria per gram of stool is less than 1 million for Bacteroides agercy and less than 1 million for Bacteroides fragilis.
Group 3: The number of bacteria per gram of stool is 1 million or more for Bacteroides agarcy and 1 million or more for Bacteroides fragilis.
Group 4: The number of bacteria per gram of stool is 1 million or more for Bacteroides aegasis and less than 1 million for Bacteroides fragilis.
The present invention also provides the allergic disease test method described above, wherein the number of bacteria in feces is measured using a primer specific for the gene sequence of each bacterial species. .
The present invention also provides an allergic disease test kit characterized by having the primer set described in the following (a) and / or (b), which is used in the above-described allergic disease test method. It is.
(A) A primer set consisting of a Bacteroides aegis specific forward primer having the base sequence of SEQ ID NO: 19 and a Bacteroides aegisie specific reverse primer having the base sequence of SEQ ID NO: 20.
(B) A primer set comprising a Bacteroides fragilis-specific forward primer having the base sequence of SEQ ID NO: 11 and a Bacteroides fragilis-specific reverse primer having the base sequence of SEQ ID NO: 12.
The present invention also relates to a method for screening an allergic disease therapeutic composition having a preventive or therapeutic effect on allergic diseases, wherein the number of Bacteroides aegasis bacteria in the stool is increased by ingestion of the test substance. And / or a screening method for a composition for treating an allergic disease, wherein the test substance is judged to have a prophylactic or therapeutic effect on an allergic disease when the number of Bacteroides fragilis bacteria decreases. It is to provide.
The present invention also provides the screening method for a composition for treating allergic diseases as described above, wherein the test substance is a pharmaceutical or food.

  The allergic disease test method of the present invention is a method using, as an index, the number of bacteria of a specific bacterial species showing a strong correlation with the symptoms of the allergic disease to be tested among the intestinal bacteria in feces. Therefore, by using the method for testing allergic diseases of the present invention, unlike conventional methods relating to antibody production, the patient can be easily suffered from allergic diseases and the risk of becoming severe without causing pain. Sex can be judged.

  The method for testing allergic diseases of the present invention is to test the presence or severity of allergic diseases using the number of Bacteroides aegy and / or Bacteroides fragilis in feces collected from a subject as an index. It is characterized by. In this way, among the intestinal bacteria belonging to the Bacteroides fragilis group, the number of bacteria in the feces derived from the subject of two types of bacterial species that are highly correlated with the symptoms of allergic diseases was measured. Thus, it is possible to judge and evaluate the presence or absence of an allergic disease in a subject and the severity of the disease.

The bacterial species belonging to the Bacteroides fragilis group highly correlated with these allergic diseases can be determined by the following method.
1. Design and synthesis of species-specific primers belonging to the Bacteroides fragilis group First, for species belonging to the Bacteroides fragilis group that have been reported to be detected from human feces, each species-specific PCR (Polymerase Chain Reaction) ) Primers were designed and synthesized. Specifically, based on 16S rRNA gene sequences of each bacterium obtained from known international base sequence databases such as DDBJ (DNA Data Bank of Japan) and NCBI (National Center for Biotechnology Information) Genbank. Using CLUSTALW ver1.83, which is a base sequence analysis software, a specific sequence site was identified, and each primer was designed using this part as a target. Primers were synthesized using a DNA synthesizer in accordance with the designed base sequence. Table 1 shows the designed nucleotide sequences of the primers. Of the primer names in the table, those with the suffix “F” such as “BaOVA-F” are forward primers, and those with the suffix “R” such as “BaOVA-R” are reverse primers. In addition, “product length (bp)” is a PCR product obtained when PCR is performed using each forward primer and reverse primer and a 16S rRNA gene of a bacterial species specifically recognized by the template as a template. Base pair length is shown.

2. Confirmation of specificity of bacterial species-specific primer In order to confirm whether the primer designed and synthesized in the above 1 is actually specific for the 16S rRNA gene of each bacterial species, a related species (standard strain and The primer reactivity was examined when the gene derived from the reference strain was used as a template.
First, bacteria of 4 genera and 18 species shown in Table 2 were anaerobically pure cultured overnight in a GAM medium (manufactured by Nissan). From each of the cells thus obtained, DNA was extracted using Dneasy Blood & Tissue kit (manufactured by QIAGEN) according to the attached protocol. The extracted DNA was dissolved in an elution buffer attached to a 200 μL kit, the concentration was measured, and the concentration was adjusted to 10 μg / mL to obtain a template DNA solution.
Next, using SYBR Premix Ex Taq (manufactured by TAKARA), each bacterial species-specific primer set (forward primer and reverse primer set) and 1 μL of each template DNA solution (10 ng template DNA) A PCR reaction solution having a liquid volume of 20 μL was prepared. These PCR reaction solutions were subjected to PCR under reaction conditions of 94 cycles at 94 ° C. for 10 seconds, 94 ° C. for 5 seconds, and 60 ° C. for 30 seconds for 35 cycles. In addition, PCR was performed using 7500 Fast Real-Time PCR system (made by Applied Biosystems). Thereafter, a melting curve was created under default conditions, and the presence or absence of nonspecific amplification was confirmed.
The results of each PCR are shown in Table 3. In the table, “+” indicates PCR positive, that is, a PCR product was obtained, and “−” indicates PCR negative, that is, a PCR product was not obtained. As a result, the primer obtained in 1 above formed a band specific to each bacterial species, and non-specific amplification was not observed.

3. Correlation analysis between Bacteroides count and total blood IgE levels in allergic disease patients with subjective symptoms and healthy subjects without subjective symptoms 33 patients with allergic diseases with subjective symptoms and 14 healthy subjects without subjective symptoms As a test subject, the correlation between the number of bacteria for each bacterial species belonging to the Bacteroides fragilis group in the collected feces and the total amount of IgE in blood was analyzed. Specifically, it was performed as follows.
(1) Measurement of the number of bacteria in each stool in stool DNA sample prepared from stool of each subject in Test Example 2 described below is used as a template DNA solution, PCR was performed under the reaction conditions. From the amount of the obtained PCR product, the number of bacteria of each bacterial species in each DNA solution was measured. The number of bacteria was measured using a calibration curve prepared by performing PCR using a DNA solution extracted in the same manner from a reference strain in which the number of bacteria was previously counted.

(2) Correlation analysis between the number of bacteria of each bacterial species and the total amount of IgE in blood Based on the number of bacteria measured in (1) above and data on the total amount of IgE in blood of each subject obtained in Test Example 1 described later Correlation analysis was performed using a known statistical analysis software SPSS ver 14.0. When the correlation coefficient was calculated by the Spearman method, it was found that Bacteroides aegyaceae and Bacteroides fragilis showed a significant positive or negative correlation with the blood total IgE level. Specifically, as shown in Table 4, Bacteroides aegea showed a strong negative correlation with the blood total IgE level. Bacteroides fragilis showed a positive correlation with the blood total IgE level.

  In addition, when these two bacterial species were combined, a stronger and significant correlation was shown with respect to the blood total IgE amount than each bacterial species alone. For example, as shown in Table 4, the number of Bacteroides fragilis bacteria that showed a positive correlation with the total blood IgE amount was divided by the number of Bacteroides agercy bacteria that showed a negative correlation with the total blood IgE amount. The ratio ("Bacteroides fragilis / Bacteroides ergacy" in the table) is such that the absolute value of the correlation coefficient is 0.45 or more and the P value is less than 0.01 with respect to the blood total IgE amount. The strongest and significant correlation was shown with respect to the amount of IgE.

  In addition, since the number of bacteria in the stool of the above two bacterial species correlates with the total amount of IgE in blood, in addition to the possibility that the growth of these bacterial species in the intestine is affected by the presence or severity of allergic diseases These bacterial species may be deeply involved in the development of allergic diseases. Therefore, these two bacterial species may be important targets not only as diagnostic markers for allergic diseases but also in the control of allergic symptoms.

(3) Correlation analysis between total bacterial count of each bacterial species and total IgE level in blood Of the 18 bacterial species whose bacterial count was measured in (1) above, the bacterial count of 14 bacterial species belonging to the Bacteroides fragilis group The total number of bacteria in the Bacteroides fragilis group was calculated. Based on the obtained total number of bacteria and the data on the total IgE in blood of each subject obtained in Test Example 1 described later, a correlation analysis is performed in the same manner as in (2) above, and the correlation coefficient is calculated by the Spearman method. Calculated. As a result, as shown in Table 4, the total bacterial count of the Bacteroides fragilis group did not show a significant correlation with the blood total IgE amount. From the above results, it is clear that the total number of Bacteroides fragilis groups in feces has a low correlation with allergic diseases and is insufficient as an index of allergic diseases.

  The allergic disease applicable to the allergic disease test method of the present invention is not particularly limited as long as it is a disease caused by an excessive antigen-antibody reaction and increases the amount of IgE in the blood. IgE whose production amount increases may be allergen-specific IgE or non-specific IgE. Examples of such allergic diseases include hay fever, atopic dermatitis, food allergy, allergic rhinitis, bronchial asthma, urticaria and the like.

  The stool provided for the allergic disease test method of the present invention is not particularly limited as long as it is collected from the subject, and may be stored immediately after the stool collection. It may be. Preservation of stool can be performed by a method that is usually performed in clinical laboratory specimens and the like. For example, room temperature storage, refrigerated storage, or frozen storage may be used. Since it can be expected that more reliable results can be obtained by suppressing fluctuations in the amount of microorganisms in the stool, it is preferably stool immediately after collection, and in the case of stool after storage, it has been refrigerated or frozen. It is preferable.

  In the present invention, the method for measuring the number of bacteria of each bacterial species in stool is not particularly limited, and the number, density, concentration, etc. of specific microorganisms in a sample in which stool or stool is treated by a conventional method are determined. Any of the methods commonly used for measurement may be used. For example, as performed in 3 (1) above, nucleic acid is extracted from feces by a conventional method, and nucleic acid amplification such as PCR is performed using the nucleic acid as a template and a primer that specifically recognizes the gene of each bacterial species. The number of bacteria can be calculated from the amount of amplification product obtained after the reaction. As the nucleic acid amplification reaction, in addition to normal PCR, real-time PCR may be performed, or RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) may be performed. By performing RT-PCR and using RNA in nucleic acid collected from stool as a template as cDNA, it is possible to increase detection sensitivity by about 1000 times. In addition, using antibodies that specifically recognize the bacteria of each bacterial species, the number of microorganisms in the diluted stool may be directly counted, using probes that specifically recognize the genes of each bacterial species, The number of bacteria can also be measured by performing a FISH (fluorescence in situ hybridization) method or the like. In order to detect and measure bacteria in stool with high accuracy and high sensitivity, the number of bacteria in stool should be measured using a specific primer (bacterial species-specific primer) for the gene sequence of each bacterial species. It is more preferable to perform a nucleic acid amplification reaction such as PCR and calculate the number of bacteria from the amount of amplification product obtained.

  Here, the bacterial species-specific primer is not particularly limited as long as it is an oligonucleotide having a base sequence that specifically recognizes the gene sequence of each bacterial species. For example, a known primer may be used, or a newly designed and synthesized primer may be used. The design and synthesis of the species-specific primer and the like may be performed using any method known in the field of gene analysis and the like. For example, it can be designed using known primer design software such as CLUSTALW or Primer 3 using gene sequence information of the target bacterial species. The gene sequence information of each bacterial species can also be obtained from analysis by a known base sequence analysis method or from a known base sequence database such as DDBJ.

  In the present invention, the species-specific primer used for measuring the number of bacteria of Bacteroides aegyaceae or Bacteroides fragilis is preferably a primer having the base sequence described in Table 1. This is because by using these primers, the target bacterial species can be clearly identified and detected from other bacterial species of the closely related species Bacteroides fragilis group. Each bacterium species-specific primer may have an additional base sequence other than the base sequences described in Table 1 as long as the specific recognition ability for the target gene is not inhibited. Examples of the additional sequence include a restriction enzyme recognition sequence. In addition, by labeling the bacterial species-specific primer with a fluorescent substance or the like, it is possible to easily detect the obtained amplification product. The primer can be labeled by a conventional method.

  In addition, it is also preferable to make a kit so as to include at least one of the Bacteroides agaric specific primer set and the Bacteroides fragilis specific primer set shown in Table 1. This is because the use of the kit makes it possible to easily test for allergic diseases. In addition to the primer set, the kit may contain a reagent used for measuring the number of bacteria using the primer set, such as a PCR reagent.

  In the method for testing allergic diseases of the present invention, the number of bacterial species which are allergic disease markers in feces derived from a subject, wherein bacteroides ergacy and bacteroides fragilis in feces are used as allergic disease markers This is a method for testing for allergic diseases using as an index. That is, when the number of bacteria of Bacteroides aegea in which the number of bacteria in stool has a negative correlation with the total amount of IgE in blood is large, or the number of bacteria in stool has a positive correlation with the amount of total IgE in blood When the number of bacteria is small, it can be determined that the subject is likely to have an allergic disease.

  Specifically, when the number of bacteria of Bacteroides aegy per gram of stool is less than the number of bacteria per gram of stool derived from non-allergic patients, or the number of bacteria of Bacteroides fragilis per gram of stool It can be determined that the subject is suffering from an allergic disease if the number of bacteria is greater than the number of bacteria per gram of stool derived from a non-affected person.

  In addition, when the number of Bacteroides aegyaceae is less than 1 million per gram of stool or when the number of Bacteroides fragilis is 1 million or more per gram of stool, the subject suffers from an allergic disease. It can be judged.

  By combining the data on the number of bacteria of these two bacterial species as appropriate, the possibility and severity of allergic diseases in the subject can be determined. For example, if the number of Bacteroides aegyace per 1 stool is less than or equal to that of Bacteroides fragilis, it is determined that the subject is suffering from an allergic disease, and the number of Bacteroides fragilis is greater than that of Bacteroides fragilis. In many cases, it can be determined that the subject does not suffer from an allergic disease.

  In addition, using the data on the number of bacteria of these bacterial species, it is possible to determine the severity when suffering from an allergic disease. For example, it can be determined that the severity of the allergic disease has increased when the number of Bacteroides aegyaceae in the feces has decreased, and that the severity of the allergic disease has decreased when the bacterial count has increased. Similarly, if the number of Bacteroides fragilis in the stool increases, it can be determined that the severity of the allergic disease has increased, and if it has decreased, it can be determined that the severity of the allergic disease has decreased.

In addition, in the following first group to fourth group, it can also be determined that the severity of allergic disease is higher in the order of the first group, the second group, the third group, and the fourth group.
First group: The number of bacteria per gram of stool is less than 1 million for Bacteroides aegasi and 1 million or more for Bacteroides fragilis.
Group 2: The number of bacteria per gram of stool is less than 1 million for Bacteroides agercy and less than 1 million for Bacteroides fragilis.
Group 3: The number of bacteria per gram of stool is 1 million or more for Bacteroides agarcy and 1 million or more for Bacteroides fragilis.
Group 4: The number of bacteria per gram of stool is 1 million or more for Bacteroides aegasis and less than 1 million for Bacteroides fragilis.

  As described above, by using the allergic disease test method of the present invention, the presence or absence of allergic disease, the risk of seriousness, etc. can be safely and easily performed without requiring invasive enemy treatment such as blood sampling or allergen sensitization. Can be inspected. In addition, the allergic disease test method of the present invention is continuously performed, and by comparing the bacterial count data of the above two bacterial species obtained at the time of each test, the allergic disease symptom is severed or ameliorated. It can be checked safely without imposing the burden of allergen sensitization for testing.

  In addition, by using the allergic disease testing method of the present invention, screening for a composition for treating allergic diseases having preventive or therapeutic effects on allergic diseases can be performed easily and safely. Specifically, the number of bacterial species of at least one of the above two bacterial species is measured on stool collected before and after ingestion of the test substance. By examining whether it fluctuates, a composition for treating allergic diseases can be screened. For example, if the test substance intake increases the number of bacteria of Bacteroides aegis and / or the number of bacteria of Bacteroides fragilis decreases in the feces, It can be judged that it has a therapeutic effect. Such a composition for allergic disease treatment obtained as a result of screening can be used as a component of pharmaceuticals, foods and the like.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Test Example 1] Measurement of blood total IgE level Blood was collected from 33 patients with allergic diseases having subjective symptoms and 14 healthy subjects without subjective symptoms, and blood total IgE levels were measured. The total amount of IgE antibody was measured by coating a commercially available IgE antigen (manufactured by Diagnostics) on a plastic 96-well plate for ELISA measurement, adding the blood of the subject thereto, washing, and then HRP-labeled anti-human IgE antibody (Diagnostics) The product was further reacted and then washed. Further, after coloring with TMB reagent (manufactured by Diagnostics), 10% sulfuric acid was added to stop the reaction, and the absorbance at 452 to 595 nm was measured with a plate reader. From the obtained measured values, the total IgE antibody amount in each blood was calculated using a calibration curve prepared using a control sample with a known concentration.

[Test Example 2] DNA extraction from stool Feces were collected from 47 subjects shown in Test Example 1, and DNA was extracted from the stool by the phenol bead method (see Non-Patent Document 2). That is, after washing 20 mg of stool twice with 1 mL of PBS, extraction buffer (100 mM Tris-HCl, 40 mM ethylenediacetic acid, pH 9.0) 450 μL, 10% SDS 50 μL, 0.1 mm diameter glass beads 300 mg, TE saturated phenol 500 μL was added, and shaken vigorously for 30 seconds at a power level of 5 using FastPrep FP100A (Bio101). After allowing to stand for 10 minutes on ice, 400 μL of phenol / chloroform was added to the supernatant obtained by centrifugation at 14,000 × g for 5 minutes, and the mixture was stirred well. Furthermore, after leaving still for 10 minutes on ice, the supernatant obtained by centrifugation at 14000 × g for 5 minutes was precipitated with isopropanol and washed with 70% ethanol. Thereafter, DNA was purified using a High Pure PCR Template Preparation kit (Roche) according to the attached protocol. A solution finally dissolved in 200 μL of TE buffer (10 mM Tris-HCl (pH 8.0) / 1 mM EDTA) was used as a DNA solution.

[Example 1]
(1) Qualitative analysis of Bacteroides eagsea and Bacteroides fragilis in patients with allergic diseases with subjective symptoms and healthy subjects without subjective symptoms The DNA solution prepared from the stool of each subject in Test Example 2 was used as a template DNA solution. PCR was performed using the primer set described in Table 1, and it was analyzed whether or not Bacteroides eggy and Bacteroides fragilis were present in each DNA solution.
Using TaKaRa Ex Taq (manufactured by Takara Co., Ltd.), prepare a PCR reaction solution of 25 μL in total volume containing each bacterial species-specific primer set (a set of forward primer and reverse primer) and 1 μL of each template DNA solution. did. These PCR reaction solutions were subjected to PCR under reaction conditions of 94 ° C. for 20 seconds, 50 ° C. for 20 seconds, and 72 ° C. for 30 seconds for 30 cycles. PCR was performed using 7500 Fast Real-Time PCR system (Applied Biosystems). Thereafter, the obtained PCR product was electrophoresed, and whether or not each bacterial species of the Bacteroides fragilis group was present in the stool was determined based on the presence or absence of a band. Specifically, electrophoresis was carried out by using a 1% agarose gel (Molecular Biology Certified Agarose; manufactured by Bio-Rad Co.) with Mupid (manufactured by Cosmo Bio) for 30 minutes and ethidium bromide (0. After staining with 5 mg / mL), the band was observed under a UV lamp. In this method, a band is detected after PCR when the number of bacteria is 1 million or more per gram of stool.

(2) Intergroup analysis of presence / absence of Bacteroides eagsea and Bacteroides fragilis and total amount of IgE in blood The results of presence / absence of Bacteroides eagsea and Bacteroides fragilis obtained in (1) above and obtained in Test Example 1 Based on the data of the blood total IgE amount of each subject, the analysis between groups was performed using statistical analysis software SPSS ver14.0. The results of the Mann-Whitney U test are shown in Tables 5 and 6. In the table, “PCR positive subject” means a subject from whom stool from which a band was detected in the above (1) and whose target bacterial species was determined to be present at 1 million or more per gram of feces was collected. “Subject” means a subject from whom stool was collected in which no band was detected and the target bacterial species was determined to be less than 1 million per gram of stool.

As is clear from these results, it was found that the total amount of IgE in blood was significantly different depending on the presence or absence of Bacteroides aegy and Bacteroides fragilis.
Moreover, when it classified into 4 groups as follows, the tendency for the blood total IgE amount to become high in order of the 1st group, the 2nd group, the 3rd group, and the 4th group was observed. In addition, each group had a low P value in comparison with the fourth group, and it became clear that the reliability of this tendency was high.
First group: The number of bacteria per gram of stool is less than 1 million for Bacteroides aegasi and 1 million or more for Bacteroides fragilis.
Group 2: The number of bacteria per gram of stool is less than 1 million for Bacteroides agercy and less than 1 million for Bacteroides fragilis.
Group 3: The number of bacteria per gram of stool is 1 million or more for Bacteroides agarcy and 1 million or more for Bacteroides fragilis.
Group 4: The number of bacteria per gram of stool is 1 million or more for Bacteroides aegasis and less than 1 million for Bacteroides fragilis.
From the above results, it is clear that Bacteroides aegasi and Bacteroides fragilis can be used as markers for allergic diseases.

[Example 2]
Statistical analysis software based on the results of the number of bacteria of Bacteroides aegy and Bacteroides fragilis in each stool obtained in 3 (1) above and the data on the total IgE in blood of each subject obtained in Test Example 1 Intergroup analysis was performed using SPSS ver14.0.
When the number of bacteria of Bacteroides fragilis was 100 million or more per gram of stool, when it was judged that PCR was positive (Bacteroides fragilis was present in stool), Mann-Whitney U test was performed. As shown in Fig. 7, it was found that the total amount of IgE in blood was significantly different depending on the presence or absence of Bacteroides fragilis.
On the other hand, when the number of Bacteroides ergacy bacteria is less than the number of Bacteroides fragilis and the number of Bacteroides fragilis is larger than that of Bacteroides fragilis, Mann- When the Whitney U test was performed, as shown in Table 8, it was found that the total IgE level in blood was significantly different in the two groups.
From the above results, the number of bacteria of Bacteroides fragilis and / or the number of bacteria of Bacteroides eagsea in stool has a strong correlation with the total amount of IgE in blood, in particular by using both in combination. It is clear that the blood total IgE amount can be determined with high accuracy.

Similarly, the number of affected persons and the number of unaffected persons in the two groups were compared. Table 9 shows the number of allergic disease affected and unaffected individuals in each group.
As a result, in the group in which the number of Bacteroides eagsea bacteria is less than or equal to that of Bacteroides fragilis, about 82% are suffering from allergic diseases. It was suggested that the presence or absence of allergic diseases can be examined with a specificity of 80% or more.

  The method for testing allergic diseases of the present invention uses non-invasive and simple determination of the presence or severity of allergic diseases by using bacteroides egyce and bacteroides fragilis in feces as allergic disease markers. Therefore, it can be used particularly in the field of diagnosis and treatment of allergic diseases.

Claims (11)

  It is characterized by examining the presence or severity of allergic diseases by using the number of Bacteroides eggerthii and / or Bacteroides fragilis as an index in feces collected from a subject. Allergic disease testing method.   2. The method for examining allergic diseases according to claim 1, wherein the subject is judged to be suffering from an allergic disease when the number of Bacteroides aegyaceae is less than 1 million per gram of stool.   2. The method for testing allergic diseases according to claim 1, wherein the subject is judged to have an allergic disease when the number of Bacteroides fragilis is 1 million or more per gram of stool.   When the number of Bacteroides aegyce per 1 stool is less than or equal to that of Bacteroides fragilis, it is determined that the subject is suffering from an allergic disease, and the number of Bacteroides fragilis is greater than the number of Bacteroides fragilis The method according to claim 1, wherein the subject is determined not to have an allergic disease.   It is judged that the severity of the allergic disease is increased when the number of Bacteroides aegyaceae in the stool is decreased, and the severity of the allergic disease is decreased when the bacterial count is increased. 2. The allergic disease test method according to 1.   The number of bacteria of Bacteroides fragilis in stool is judged to be increased when the number of Bacteroides fragilis is increased, and when the number is decreased, the degree of severity of allergic disease is decreased. 2. The allergic disease test method according to 1. The allergy according to claim 1, wherein in the following first group to fourth group, it is determined that the severity of allergic disease is higher in the order of the first group, the second group, the third group, and the fourth group. Sexual disease testing method.
First group: The number of bacteria per gram of stool is less than 1 million for Bacteroides aegasi and 1 million or more for Bacteroides fragilis.
Group 2: The number of bacteria per gram of stool is less than 1 million for Bacteroides agercy and less than 1 million for Bacteroides fragilis.
Group 3: The number of bacteria per gram of stool is 1 million or more for Bacteroides agarcy and 1 million or more for Bacteroides fragilis.
Group 4: The number of bacteria per gram of stool is 1 million or more for Bacteroides aegasis and less than 1 million for Bacteroides fragilis.   The method for testing allergic diseases according to any one of claims 1 to 7, wherein the number of bacteria in the stool is measured using a primer specific for the gene sequence of each bacterial species. A kit for testing allergic diseases, comprising the primer set described in (a) and / or (b) below, which is used in the method for testing allergic diseases according to claim 8.
(A) A primer set consisting of a Bacteroides aegis specific forward primer having the base sequence of SEQ ID NO: 19 and a Bacteroides aegisie specific reverse primer having the base sequence of SEQ ID NO: 20.
(B) A primer set comprising a Bacteroides fragilis-specific forward primer having the base sequence of SEQ ID NO: 11 and a Bacteroides fragilis-specific reverse primer having the base sequence of SEQ ID NO: 12. A screening method for a composition for treating allergic diseases having a preventive or therapeutic effect on allergic diseases, comprising:
When the test substance intake increases the number of Bacteroides aegasis bacteria and / or decreases the number of Bacteroides fragilis bacteria, the test substance is effective in preventing or treating allergic diseases. A screening method for a composition for treating allergic diseases, characterized in that the composition is judged to have.   The method for screening a composition for treating allergic diseases according to claim 10, wherein the test substance is a pharmaceutical or a food.