JP2017201894A - Diabetes examination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diabetes examination method.SOLUTION: There is provided a diabetes examination method comprising: obtaining end labeled restriction enzyme fragments by a T-RFLP method targeting the 16S ribosomal RNA gene of a bacterium in the whole intestinal flora included in a sample collected from a subject; and determining the presence or absence of type 1 diabetes and/or the presence or absence of type 2 diabetes, based on the abundance ratio of the end labeled restriction enzyme fragments having a specific base length to the total amount of end labeled restriction enzyme fragments obtained by the T-RFLP method.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for testing diabetes.

Diabetes is a common disease with a high incidence. In order to diagnose diabetes, it is necessary to examine the 75 g oral glucose tolerance test (75 gOGTT) and hemoglobin A _1C (HbA _1C ), unless the fasting blood glucose level is 126 mg / dL or more. Although HbA _1C can be tested using peripheral blood and the burden on the patient is relatively small, there is a problem that the test value is affected by anemia or the like. On the other hand, 75 gOGTT requires 2 hours for inspection, and is not a simple inspection and is not sufficiently high in reproducibility. In addition, since 75 gOGTT requires 2 hours for the examination, there are many people who do not consult a medical institution even if a secondary examination is instructed, thus preventing early treatment intervention.
As a test for screening for diabetes, fasting blood glucose level and / or HbA _1C are often tested. However, even when the fasting blood glucose level does not exceed the reference value, there is a case where 75 gOGTT exceeds the reference value and is diagnosed as being diabetic, and the fasting blood glucose level is sufficient as a screening test I can't say that. Moreover, diabetes may not be detected even if HbA _1C and fasting blood glucose level are combined.

  The differential diagnosis between type 1 diabetes and type 2 diabetes is performed by comprehensively judging the clinical course and pathology, and the presence or absence of pancreatic autoantibodies such as anti-glutamic acid decarboxylase antibody (anti-GAD antibody). Although anti-GAD antibodies are important as diagnostic markers for type 1 diabetes, they are not always positive in type 1 diabetic patients, and there are also type 1 diabetic patients whose anti-GAD antibodies are below the reference value. Therefore, there are many cases where it is difficult to distinguish between type 1 diabetes and type 2 diabetes.

In recent years, it has been clarified that the intestinal microflora changes due to various diseases, and research on the relation between specific bacterial species and fungi and diseases is underway. In relation to diabetes, intestinal bacteria have been identified that may be associated with the onset of diabetes in diabetes model animals (Patent Document 1). However, it is said that there are about 1500 types of intestinal bacteria in the human intestinal flora, but of these, only about 300 species have been identified. For unidentified fungal species, it is currently impossible to identify the bacterial species even if the genome is decoded. Whether or not unidentified bacterial species are associated with diabetes has not been reported, and the relationship between many unidentified bacterial species and diabetes is unknown.
If a search is made for the relationship between diabetes and the gut microbiota and a new test method is provided, it is considered that the test can be an alternative to the existing test method or a test that supplements the existing test method.

Republished Patent Publication No. 2013-146319

  Although diabetes is an important disease with a high morbidity, a time-consuming test such as 75 gOGTT may be required for diagnosis of diabetes. Therefore, a new test that is simple and less invasive is desired. It is an object of the present invention to provide a new method for testing for diabetes that is simple and has low invasiveness to determine the presence or absence of type 1 diabetes and / or the presence or absence of type 2 diabetes.

Means for solving the above problems are as follows.
<1> Obtaining an end-labeled restriction enzyme fragment by the T-RFLP method for the 16S ribosomal RNA gene of the entire gut microbiota contained in the sample collected from the subject, and by the T-RFLP method Diabetes comprising determining the presence or absence of type 1 diabetes and / or the presence or absence of type 2 diabetes based on the abundance ratio of end label restriction enzyme fragments having a specific base length to the total amount of the obtained end-labeled restriction enzyme fragments Inspection method.
<2> The T-RFLP method includes obtaining an end-labeled restriction enzyme fragment using at least one restriction enzyme selected from the group consisting of BslI, HaeIII, MspI, and AluI. Diabetes testing method.
<3> The method for testing diabetes according to <1> or <2>, wherein the sample is feces.
<4> The determination includes classifying subjects according to a decision tree generated by a data mining method, wherein the data mining method is type 1 diabetes, type 2 diabetes, or type 1 The target variable is whether it is diabetes or type 2 diabetes, and the abundance ratio of the end-labeled restriction enzyme fragment having a specific base length relative to the total amount of the end-labeled restriction enzyme fragment is used as an explanatory variable. <1> to <3> The method for testing diabetes according to any one of 3>.
<5> The method for testing diabetes according to <4>, wherein the data mining method is a C & RT method.

<6> The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 1, and cleaving the obtained amplification product with BslI. Any one of <1> to <5>, including at least one of the conditions (1) to (13) of the abundance ratio of the end-labeled restriction enzyme fragments having a specific base length to the total amount of the end-labeled restriction enzyme fragments shown The method for testing diabetes according to any one of the above.
(1) Type 1 diabetes when the abundance ratio of fragments having a base length of 366 bp is not less than a predetermined threshold and the abundance ratio of fragments having a base length of 168 bp is less than the predetermined threshold Is determined.
(2) Fragment having an abundance ratio of fragments having a base length of 366 bp equal to or greater than a predetermined threshold, an abundance ratio of fragments having a base length of 168 bp being equal to or greater than a predetermined threshold, and a base length of 505 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 1 diabetes.
(3) Fragment in which the abundance ratio of fragments having a base length of 366 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 754 bp is greater than or equal to a predetermined threshold, and the base length is 968 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 1 diabetes.
(4) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 317 bp is greater than or equal to a predetermined threshold value, it is determined as having type 1 diabetes.
(5) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp The abundance ratio of fragments with a base length of 650 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments with a base length of 106 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(6) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being equal to or greater than a predetermined threshold, and a base length of 968 bp Is greater than or equal to a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 366 bp is less than the predetermined threshold value, it is determined as having type 2 diabetes.
(7) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp Is greater than or equal to a predetermined threshold, and when the abundance of a fragment having a base length of 650 bp is less than the predetermined threshold, it is determined that the subject has type 2 diabetes.
(8) A fragment having a base length of 366 bp in which the abundance ratio is not less than a predetermined threshold, a fragment having a base length of 168 bp is not less than a predetermined threshold, and a base length is 505 bp The abundance ratio of fragments whose base length is 332 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 106 bp is less than the predetermined threshold. Is determined to be type 2 diabetes.
(9) Fragment having an abundance ratio of fragments having a base length of 366 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 168 bp being not less than a predetermined threshold, and having a base length of 505 bp Is greater than or equal to a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 332 bp is greater than or equal to a predetermined threshold value, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being equal to or greater than a predetermined threshold, and a base length of 968 bp If the abundance ratio of the fragment is equal to or greater than a predetermined threshold and the abundance ratio of a fragment having a base length of 366 bp is equal to or greater than the predetermined threshold, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(11) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 317 bp is less than the predetermined threshold value, it is determined that the type is not type 1 diabetes or type 2 diabetes.
(12) Fragment having an abundance ratio of fragments having a base length of 366 bp is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 168 bp is not less than a predetermined threshold, and a base length is 505 bp The abundance ratio of fragments having a base length of 332 bp is less than a predetermined threshold, and the abundance ratio of fragments having a base length of 106 bp is not less than a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
(13) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp The abundance ratio of fragments having a base length of 650 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 106 bp is greater than or equal to a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
<7> The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined threshold values of the conditions (1) to (13) according to <6> are as follows: The method for testing diabetes according to <6>, which is defined as shown in (1) to (13).
(1) When the abundance ratio of a fragment having a base length of 366 bp is 2.281 or more and the abundance ratio of a fragment having a base length of 168 bp is less than 3.081, it is determined as having type 1 diabetes.
(2) The abundance ratio of fragments with a base length of 366 bp is 2.281 or more, the abundance ratio of fragments with a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments with a base length of 505 bp is If it is less than 1.859, it is determined that it is type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is If it is less than 0.560, it is determined as type 1 diabetes.
(4) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a base length of less than 4.644 and a base length of 317 bp is 17.401 or more, it is determined that the patient has type 1 diabetes.
(5) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is It is type 1 diabetes when the abundance ratio of fragments having a base length of 650 bp is 0.300 or more and the abundance ratio of fragments having a base length of 106 bp is less than 0.778. Is determined.
(6) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is When the abundance ratio of fragments having a length of 0.560 or more and a base length of 366 bp is less than 36.992, it is determined as having type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a length of 4.644 or more and a base length of 650 bp is less than 0.300, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is If the abundance ratio of a fragment having a base length of 332 bp is less than 6.231 and the abundance ratio of a fragment having a base length of 106 bp is less than 0.181, it is type 2 diabetes Is determined.
(9) The abundance ratio of fragments with a base length of 366 bp is 2.281 or more, the abundance ratio of fragments with a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments with a base length of 505 bp is When the abundance ratio of a fragment having a length of 1.859 or more and a base length of 332 bp is 6.231 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is When the abundance ratio of fragments having a length of 0.560 or more and a base length of 366 bp is 36.992 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(11) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of the fragment having a base length of less than 4.644 and a base length of 317 bp is less than 17.401, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(12) The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is When the abundance ratio of fragments having a base length of 332 bp is less than 6.231 and the abundance ratio of fragments having a base length of 106 bp is 0.181 or more, it is 2 in type 1 diabetes. It is determined that it is not type 2 diabetes.
(13) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a base length of 650 bp is not less than 0.300 and the abundance ratio of fragments having a base length of 106 bp is not less than 0.778, 2 in type 1 diabetes It is determined that it is not type 2 diabetes.
<8> Diabetes test method according to <6> or <7>, comprising any two or more of the conditions (1) to (13) according to <6> or <7> Method.

<9> The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 1, and cleaving the obtained amplification product with HaeIII. Any one of <1> to <5>, including at least one of the conditions (1) to (11) of the abundance ratio of the end-labeled restriction enzyme fragments having a specific base length to the total amount of the end-labeled restriction enzyme fragments The method for testing diabetes according to any one of the above.
(1) Fragment having an abundance ratio of fragments having a base length of 85 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 315 bp being less than the predetermined threshold, and a base length of 58 bp Is less than a predetermined threshold, it is determined that the patient has type 1 diabetes.
(2) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is not less than a predetermined threshold, and the base length is 236 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 203 bp is less than a predetermined threshold value, it is determined as having type 1 diabetes.
(3) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is greater than or equal to a predetermined threshold, and the base length is 236 bp The abundance ratio of fragments whose base length is 265 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 45 bp is greater than or equal to the predetermined threshold. Is determined to be type 1 diabetes.
(4) Fragment in which the abundance ratio of fragments having a base length of 85 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 315 bp is not less than a predetermined threshold, and the base length is 79 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 2 diabetes.
(5) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is not less than a predetermined threshold, and the base length is 236 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 203 bp is equal to or greater than a predetermined threshold value, it is determined as having type 2 diabetes.
(6) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is not less than a predetermined threshold, and the base length is 236 bp The abundance ratio of fragments having a base length of 265 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is less than the predetermined threshold. Is determined to be type 2 diabetes.
(7) When the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold value and the abundance ratio of fragments having a base length of 177 bp is less than a predetermined threshold value, 2 even in type 1 diabetes It is determined that it is not type 2 diabetes.
(8) Fragment having an abundance ratio of fragments having a base length of 85 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 315 bp being not less than a predetermined threshold, and having a base length of 79 bp When the abundance ratio is equal to or greater than a predetermined threshold value, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(9) Fragment in which the abundance ratio of fragments having a base length of 85 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 315 bp is less than the predetermined threshold, and the base length is 58 bp When the abundance ratio is equal to or greater than a predetermined threshold value, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) Fragment having an abundance ratio of fragments having a base length of 85 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 177 bp being equal to or greater than a predetermined threshold, and a base length being 236 bp The abundance ratio of fragments having a base length of 265 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is greater than or equal to a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
(11) Fragment having an abundance ratio of fragments having a base length of 85 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 177 bp being equal to or greater than a predetermined threshold, and a base length being 236 bp The abundance ratio of fragments whose base length is 265 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 45 bp is less than the predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
<10> The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined threshold values of the conditions (1) to (11) according to claim 9 are as follows: The method for testing diabetes according to <9>, which is defined as shown in (1) to (10).
(1) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is less than 7.941, and the abundance ratio of fragments having a base length of 58 bp is When it is less than 0.114, it is determined as type 1 diabetes.
(2) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of less than 4.140 and a base length of 203 bp is less than 2.636, it is determined as having type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4. Type 1 diabetes when the abundance ratio of fragments having a base length of 265 bp is less than 38.276 and the abundance ratio of fragments having a base length of 45 bp is 1.328 or more Is determined.
(4) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is When it is less than 0.856, it is determined as type 2 diabetes.
(5) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of less than 4.140 and a base length of 203 bp is 2.636 or more, it is determined as type 2 diabetes.
(6) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of 265 bp is 38.276 or more and the abundance ratio of fragments having a base length of 45 bp is less than 1.258, it is type 2 diabetes Is determined.
(7) When the abundance ratio of a fragment having a base length of 85 bp is less than 0.392 and the abundance ratio of a fragment having a base length of 177 bp is less than 4.067, neither type 1 diabetes nor type 2 diabetes Is determined.
(8) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is When it is 0.856 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(9) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is less than 7.941, and the abundance ratio of fragments having a base length of 58 bp is When it is 0.114 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4. When the abundance ratio of fragments having a base length of 265 bp is 38.276 or more and the abundance ratio of fragments having a base length of 45 bp is 1.258 or more, it is 2 in type 1 diabetes. It is determined that it is not type 2 diabetes.
(11) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4. If the abundance ratio of fragments having a base length of 265 bp is less than 38.276 and the abundance ratio of fragments having a base length of 45 bp is less than 1.328, it is 2 for type 1 diabetes. It is determined that it is not type 2 diabetes.
<11> A method for testing diabetes according to <9> or <10>, wherein the method comprises any two or more of the conditions (1) to (11) according to <9> or <10>. Method.

<12> The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 3, and cleaving the obtained amplification product with MspI. Any one of <1> to <5>, including at least one of the conditions (1) to (12) of the abundance ratio of the end-labeled restriction enzyme fragments having a specific base length to the total amount of the end-labeled restriction enzyme fragments The method for testing diabetes according to any one of the above.
(1) Fragment having an abundance ratio of fragments having a base length of 293 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 457 bp being equal to or greater than a predetermined threshold, and a base length being 120 bp When the abundance ratio is equal to or greater than a predetermined threshold, it is determined that the patient has type 1 diabetes.
(2) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp, a ratio of fragments having a base length of 150 bp and a base length of 194 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 88 bp is less than a predetermined threshold value, it is determined as having type 1 diabetes.
(3) Fragment having a base length of 293 bp that is less than a predetermined threshold, a base length of 457 bp being greater than or equal to a predetermined threshold, and a base length of 120 bp Is less than a predetermined threshold value, and a fragment having a base length of 81 bp is less than the predetermined threshold value, it is determined as having type 1 diabetes.
(4) A fragment whose base length is 293 bp or more is a predetermined threshold or more, a fragment whose base length is 150 bp or more is a predetermined threshold or more and a base length is 194 bp The abundance ratio of fragments having a base length of 390 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 200 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(5) Fragment having an abundance ratio of fragments having a base length of 293 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 150 bp being not less than a predetermined threshold, and a base length of 194 bp The abundance ratio of fragments whose base length is 390 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 45 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(6) Type 2 diabetes when the abundance ratio of fragments having a base length of 293 bp is equal to or greater than a predetermined threshold and the abundance ratio of fragments having a base length of 150 bp is less than the predetermined threshold Is determined.
(7) Fragment having an abundance ratio of fragments having a base length of 293 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 457 bp being equal to or greater than a predetermined threshold, and a base length being 120 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 81 bp is equal to or greater than the predetermined threshold value, it is determined as having type 2 diabetes.
(8) Fragment having an abundance ratio of fragments having a base length of 293 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 150 bp being not less than a predetermined threshold, and having a base length of 194 bp The abundance ratio of fragments having a base length of 390 bp is less than a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is not less than a predetermined threshold. Is determined to be type 2 diabetes.
(9) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp and a base ratio of 150 bp and a fragment having a base length of 194 bp The abundance ratio of fragments having a base length of 88 bp is not less than a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is not less than a predetermined threshold. Is determined to be type 2 diabetes.
(10) When the abundance ratio of fragments having a base length of 293 bp is less than a predetermined threshold value and the abundance ratio of fragments having a base length of 457 bp is less than a predetermined threshold value, 2 even in type 1 diabetes It is determined that it is not type 2 diabetes.
(11) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp, a ratio of fragments having a base length of 150 bp and a base length of 194 bp The abundance ratio of fragments having a base length of 390 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 200 bp is greater than or equal to a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
(12) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp, a ratio of fragments having a base length of 150 bp and a base length of 194 bp The abundance ratio of fragments having a base length of 88 bp is greater than or equal to a predetermined threshold and the abundance ratio of fragments having a base length of 45 bp is less than a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
<13> The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined threshold values of the conditions (1) to (12) according to <12> are as follows: The method for examining diabetes according to <12>, which is defined as shown in (1) to (12).
(1) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When it is 0.312 or more, it is determined that it is type 1 diabetes.
(2) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is When the abundance ratio of fragments having a length of less than 2.617 and a base length of 88 bp is less than 1.179, it is determined as having type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When the abundance ratio of a fragment having a base length of less than 0.312 and a base length of 81 bp is less than 7.009, it is determined as having type 1 diabetes.
(4) The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2. Type 1 diabetes when the abundance ratio of fragments having a base length of 390 bp is 0.750 or more and the abundance ratio of fragments having a base length of 200 bp is less than 0.542 Is determined.
(5) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is If the abundance ratio of fragments having a base length of 390 bp is less than 0.750 and the abundance ratio of fragments having a base length of 45 bp is less than 1.263, it is type 1 diabetes Is determined.
(6) When the abundance ratio of a fragment having a base length of 293 bp is 9.740 or more and the abundance ratio of a fragment having a base length of 150 bp is less than 3.879, it is determined as having type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When the abundance ratio of a fragment having a base length of less than 0.312 and a base length of 81 bp is 7.009 or more, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2. Type 2 diabetes when the abundance ratio of fragments having a base length of 390 bp is less than 0.750 and the abundance ratio of fragments having a base length of 45 bp is 1.263 or more Is determined.
(9) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is It is type 2 diabetes when the abundance ratio of fragments having a base length of less than 2.617, a base length of 88 bp is 1.179 or more, and the abundance ratio of fragments having a base length of 45 bp is 1.688 or more Is determined.
(10) When the abundance ratio of fragments having a base length of 293 bp is less than 9.740 and the abundance ratio of fragments having a base length of 457 bp is less than 1.620, neither type 1 diabetes nor type 2 diabetes Is determined.
(11) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is 2. If the abundance ratio of fragments having a base length of 390 bp is 0.750 or more and the abundance ratio of fragments having a base length of 200 bp is 0.542 or more, it is 2 even in type 1 diabetes. It is determined that it is not type 2 diabetes.
(12) The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is If the abundance ratio of fragments having a base length of less than 2.617, a base length of 88 bp is 1.179 or more, and the abundance ratio of fragments having a base length of 45 bp is less than 1.688, even in type 1 diabetes, 2 It is determined that it is not type 2 diabetes.
<14> A method for testing diabetes according to <12> or <13>, wherein the method includes any two or more of the conditions (1) to (12) according to <12> or <13>. Method.

<15> The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 3, and cleaving the obtained amplification product with AluI, and the determination is shown below. Any one of <1> to <5>, including at least one of the conditions (1) to (10) of the abundance ratio of the end-labeled restriction enzyme fragments having a specific base length to the total amount of the end-labeled restriction enzyme fragments The method for testing diabetes according to any one of the above.
(1) Type 1 diabetes when the abundance ratio of fragments having a base length of 44 bp is less than a predetermined threshold and the abundance ratio of fragments having a base length of 58 bp is less than a predetermined threshold Is determined.
(2) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is not less than a predetermined threshold, and the base length is 76 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 1 diabetes.
(3) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is less than the predetermined threshold, and the base length is 487 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 83 bp is less than a predetermined threshold value, it is determined as having type 1 diabetes.
(4) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is less than the predetermined threshold, and the base length is 487 bp The abundance ratio of fragments having a base length of 203 bp is greater than or equal to a predetermined threshold and the abundance ratio of fragments having a base length of 830 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(5) Type 2 diabetes when the abundance ratio of fragments having a base length of 44 bp is less than a predetermined threshold and the abundance ratio of fragments having a base length of 58 bp is not less than a predetermined threshold Is determined.
(6) Fragment having an abundance ratio of fragments having a base length of 44 bp is equal to or greater than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp is not less than a predetermined threshold, and a base length is 76 bp If the abundance ratio is equal to or greater than a predetermined threshold value, it is determined that the patient has type 2 diabetes.
(7) Fragment having an abundance ratio of fragments having a base length of 44 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp being less than the predetermined threshold, and a base length of 487 bp Is greater than or equal to a predetermined threshold value, and a fragment having a base length of 203 bp is less than the predetermined threshold value, it is determined that the subject has type 2 diabetes.
(8) Fragment having an abundance ratio of fragments having a base length of 44 bp is equal to or greater than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp is equal to or greater than a predetermined threshold, and a base length is 76 bp When the abundance ratio is equal to or greater than a predetermined threshold, it is determined that the person is a healthy person.
(9) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is less than the predetermined threshold, and the base length is 487 bp If the abundance ratio of the fragment is less than a predetermined threshold and the abundance ratio of the fragment having a base length of 83 bp is equal to or greater than the predetermined threshold, it is determined that the person is a healthy person.
(10) Fragment having an abundance ratio of fragments having a base length of 44 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp being less than the predetermined threshold, and a base length of 487 bp The abundance ratio of fragments having a base length of 203 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 830 bp is greater than or equal to a predetermined threshold. When it is, it determines with it being a healthy person.
<16> The diabetes according to <15>, wherein the predetermined threshold values of the conditions (1) to (10) according to <15> are determined as shown in the following (1) to (10), respectively: Inspection method.
(1) When the abundance ratio of a fragment having a base length of 44 bp is less than 9.028 and the abundance ratio of a fragment having a base length of 58 bp is less than 1.062, it is determined as having type 1 diabetes.
(2) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is If it is less than 4.11, it is determined that the patient has type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of a fragment having a base length of less than 0.762 and a base length of 83 bp is less than 9.744, it is determined as having type 1 diabetes.
(4) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp Is 0.762 or more, the abundance ratio of fragments having a base length of 203 bp is 0.749 or more, and the abundance ratio of fragments having a base length of 830 bp is less than 0.317, Judge that there is.
(5) When the abundance ratio of a fragment having a base length of 44 bp is less than 9.028 and the abundance ratio of a fragment having a base length of 58 bp is 1.062 or more, it is determined as having type 2 diabetes.
(6) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is When it is 0.405 or more, it is determined that it is type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of fragments having a length of 0.762 or more and a base length of 203 bp is less than 0.749, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is 4. If it is 121 or more, it is determined that the person is healthy.
(9) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is If the abundance ratio of fragments having a length of less than 0.762 and a base length of 83 bp is 9.744 or more, it is determined that the subject is a healthy person.
(10) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of fragments having a base length of 0.762 or more is 0.749 or more and the abundance ratio of fragments having a base length of 830 bp is 0.317 or more, judge.
<17> Diabetes test method according to <15> or <16>, comprising any two or more of the conditions (1) to (10) according to <15> or <16> Method.

ADVANTAGE OF THE INVENTION According to this invention, the test method of diabetes which determines the presence or absence of the new type 1 diabetes and / or the presence or absence of type 2 diabetes which are simple and low in invasiveness can be provided. This inspection method can reduce the burden on the examinee and is expected to improve the consultation rate.
Further, according to the present invention, it is possible to provide a method for testing diabetes with a new mechanism by analyzing the entire intestinal bacterial flora including unidentified bacteria.

An example of the result of T-RFLP method is shown. The example of a decision tree in the test | inspection method of diabetes concerning one Embodiment of this invention using BslI is shown. The example of a decision tree in the test | inspection method of diabetes concerning one Embodiment of this invention using HaeIII is shown. The example of a decision tree in the test | inspection method of diabetes concerning one Embodiment of this invention using MspI is shown. The example of a decision tree in the test | inspection method of diabetes concerning one Embodiment of this invention using AluI is shown.

Hereinafter, modes for carrying out the present invention will be described. However, the present invention is not limited to the following embodiments.
The test method of the present invention is obtained by performing the T-RFLP method on the 16S ribosomal RNA gene of the bacteria in the entire intestinal flora contained in the sample collected from the subject, and obtained by the T-RFLP method. And determining the presence or absence of type 1 diabetes and / or the presence or absence of type 2 diabetes based on the fragment length and the abundance ratio of the end-labeled restriction enzyme fragment.

In the present specification, a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In addition, when referring to the amount of a certain component in the composition in the present specification, when there are a plurality of substances corresponding to the component in the composition, the amount is determined in the composition unless otherwise defined. Means the total amount of the plurality of substances present.
In addition, in this specification, the term “process” is not limited to an independent process, and even if it cannot be clearly distinguished from other processes, the term “process” is used if the intended purpose of the process is achieved. included.
Moreover, in this specification, "bacteria of the whole intestinal flora" means a group of bacteria contained in the intestinal flora, not individual bacteria contained in the intestinal flora of the subject.

<Collecting bacteria from the entire intestinal flora>
Since the bacteria contained in feces reflect the type and amount of bacteria in the entire intestinal flora, feces are generally used for analysis of the intestinal flora. Therefore, by collecting a part of the feces of the subject, it is possible to collect the bacteria of the entire intestinal flora. Instead of feces, the contents of the intestine of the subject may be collected and used as a sample.
The collected sample such as stool may be suspended in, for example, a Tris-EDTA buffer and stored until the T-RFLP method is performed.

<Nucleic acid extraction>
Extraction of nucleic acid from bacteria can be performed by a conventionally known method such as a bead-phenol method or a benzyl chloride method. For example, after damaging a bacterial cell wall that inhibits nucleic acid extraction, the nucleic acid may be precipitated using an alcohol such as ethanol or isopropanol. Examples of the method of damaging the bacterial cell wall include a method of lysing the cell wall with an enzyme, a method of crushing the cells by vigorous vibration, and a method of crushing the cells using a homogenizer. In the method of crushing microbial cells by vigorously vibrating, the cells can be more efficiently crushed by using glass beads, zirconia beads or the like when vigorously vibrated. The collected stool may be washed by suspending it in water or a buffer and centrifuging it before extracting the nucleic acid.
Extraction of nucleic acids from bacteria can also be performed using a commercially available kit. Examples thereof include PowerFecal DNA Isolation Kit (MO BIO Laboratories) and QIAamp DNA Stool (registered trademark, Qiagen). Moreover, you may carry out by the method as described in a well-known literature (Appl Environ Microbiol 69: 1251-1262, 2003 etc.).
The extracted nucleic acid may be further washed and / or purified by a method such as precipitation using 70% ethanol. In addition, treatment for preventing inhibition of nucleic acid amplification such as heat treatment may be performed before or after nucleic acid extraction.

<End-labeled restriction enzyme fragment length polymorphism (T-RFLP) method>
The T-RFLP method targeting 16S ribosomal RNA (16SrRNA) gene generally includes the following (1) to (3). However, there is no limitation on including other steps. (1) Nucleic acid amplification is performed using a labeled primer targeting a sequence common to all bacteria on the 16S rRNA gene. (2) Cleave the ends of the amplification products with restriction enzymes. (3) Electrophoresis using a capillary electrophoresis apparatus or the like to measure the fragment length and abundance ratio of the end-labeled fragments.
The T-RFLP method is a publicly known method, Kobayashi T. et al., Biosci Microbiota Food Health. 2013; 32 (4): pp129-37 and Nagashima K. et al., Appl Environ Microbiol. 2003, 69, 1251 It can also be done with reference to -1262.

(1) Nucleic acid amplification The nucleic acid amplification method is not particularly limited. Examples of the nucleic acid amplification method include a PCR method, a NASBA (Nucleic Acid Sequence Based Amplification) method, a TMA (Transcription-Mediated Amplification) method, an SDA (Strand Displacement Amplification) method, and the PCR method is preferred.

  Hereinafter, amplification will be described by taking the PCR method as an example, but the present invention is not limited to this example.

  First, a PCR reaction solution containing a template nucleic acid, a forward primer, and a reverse primer is prepared. The addition ratio of various primers in the PCR reaction solution is not particularly limited.

  The primer set (forward primer and reverse primer) used for nucleic acid amplification is preferably a primer set capable of amplifying bacterial 16S rRNA gene regardless of bacterial species. A person skilled in the art can design an appropriate primer set based on the base sequence of a known bacterial 16S rRNA gene region. A primer that binds to a 16S rRNA gene region having a common base sequence regardless of bacterial species is known in the art as a universal primer. Therefore, those skilled in the art can select an appropriate forward primer and reverse primer from these universal primers, perform nucleic acid amplification, and use it for the T-RLFP method.

As such a primer set, those shown in Table 1 shown below are particularly preferable. In Table 1, “name” is the name of a universal primer usually used in this field. For example, when the T-RFLP method is performed using BslI or HaeIII as a restriction enzyme, 516f (SEQ ID NO: 1) and 1510r (SEQ ID NO: 2) are preferably used in combination, and MspI or AluI is used as a restriction enzyme. When the T-RFLP method is used, 27f (SEQ ID NO: 3) and 1510r are preferably used in combination. It is also preferable to use 35f (SEQ ID NO: 4) instead of 27f. When 35f is used, the fragment length of the restriction enzyme terminal fragment obtained by the T-RFLP method is 10 bp shorter than when 27f is used.
The reverse primer combined with each of the forward primers 27f, 35f, or 516f may be any reverse primer that binds to the complementary strand side downstream (3 ′ side) from each of 27f, 35f, or 516f. Examples thereof include, but are not limited to, 926r (5′-CCGTCAATTCCCTTTGAGTTT-3 ′) (SEQ ID NO: 5), 1406r (5′-ACGGGCGGTGTGTAC-3 ′) (SEQ ID NO: 6).

  At least one of the forward primer and the reverse primer used for nucleic acid amplification is labeled. The forward primer is preferably labeled. The kind of labeling substance is not particularly limited, and a known label may be used. For example, 6-carboxy-2 ′, 4 ′, 7 ′, 4,7-hexachlorofluorescein (HEX), 6-carboxyfluorescein (FAM) and 6-carboxy-4,7,2 ′, 7′-tetrachlorofluorescein Examples thereof include fluorescent dyes such as (TET), 5 (6) -carboxy-X-rhodamine (ROX), Cy5, and Cy3.

Methods for labeling primers are well known in the art. Specifically, the labeling substance may be bound to the oligonucleotide contained in the primer. From the viewpoint of the efficiency of fluorescent labeling and nucleic acid amplification efficiency, it is preferable that the fluorescently labeled base is present at any one of the first to third positions from the 5 ′ end of the primer. Is particularly preferred. The fluorescent labeling substance may be bound directly to the oligonucleotide or indirectly via a linker or the like.
The binding of the fluorescent labeling substance to the oligonucleotide can also be performed using a commercially available labeling kit such as OliGro (registered trademark) manufactured by Mercer Gene Technologies.

  Other composition components in the PCR reaction solution are not particularly limited, and include conventionally known components, and the ratio thereof is not particularly limited. Examples of other composition components include nucleotides such as DNA polymerase and nucleoside triphosphate (dNTP), solvents, and the like. In the PCR reaction solution, the order of adding each composition component is not limited.

The DNA polymerase used for nucleic acid amplification is not particularly limited. For example, a conventionally known heat-resistant bacterium-derived polymerase can be used. Specific examples include DNA polymerase derived from Thermus aquaticus (see US Pat. Nos. 4,889,818 and 5,079,352) (Taq polymerase (trade name)), Thermus thermophilus (Thermus thermophilus). ) -Derived DNA polymerase (see WO91 / 09950) (rTth DNA polymerase), Pyrococcus furiosus-derived DNA polymerase (see WO92 / 96889) (Pfu DNA polymerase; manufactured by Strategene) ), DNA polymerase derived from Thermococcus litoralis (see European Patent No. 0455430) (Vent (trademark); manufactured by New England Biolabs) and the like are commercially available. Of these, thermostable DNA polymerase derived from Thermus aquaticus is preferred.
The addition ratio of DNA polymerase in the PCR reaction solution may be a ratio that is usually used in the art for the purpose of amplifying the target nucleic acid.

Examples of nucleoside triphosphates generally include dNTPs (eg, dATP, dGTP, dCTP, dTTP, dUTP, etc.). The addition ratio of dNTP in the PCR reaction solution may be a ratio that is usually used in the art for the purpose of amplifying the target nucleic acid.
As the solvent, Tris-HCl, Tricine, MES (2-morpholinoethanesulfonic acid), MOPS (3-morpholinopropanesulfonic acid), HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), CAPS (N-cyclohexyl-3-) For example, a buffer solution such as aminopropanesulfonic acid may be used, and a commercially available buffer solution for PCR, a buffer solution attached to the PCR kit, or the like may be used as it is.
The PCR reaction solution may contain glycerol, heparin, betaine, NaN ₃ , KCl, MgCl ₂ , MgSO _{4 and the} like.

PCR usually involves (i) dissociation of double-stranded nucleic acid into single-stranded nucleic acid (dissociation step), (ii) annealing of primer to template nucleic acid (annealing step), and (iii) nucleic acid from primer by DNA polymerase. It includes 3 steps of sequence extension (extension step). The conditions for each step are not particularly limited. The conditions for the dissociation step are preferably, for example, 90 ° C. to 99 ° C. and 1 second to 120 seconds, and more preferably 92 ° C. to 95 ° C. and 1 second to 60 seconds. The conditions for the annealing step are, for example, preferably 40 ° C to 70 ° C, 1 second to 300 seconds, and more preferably 45 ° C to 60 ° C, 5 seconds to 60 seconds. In addition, the conditions for the elongation step are preferably, for example, 50 ° C. to 80 ° C., 1 second to 300 seconds, and more preferably 50 ° C. to 80 ° C., 5 seconds to 120 seconds. The number of cycles is not particularly limited. For example, 10 cycles or more are preferable with 3 steps as 1 cycle. The upper limit is not particularly limited.
For example, the total is 100 cycles or less, preferably 70 cycles or less, more preferably 50 cycles or less. What is necessary is just to control automatically the temperature change of each process using a thermal cycler etc., for example. The annealing process and the extension process may be performed under the same temperature condition, and PCR may be performed in two processes.

(2) Cleavage with a restriction enzyme The restriction enzyme used in the T-RFLP method is not particularly limited as long as it can cleave the amplified 16S rRNA gene region. A person skilled in the art can predict the sequence of a bacterial amplification product whose gene sequence of 16S rRNA is known from the sequence of the primers used. Furthermore, the restriction enzyme recognition site contained in the amplification product can be known from the sequence of the amplification product.
Examples of restriction enzymes used in the T-RFLP method include BslI, HaeIII, MspI, AluI, HhaI, MbolI, MaeII, HpaII, RsaI and the like. BslI, HaeIII, MspI and AluI are particularly preferred.
The restriction enzyme treatment can be performed by a known method. What is necessary is just to mix the reaction liquid containing the restriction enzyme and buffer solution of a suitable unit number, and the amplified nucleic acid, and to incubate at the temperature and time suitable for the restriction enzyme to be used.

(3) Measurement of fragment length and abundance ratio of end-labeled fragments The fragment length and abundance ratio of end-labeled restriction enzyme fragments obtained by the T-RFLP method are measured. The fragment length of the end-labeled fragment can be measured by a method known to those skilled in the art. For example, a sample cleaved with a restriction enzyme may be subjected to capillary electrophoresis together with a base length standard, and may be obtained from a standard curve created from the base length standard based on the migration distance. The abundance ratio of the end-labeled fragments can be measured by methods known to those skilled in the art. For example, it may be grasped from the signal intensity of the label of the end label fragment. The fragment length and abundance ratio of the end-labeled fragment may be measured, for example, by detecting the label of the end-labeled fragment using a capillary electrophoresis apparatus of a DNA sequencer. For example, capillary electrophoresis is performed using ABI PRISM 3130x1 manufactured by Thermo Fisher Scientific, GeneMapper ver. The result is analyzed using 4.0, and the fragment length of the end-labeled fragment, the peak area of each fragment length, and the fluorescence intensity can be obtained.

FIG. 1 shows an example in which the results of capillary electrophoresis of four samples using ABI PRISM 3130 × 1 manufactured by Thermo Scientific are shown as the relationship between the fragment length of the obtained end-labeled fragment and the fluorescence intensity.
The abundance ratio of a certain end-labeled fragment is a peak corresponding to the target end-labeled fragment using the fragment length of the end-labeled fragment and the intensity of the labeled signal based on the results obtained by capillary electrophoresis as shown in FIG. And the total area of all peaks can be calculated using the following formula.

Abundance ratio of target end-labeled fragment (%) = peak area corresponding to target end-labeled fragment / total peak area × 100

Thermo Fisher Scientific, GeneMapper ver. 4.0 is preferably used for calculating the peak area.

<Generation of decision tree by data mining method>
In one embodiment of the present invention, when generating a decision tree that classifies subjects according to a decision tree generated by a data mining method, the algorithm of the data mining method is not particularly limited. As the algorithms, the classification and regression trees (C & RT) method, the Chi-squared automatic interaction detection (CHAID) method, the quick, unambiguous, efficient, and static method (UE) are widely used. Illustrated.
The C & RT method is a particularly preferred algorithm. The generation of decision trees by the C & RT method is described below.
The C & RT method is a method of constructing a decision tree by dividing an object into two in order to create a data subset as homogeneous as possible with respect to an objective variable. The objective variable is diagnostic information (type 1 diabetic patient, type 2 diabetic patient, healthy person (one who is neither type 1 diabetes nor type 2 diabetes)), and the explanatory variable is the fragment length and abundance ratio of restriction enzyme fragments.
Specifically, when the impurity of data (Gini coefficient) is calculated and the original data (parent node data) is divided into two subsets (child node data), the impurity of the child node is The degree of improvement indicating how much improvement has been made with respect to the impurity is established as an evaluation standard for the classification work.
Then, the process of recursively searching for the separation point (the classification variable and its value) that maximizes the improvement degree is repeated until the stop rule is satisfied (Japanese Patent No. 5645761).

  The impurity g (t) and the improvement degree f (t) are calculated as follows.

  However, in Equation 1 and Equation 2,

It is.

Here, π (j) is the prior probability of category j, Nj (t) is the number of cases of category j at node t, and Nj is the number of cases of category j at the root node. In addition, PL is a ratio of cases transmitted to the first child node at the node t, and PR is a ratio of cases transmitted to the second child node at the node t.
In the C & RT method, separation is performed so that the improvement degree f (t) is maximized. That is, fractionation is performed so that the impurity is most greatly reduced.
It should be noted that the generation of a decision tree by the C & RT method may use a method of using Twining, least square deviation (LSD), or the like in addition to using the Gini coefficient as a branching condition as described above.

At each node in the generated decision tree, the leaf node located at the end of the decision tree that does not have a child node on the downstream side has an impurity of 0 and is a type 1 diabetic patient, a type 2 diabetic patient, or a healthy person ( This is a node that includes only one of those who are neither type 1 diabetes nor type 2 diabetes (except the node located at the end as a result of stopping the recursive calculation). By following the path from the root node located at the most upstream of the decision tree to the leaf node containing only one of type 1 diabetic patient, type 2 diabetic patient or healthy person, clinical information of the leaf node (type 1 diabetes Patients, type 2 diabetics or healthy subjects). In order to trace the route from a root node to a leaf node for a subject, the result of the subject's T-RFLP method satisfies all the conditions set at the branch point from the root node to the leaf node in the decision tree. You can confirm. When tracing a route from a root node to a leaf node for a subject, the order in which the conditions are applied may be any as long as all of the conditions set at the branch points from the root node to the leaf node are satisfied.
The decision tree generated from the result of the T-RFLP method using BslI shown in FIG. 2 will be specifically described as an example. Node 4 is a leaf node that includes only type 1 diabetic patients, and the root node includes all subjects. When the subject contained in the root node has a fragment abundance of 366 bp in the fragment length of the restriction enzyme fragment obtained by cutting the bacteria contained in the entire intestinal flora with BslI is 2.281 or more It is classified as node 1 which is a child node. Next, subjects included in node 1 are classified into leaf node 4 including only type 1 diabetic patients when the abundance ratio of fragments having a fragment length of 168 bp is less than 3.081. From this result, in a certain subject, the abundance ratio of the restriction enzyme fragment cleaved with BsII is 366 bp and the fragment abundance is 281 bp and the abundance of the fragment of 168 bp is 3 If it is less than 0.081, it can be determined that it is type 1 diabetes.

In the generated decision tree, the condition for branching from the parent node to the child node is a combination of the fragment length of the end label restriction enzyme fragment and the abundance ratio. Among these, the abundance ratio of the lengths of the end-labeled restriction enzyme fragments is called a threshold value. The threshold value that is a condition for branching to a child node in each node obtained when generating a decision tree can be applied as a predetermined threshold value to other subjects. Using a predetermined threshold obtained by generating a decision tree, whether the subject is a type 1 diabetes, a type 2 diabetes, or a subject other than the subject who is the target when generating the decision tree, or It can be determined that neither type 1 diabetes nor type 2 diabetes is present.
In addition, the fragment length of the end-labeled restriction enzyme fragment can also be applied to subjects other than the subject who was the subject when generating the decision tree.

  One route from the root node to the leaf node may be used alone or in combination with another route. For example, when all leaf nodes are used in one decision tree, the presence or absence of type 1 diabetes and the presence or absence of type 2 diabetes can be determined for all subjects.

  For the generation of the decision tree, the abundance ratio of the end-labeled restriction enzyme fragments used as explanatory variables may be only those obtained from the T-RFLP method using one type of restriction enzyme, The abundance ratios of the end-labeled restriction enzyme fragments obtained from the T-RFLP method performed separately using two types of restriction enzymes may be used together.

  End-labeling by a T-RFLP method using a specific leaf node of a decision tree generated using the abundance ratio of fragment lengths of end-labeling restriction enzyme fragments by one type of restriction enzyme and T-RFLP method You may use combining the determination by the specific leaf node of the decision tree produced | generated using the abundance ratio of the fragment length of a restriction enzyme fragment | piece. One decision can complement the other decision. Further, the number of leaf nodes applied to the determination is not particularly limited, and a route from a plurality of root routes to the leaf nodes may be used. The plurality of leaf nodes used for the combination may include a leaf node including a type 1 diabetic patient, a type 2 diabetes leaf node, a leaf node including a healthy person, or a part or all of them.

[Example of analysis]
Hereinafter, the present invention will be specifically described with analysis examples, but the present invention is not limited to these analysis examples.

[Analysis Example 1]
<Subject>
The subjects were 11 type 1 diabetic patients within 11 years of onset, 11 type 2 diabetic patients, and 31 healthy people diagnosed as not having type 1 diabetes or type 2 diabetes.

<Collecting bacteria and extracting DNA from the entire intestinal flora>
500 mg of the subject's stool was collected and suspended in 9 volumes of sterile distilled water. After standing for 10 minutes, 0.1 mL of the obtained suspension was added to 0.9 mL of sterile distilled water, and centrifuged at 18000 × g for 5 minutes. The pellet was washed with 1 mL of sterile distilled water, further centrifuged, and then suspended in 250 μL of a solution containing 40 mM EDTA and 100 mM Tris-HCl (pH 9.0). The obtained suspension was transferred to a test tube containing 0.6 g of glass beads (diameter 0.1 mm), and the stool was crushed using a bead crusher. To 100 μL of the obtained suspension, 150 μL of benzyl chloride and 50 μL of 10% sodium dodecyl sulfate were added and vigorously shaken at 50 ° C. for 30 minutes. Subsequently, 150 μL of 3M sodium acetate was added, and the mixture was ice-cooled for 15 minutes, and then centrifuged at 18000 × g for 10 minutes. The upper layer was recovered and an equal volume of isopropanol was added to precipitate the DNA. The pellet was washed with 70% ethanol, then the DNA pellet was dried and dissolved in 100 μL of Tris EDTA (TE) buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA) containing 50 μg RNase per mL. And incubated at 37 ° C. for 30 minutes. Next, DNA was purified using illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Japan, Inc.), and the obtained DNA was quantified according to a conventional method.

<PCR method>
PCR was performed using the extracted DNA as a template. 1 × PCR buffer, 200 μM each of dATP, dTTP, dGTP and dCTP nucleotide-3 phosphate, 1.5 mM MgCl ₂ , 0.1 μM each of forward and reverse primers, and 10 ng of DNA extracted from stool, 0 A PCR reaction solution containing 5 U HotStarTaq DNA polymerase (Qiagen Co., Ltd.) was prepared. Universal primer 516f (5′-TGCCAGCAGCCCCGGGTA-3 ′) labeled with FAM (registered trademark, Thermo Fisher Scientific Co., Ltd.) was used as a forward primer, and universal primer 1510r (5′-GGTCACTCTGTTACGACTTT-3 ′) was used as a reverse primer. . 20 μL of PCR reaction solution was used per test.
The PCR method was carried out at 95 ° C. for 15 minutes, followed by 30 cycles of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 1 minute, followed by a final cycle of 72 ° C. for 10 minutes. A typical elongation reaction was performed.

<Purification of PCR product and treatment with restriction enzyme BslI>
20 μL of the PCR product was purified using a MultiScreenFB filter plate (Merck). 0.5 U of BslI (trade name: Fast Digest BslI, Thermo Fisher Scientific, USA) was added to 10 μL of the purified PCR product, and restriction enzyme treatment was performed at 37 ° C. for 10 minutes.

<T-RFLP method>
2 μL of the PCR product cleaved with the restriction enzyme was mixed with 4 μl diluted GeneScan 2500 size standard (Thermo Fisher Scientific, USA) 0.5 μL and deionized formamide 12 μL, and immediately denatured at 96 ° C. for 2 minutes. It was ice-cooled and used as a sample for capillary electrophoresis. Capillary electrophoresis was performed using an ABI PRISM 3130x1 (Thermo Fisher Scientific, USA) with an injection time of 40 seconds and a migration time of 40 minutes, and the fragment length and peak area of the restriction enzyme fragment labeled with HEX were determined using ABI PRISM 3130x1. GeneMapper ver. Calculated using 4.0 software. An example of the results obtained is shown in FIG.

<Data mining>
Correspondence between the fragment length of the end-labeled restriction enzyme fragment obtained by the T-RFLP method and its abundance ratio and clinical information of subjects (type 1 diabetes, type 2 diabetes or healthy people who are not type 1 diabetes or type 2 diabetes) In addition, data mining was performed by the C & RT method to generate a decision tree. The abundance ratio can be calculated as described above.

<Results of Analysis Example 1>
The obtained decision tree is shown in FIG. Type 1 diabetic patients were included in Node 4, Node 8, Node 10, Node 19, and Node 24. Type 2 diabetic patients were included in node 18 and node 22. Healthy people were included in node 13, node 15, node 20, node 12, and node 13.

  Node 4 includes only patients with type 1 diabetes. This is based on the T-RFLP method using BslI as a restriction enzyme, the abundance ratio of fragments having a base length of 366 bp is 2.281 or more, and the abundance ratio of fragments having a fragment length of 168 bp is less than 3.081 This indicates that it can be determined that the patient has type 1 diabetes. Similarly, the abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp Is less than 1.859 (node 8), it can be determined that it is type 1 diabetes. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is 0.560. If it is less than (Node 10), it indicates that it can be determined that it is type 1 diabetes. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is 4.644. If the abundance ratio of fragments having a base length of 317 bp is 17.401 or more (node 19), it can be determined that it is type 1 diabetes. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is 4.644. When the abundance ratio of fragments having a base length of 650 bp is 0.300 or more and the abundance ratio of fragments having a base length of 106 bp is less than 0.778 (node 24), type 1 diabetes Indicates that it can be determined that there is. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is 0.560. As described above, when the abundance ratio of a fragment having a base length of 366 bp is less than 36.992 (node 16), it can be determined that it is type 2 diabetes. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is 4.644. As described above, when the abundance ratio of a fragment having a base length of 650 bp is less than 0.300 (node 18), it can be determined that it is type 2 diabetes. The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is 1.859. When the abundance ratio of fragments having a base length of 332 bp is less than 6.231 and the abundance ratio of fragments having a base length of 106 bp is less than 0.181 (node 22), Indicates that it can be determined that there is. The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is 1.859. As described above, when the abundance ratio of a fragment having a base length of 332 bp is 6.231 or more (node 13), it can be determined that neither type 1 diabetes nor type 2 diabetes can be determined. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is 0.560. As described above, when the abundance ratio of a fragment having a base length of 366 bp is 36.992 or more (node 15), it can be determined that neither type 1 diabetes nor type 2 diabetes can be determined. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is 4.644. When the abundance ratio of fragments having a base length of 317 bp is less than 17.401 (node 20), it can be determined that neither type 1 diabetes nor type 2 diabetes can be determined. The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is 1.859. When the abundance ratio of fragments having a base length of 332 bp is less than 6.231 and the abundance ratio of fragments having a base length of 106 bp is 0.181 or more (node 21), even in type 1 diabetes It shows that it can be determined that it is not type 2 diabetes. The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is 4.644. When the abundance ratio of fragments having a base length of 650 bp is 0.300 or more and the abundance ratio of fragments having a base length of 106 bp is 0.778 or more (node 23), even in type 1 diabetes It shows that it can be determined that it is not type 2 diabetes.

All type 1 diabetic patients are included in either node 4, node 8, node 10, node 19, or node 24, and all the type 1 diabetic patients can be found by following the route from the root node of the decision tree to these nodes. Could be determined to be type 1 diabetic. All type 2 diabetic patients are included in node 18 or node 22, and it is possible to determine that all type 2 diabetic patients are type 2 diabetic patients by following a route from the root node of the decision tree to these nodes. there were. In addition, all healthy persons are included in any one of the nodes 13, 15, 21, and 23, and all healthy persons are healthy persons by following a route from the root node of the decision tree to these nodes. It was possible to determine.
Further, there are 6 of 11 patients with type 1 diabetes included in node 19, and in the decision tree shown in FIG. 2, determination is made with 55% sensitivity and 100% specificity only by the route from the root node to node 19. I was able to. There are 9 of the 11 type 2 diabetic patients included in the node 18, and in the decision tree shown in FIG. 2, it is possible to determine sensitivity 82% and specificity 100% only by the route from the root node to the node 18. did it. Further, 19 healthy persons included in the node 13 are 19 out of 31 persons. In the decision tree shown in FIG. 2, it is possible to determine the sensitivity 61% and the specificity 100% only by the route from the root node to the node 13. did it.

[Analysis Examples 2 to 4]
As restriction enzymes, HaeIII (trade name: Fast Digest HaeIII, Thermo Fisher Scientific, USA) (analysis example 2), MspI (trade name: Fast Digest MspI, Thermo Fisher Scientific analysis company, USA, USA) T-RFLP method was performed using AluI (trade name: Fast Digest AluI, Thermo Fisher Scientific, USA) (Analysis Example 4). When HaeIII was used as a restriction enzyme, the same forward primer and reverse primer as in Analysis Example 1 were used. When MspI or AluI is used as a restriction enzyme, FAM (universal primer 27f (5′-AGAGTTGATCMTGGCTCAG-3 ′) labeled as a forward primer and universal primer 1510r (5′-GGTTACCCTGTTACGACTTT-3 ′) as a reverse primer are used. used.
The treatment conditions of the amplification product with each restriction enzyme were the same as those in Analysis Example 1.

<Results of Analysis Example 2>
The obtained decision tree is shown in FIG. Type 1 diabetic patients were included in Node 10, Node 16, and Node 19. Type 2 diabetic patients were included in Node 8, Node 15, and Node 18. Healthy people were included in node 6, node 7, node 9, node 17 and node 20.

  Node 10 includes only type 1 diabetic patients. This is based on the T-RFLP method using HaeIII as a restriction enzyme, the abundance ratio of fragments having a base length of 85 bp is 0.392 or more, and the abundance ratio of fragments having a base length of 315 bp is less than 7.941 When the abundance ratio of fragments having a base length of 58 bp is less than 0.114, it indicates that type 1 diabetes can be determined. Similarly, the abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp Is less than 4.140, and the abundance ratio of fragments having a base length of 203 bp is less than 2.636, it can be determined that it is type 1 diabetes (node 16). The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4.140. As described above, when the abundance ratio of a fragment having a base length of 265 bp is less than 38.276 and the abundance ratio of a fragment having a base length of 45 bp is 1.328 or more, it can be determined that it is type 1 diabetes. (Node 19). The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is 0.856. If it is less than that, it indicates that it can be determined that it is type 2 diabetes (node 8). The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4.140. When the abundance ratio of fragments having a base length of 203 bp is less than or equal to 2.636, it indicates that it can be determined as type 2 diabetes (node 15). The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4.140. As described above, when the abundance ratio of a fragment having a base length of 265 bp is 38.276 or more and the abundance ratio of a fragment having a base length of 45 bp is less than 1.258, it can be determined that the subject has type 2 diabetes. (Node 18). When the abundance ratio of a fragment with a base length of 85 bp is less than 0.392 and the abundance ratio of a fragment with a base length of 177 bp is less than 4.067, it can be determined that neither type 1 diabetes nor type 2 diabetes is present. (Node 6). The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is 0.856. If this is the case, it can be determined that neither type 1 diabetes nor type 2 diabetes can be determined (node 7). The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is less than 7.941, and the abundance ratio of fragments having a base length of 58 bp is 0.114. If this is the case, it can be determined that neither type 1 diabetes nor type 2 diabetes can be determined (node 9). The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4.140. When the abundance ratio of fragments having a base length of 265 bp is 38.276 or more and the abundance ratio of fragments having a base length of 45 bp is 1.258 or more, both type 1 diabetes and type 2 diabetes This indicates that it can be determined that there is not (node 17). The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4.140. When the abundance ratio of fragments having a base length of 265 bp is less than 38.276 and the abundance ratio of fragments having a base length of 45 bp is less than 1.328, both type 1 diabetes and type 2 diabetes This indicates that it can be determined that there is not (node 20).

All type 1 diabetic patients are included in either node 10, node 16, or node 19, and if a path from the root node of the decision tree to these nodes is followed, all type 1 diabetic patients are type 1 diabetic patients. It was possible to determine that. All type 2 diabetic patients are included in either node 8, node 15 or node 18, and if a path from the root node of the decision tree to these nodes is followed, all type 2 diabetic patients are type 2 diabetic patients. It was possible to determine. In addition, all healthy persons are included in any of node 6, node 7, node 9, node 17, or node 20, and all healthy persons are healthy by following the route from the root node of the decision tree to these nodes. It was possible to determine that the person was a person.
Further, 7 of 11 patients with type 1 diabetes included in node 16 are determined. In the decision tree shown in FIG. 3, determination is made with a sensitivity of 64% and a specificity of 100% only by the route from the root node to node 16. I was able to. There are 8 type 11 diabetic patients included in the node 18, and in the decision tree shown in FIG. 3, it is possible to determine a sensitivity of 73% and a specificity of 100% only by the route from the root node to the node 18. did it. In addition, there are 22 of 31 healthy persons included in node 7, and in the decision tree shown in FIG. 3, it is possible to determine sensitivity 71% and specificity 100% only by the route from the root node to node 7. did it.

<Results of Analysis Example 3>
The obtained decision tree is shown in FIG. Type 1 diabetic patients were included in Node 9, Node 14, Node 16, Node 18, and Node 20. Type 2 diabetic patients were included in Node 4, Node 15, Node 19, and Node 21. Healthy people were included in node 6, node 17, and node 22.

  Node 9 includes only patients with type 1 diabetes. This is based on the T-RFLP method using MspI as a restriction enzyme, the abundance ratio of fragments having a base length of 293 bp is less than 9.740, and the abundance ratio of fragments having a base length of 457 bp is 1.620 or more. When the abundance ratio of a fragment having a base length of 120 bp is 0.312 or more, it indicates that it can be determined that it is type 1 diabetes. Similarly, the abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp Is less than 2.617, and the abundance ratio of fragments having a base length of 88 bp is less than 1.179, it can be determined that it is type 1 diabetes (node 14). The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is 0.312. If the abundance ratio of fragments having a base length of 81 bp is less than 7.009, it can be determined that the patient has type 1 diabetes (node 16). The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2.617. As described above, when the abundance ratio of a fragment having a base length of 390 bp is 0.750 or more and the abundance ratio of a fragment having a base length of 200 bp is less than 0.542, it can be determined as having type 1 diabetes. (Node 18). The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2.617. As described above, when the abundance ratio of a fragment having a base length of 390 bp is less than 0.750 and the abundance ratio of a fragment having a base length of 45 bp is less than 1.263, it can be determined as having type 1 diabetes. (Node 20). When the abundance ratio of a fragment having a base length of 293 bp is 9.740 or more and the abundance ratio of a fragment having a base length of 150 bp is less than 3.879, this indicates that it can be determined as type 2 diabetes (node) 4). The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is 0.312. When the abundance ratio of fragments having a base length of 81 bp is 7.009 or more, it is indicated that it can be determined that it is type 2 diabetes (node 15). The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2.617. As described above, when the abundance ratio of a fragment having a base length of 390 bp is less than 0.750 and the abundance ratio of a fragment having a base length of 45 bp is 1.263 or more, it can be determined as having type 2 diabetes. (Node 19). The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2.617. If the abundance ratio of a fragment having a base length of 88 bp is 1.179 or more and the abundance ratio of a fragment having a base length of 45 bp is 1.688 or more, it can be determined as having type 2 diabetes. (Node 21). When the abundance ratio of fragments having a base length of 293 bp is less than 9.740 and the abundance ratio of fragments having a base length of 457 bp is less than 1.620, it can be determined that neither type 1 diabetes nor type 2 diabetes is present. (Node 6). The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2.617. When the abundance ratio of fragments having a base length of 390 bp is 0.750 or more and the abundance ratio of fragments having a base length of 200 bp is 0.542 or more, both type 1 diabetes and type 2 diabetes This indicates that it can be determined that there is not (node 17). The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2.617. When the abundance ratio of fragments having a base length of 88 bp is 1.179 or more and the abundance ratio of fragments having a base length of 45 bp is less than 1.688, both type 1 diabetes and type 2 diabetes This indicates that it can be determined that there is not (node 22).

All type 1 diabetic patients are included in any of node 9, node 14, node 16, node 18, or node 20, and all the type 1 diabetics can be found by following the path from the root node of the decision tree to these nodes. It was possible to determine that the patient was a type 1 diabetes patient. 10 type 2 diabetic patients out of 11 are included in any of node 4, node 15, node 19, or node 21, and follow the path from the root node of the decision tree to these nodes. It was possible to determine that the patient was a type 2 diabetic. In addition, all healthy persons are included in any one of the nodes 6, 11, and 22, and if a route from the root node of the decision tree to these nodes is traced, all the healthy persons are determined to be healthy persons. It was possible.
In addition, there are 5 of 11 patients with type 1 diabetes included in node 9, and in the decision tree shown in FIG. 4, determination is made with 45% sensitivity and 100% specificity only by the route from the root node to node 9. I was able to. There are 5 of 11 patients with type 2 diabetes included in node 15, and in the decision tree shown in FIG. 4, it is possible to determine sensitivity 45% and specificity 100% only by the route from the root node to node 15. did it. In addition, there are 27 healthy persons included in the node 17, and in the decision tree shown in FIG. 4, it is possible to determine sensitivity 87% and specificity 96% only by the route from the root node to the node 17. did it.

<Result of Analysis Example 4>
The obtained decision tree is shown in FIG. Type 1 diabetic patients were included in Node 6, Node 12, Node 16, and Node 18. Type 2 diabetic patients were included in Node 5, Node 7, and Node 14. Healthy people were included in node 11, node 15, and node 17.

  Node 6 includes only patients with type 1 diabetes. This is based on the T-RFLP method using AluI as a restriction enzyme, the abundance ratio of fragments having a base length of 44 bp is less than 9.028, and the abundance ratio of fragments having a base length of 58 bp is less than 1.062 When it is, it shows that it can determine with having type 1 diabetes. Similarly, the abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp Indicates that it can be determined that the patient has type 1 diabetes (node 12). The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is 0.762. If the abundance ratio of the fragment having a base length of less than 83 bp is less than 9.744, it can be determined that it is type 1 diabetes (node 16). The abundance ratio of fragments having a base length of 44 bp is not less than 9.028 value, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is 0.00. If the abundance ratio of fragments having a base length of 203 bp is 0.749 or more and the abundance ratio of fragments having a base length of 830 bp is less than 0.317, it is determined as type 1 diabetes Indicates that it can be done (node 18). When the abundance ratio of a fragment having a base length of 44 bp is less than 9.028 and the abundance ratio of a fragment having a base length of 58 bp is 1.062 or more, this indicates that it can be determined as type 2 diabetes (node 5). The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is 0.405. If this is the case, it can be determined that it is type 2 diabetes (node 7). The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is 0.762. As described above, when the abundance ratio of a fragment having a base length of 203 bp is less than 0.749, it can be determined that it is type 2 diabetes (node 14). The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is 4.121. When it is above, it shows that it can determine with being a healthy person (node 11). The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is 0.762. If the abundance ratio of fragments having a base length of 83 bp is 9.744 or more, it indicates that it can be determined to be a healthy person (node 15). The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is 0.762. That is, when the abundance ratio of fragments having a base length of 203 bp is 0.749 or more and the abundance ratio of fragments having a base length of 830 bp is 0.317 or more, it can be determined that the subject is a healthy person. Shown (node 17).

10 out of 11 type 1 diabetic patients are included in any of node 6, node 12, node 16, or node 18 and follow the route from the root node of the decision tree to these nodes. Could be determined to be type 1 diabetic. Nine out of eleven patients with type 2 diabetes are included in either node 5, node 7, or node 14 and follow the path from the root node of the decision tree to these nodes. It was possible to determine that the patient was diabetic. Further, all healthy persons are included in any one of the node 11, the node 15, or the node 17, and if a path from the root node of the decision tree to these nodes is traced, all the healthy persons are determined to be healthy persons. It was possible.
Further, the number of type 1 diabetic patients included in node 16 is five out of eleven patients, and in the decision tree shown in FIG. 5, determination is made with 45% sensitivity and 100% specificity only by the route from the root node to node 16. I was able to. There are 5 of 11 patients with type 2 diabetes mellitus included in node 5, and in the decision tree shown in FIG. 5, it is possible to make a determination of 45% sensitivity and 100% specificity only by the route from the root node to node 5. did it. In addition, the healthy persons included in the node 11 were 19 out of 31 persons, and the sensitivity 61% and the specificity 100% could be determined only by the route from the root node to the node 11.

Claims (17)

Obtaining an end-labeled restriction enzyme fragment by the T-RFLP method for the bacterial 16S ribosomal RNA gene of the entire intestinal flora contained in the sample collected from the subject; and
The presence or absence of type 1 diabetes and / or the presence or absence of type 2 diabetes is determined based on the abundance ratio of end-labeled restriction enzyme fragments having a specific base length to the total amount of end-labeled restriction enzyme fragments obtained by the T-RFLP method. To do,
Diabetes testing method including.   The test for diabetes according to claim 1, wherein the T-RFLP method comprises obtaining an end-labeled restriction enzyme fragment using at least one restriction enzyme selected from the group consisting of BslI, HaeIII, MspI and AluI. Method.   The method for testing diabetes according to claim 1 or 2, wherein the sample is feces.   The determination includes classifying subjects according to a decision tree generated by a data mining method, wherein the data mining method is type 1 diabetes, type 2 diabetes, or even type 1 diabetes 2 The target variable is whether it is not type 2 diabetes, and the abundance ratio of end-labeled restriction enzyme fragments having a specific base length to the total amount of the end-labeled restriction enzyme fragments is used as an explanatory variable. The method for testing diabetes according to any one of the above.   The method for testing diabetes according to claim 4, wherein the data mining method is a C & RT method. The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 1, and cleaving the obtained amplification product with BslI. 6. The method according to claim 1, comprising at least one of the conditions (1) to (13) for the abundance ratio of the end-labeled restriction enzyme fragment having a specific base length relative to the total amount of the labeled restriction enzyme fragment. 2. Diabetes testing method described in 1.
(1) Type 1 diabetes when the abundance ratio of fragments having a base length of 366 bp is not less than a predetermined threshold and the abundance ratio of fragments having a base length of 168 bp is less than the predetermined threshold Is determined.
(2) Fragment having an abundance ratio of fragments having a base length of 366 bp equal to or greater than a predetermined threshold, an abundance ratio of fragments having a base length of 168 bp being equal to or greater than a predetermined threshold, and a base length of 505 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 1 diabetes.
(3) Fragment in which the abundance ratio of fragments having a base length of 366 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 754 bp is greater than or equal to a predetermined threshold, and the base length is 968 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 1 diabetes.
(4) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 317 bp is greater than or equal to a predetermined threshold value, it is determined as having type 1 diabetes.
(5) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp The abundance ratio of fragments with a base length of 650 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments with a base length of 106 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(6) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being equal to or greater than a predetermined threshold, and a base length of 968 bp Is greater than or equal to a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 366 bp is less than the predetermined threshold value, it is determined as having type 2 diabetes.
(7) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp Is greater than or equal to a predetermined threshold, and when the abundance of a fragment having a base length of 650 bp is less than the predetermined threshold, it is determined that the subject has type 2 diabetes.
(8) A fragment having a base length of 366 bp in which the abundance ratio is not less than a predetermined threshold, a fragment having a base length of 168 bp is not less than a predetermined threshold, and a base length is 505 bp The abundance ratio of fragments whose base length is 332 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 106 bp is less than the predetermined threshold. Is determined to be type 2 diabetes.
(9) Fragment having an abundance ratio of fragments having a base length of 366 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 168 bp being not less than a predetermined threshold, and having a base length of 505 bp Is greater than or equal to a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 332 bp is greater than or equal to a predetermined threshold value, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being equal to or greater than a predetermined threshold, and a base length of 968 bp If the abundance ratio of the fragment is equal to or greater than a predetermined threshold and the abundance ratio of a fragment having a base length of 366 bp is equal to or greater than the predetermined threshold, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(11) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 317 bp is less than the predetermined threshold value, it is determined that the type is not type 1 diabetes or type 2 diabetes.
(12) Fragment having an abundance ratio of fragments having a base length of 366 bp is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 168 bp is not less than a predetermined threshold, and a base length is 505 bp The abundance ratio of fragments having a base length of 332 bp is less than a predetermined threshold, and the abundance ratio of fragments having a base length of 106 bp is not less than a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
(13) Fragment having an abundance ratio of fragments having a base length of 366 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 754 bp being less than a predetermined threshold, and a base length of 749 bp The abundance ratio of fragments having a base length of 650 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 106 bp is greater than or equal to a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes. The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined thresholds of the conditions (1) to (13) according to claim 6 are the following (1) The method for examining diabetes according to claim 6, which is defined as shown in (13).
(1) When the abundance ratio of a fragment having a base length of 366 bp is 2.281 or more and the abundance ratio of a fragment having a base length of 168 bp is less than 3.081, it is determined as having type 1 diabetes.
(2) The abundance ratio of fragments with a base length of 366 bp is 2.281 or more, the abundance ratio of fragments with a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments with a base length of 505 bp is If it is less than 1.859, it is determined that it is type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is If it is less than 0.560, it is determined as type 1 diabetes.
(4) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a base length of less than 4.644 and a base length of 317 bp is 17.401 or more, it is determined that the patient has type 1 diabetes.
(5) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is It is type 1 diabetes when the abundance ratio of fragments having a base length of 650 bp is 0.300 or more and the abundance ratio of fragments having a base length of 106 bp is less than 0.778. Is determined.
(6) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is When the abundance ratio of fragments having a length of 0.560 or more and a base length of 366 bp is less than 36.992, it is determined as having type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a length of 4.644 or more and a base length of 650 bp is less than 0.300, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is If the abundance ratio of a fragment having a base length of 332 bp is less than 6.231 and the abundance ratio of a fragment having a base length of 106 bp is less than 0.181, it is type 2 diabetes Is determined.
(9) The abundance ratio of fragments with a base length of 366 bp is 2.281 or more, the abundance ratio of fragments with a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments with a base length of 505 bp is When the abundance ratio of a fragment having a length of 1.859 or more and a base length of 332 bp is 6.231 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is 1.226 or more, and the abundance ratio of fragments having a base length of 968 bp is When the abundance ratio of fragments having a length of 0.560 or more and a base length of 366 bp is 36.992 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(11) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of the fragment having a base length of less than 4.644 and a base length of 317 bp is less than 17.401, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(12) The abundance ratio of fragments having a base length of 366 bp is 2.281 or more, the abundance ratio of fragments having a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments having a base length of 505 bp is When the abundance ratio of fragments having a base length of 332 bp is less than 6.231 and the abundance ratio of fragments having a base length of 106 bp is 0.181 or more, it is 2 in type 1 diabetes. It is determined that it is not type 2 diabetes.
(13) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a base length of 650 bp is not less than 0.300 and the abundance ratio of fragments having a base length of 106 bp is not less than 0.778, 2 in type 1 diabetes It is determined that it is not type 2 diabetes. The method for testing diabetes according to claim 7, comprising at least one of the following (4), (7), and (9).
(4) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a base length of less than 4.644 and a base length of 317 bp is 17.401 or more, it is determined that the patient has type 1 diabetes.
(7) The abundance ratio of fragments having a base length of 366 bp is less than 2.281, the abundance ratio of fragments having a base length of 754 bp is less than 1.226, and the abundance ratio of fragments having a base length of 749 bp is When the abundance ratio of fragments having a length of 4.644 or more and a base length of 650 bp is less than 0.300, it is determined as having type 2 diabetes.
(9) The abundance ratio of fragments with a base length of 366 bp is 2.281 or more, the abundance ratio of fragments with a base length of 168 bp is 3.081 or more, and the abundance ratio of fragments with a base length of 505 bp is When the abundance ratio of a fragment having a length of 1.859 or more and a base length of 332 bp is 6.231 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present. The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 1, and cleaving the obtained amplification product with HaeIII. 6. The method according to claim 1, comprising at least one of the conditions (1) to (11) of the abundance ratio of the end-labeled restriction enzyme fragment having a specific base length relative to the total amount of the labeled restriction enzyme fragment. 2. Diabetes testing method described in 1.
(1) Fragment having an abundance ratio of fragments having a base length of 85 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 315 bp being less than the predetermined threshold, and a base length of 58 bp Is less than a predetermined threshold, it is determined that the patient has type 1 diabetes.
(2) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is not less than a predetermined threshold, and the base length is 236 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 203 bp is less than a predetermined threshold value, it is determined as having type 1 diabetes.
(3) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is greater than or equal to a predetermined threshold, and the base length is 236 bp The abundance ratio of fragments whose base length is 265 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 45 bp is greater than or equal to the predetermined threshold. Is determined to be type 1 diabetes.
(4) Fragment in which the abundance ratio of fragments having a base length of 85 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 315 bp is not less than a predetermined threshold, and the base length is 79 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 2 diabetes.
(5) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is not less than a predetermined threshold, and the base length is 236 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 203 bp is equal to or greater than a predetermined threshold value, it is determined as having type 2 diabetes.
(6) Fragment in which the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold, the abundance ratio of fragments having a base length of 177 bp is not less than a predetermined threshold, and the base length is 236 bp The abundance ratio of fragments having a base length of 265 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is less than the predetermined threshold. Is determined to be type 2 diabetes.
(7) When the abundance ratio of fragments having a base length of 85 bp is less than a predetermined threshold value and the abundance ratio of fragments having a base length of 177 bp is less than a predetermined threshold value, 2 even in type 1 diabetes It is determined that it is not type 2 diabetes.
(8) Fragment having an abundance ratio of fragments having a base length of 85 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 315 bp being not less than a predetermined threshold, and having a base length of 79 bp When the abundance ratio is equal to or greater than a predetermined threshold value, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(9) Fragment in which the abundance ratio of fragments having a base length of 85 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 315 bp is less than the predetermined threshold, and the base length is 58 bp When the abundance ratio is equal to or greater than a predetermined threshold value, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) Fragment having an abundance ratio of fragments having a base length of 85 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 177 bp being equal to or greater than a predetermined threshold, and a base length being 236 bp The abundance ratio of fragments having a base length of 265 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is greater than or equal to a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
(11) Fragment having an abundance ratio of fragments having a base length of 85 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 177 bp being equal to or greater than a predetermined threshold, and a base length being 236 bp The abundance ratio of fragments whose base length is 265 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 45 bp is less than the predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes. The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined thresholds of the conditions (1) to (11) according to claim 9 are the following (1) The method for testing diabetes according to claim 9, which is defined as shown in (10).
(1) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is less than 7.941, and the abundance ratio of fragments having a base length of 58 bp is When it is less than 0.114, it is determined as type 1 diabetes.
(2) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of less than 4.140 and a base length of 203 bp is less than 2.636, it is determined as having type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4. Type 1 diabetes when the abundance ratio of fragments having a base length of 265 bp is less than 38.276 and the abundance ratio of fragments having a base length of 45 bp is 1.328 or more Is determined.
(4) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is When it is less than 0.856, it is determined as type 2 diabetes.
(5) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of less than 4.140 and a base length of 203 bp is 2.636 or more, it is determined as type 2 diabetes.
(6) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of 265 bp is 38.276 or more and the abundance ratio of fragments having a base length of 45 bp is less than 1.258, it is type 2 diabetes Is determined.
(7) When the abundance ratio of a fragment having a base length of 85 bp is less than 0.392 and the abundance ratio of a fragment having a base length of 177 bp is less than 4.067, neither type 1 diabetes nor type 2 diabetes Is determined.
(8) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is When it is 0.856 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(9) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is less than 7.941, and the abundance ratio of fragments having a base length of 58 bp is When it is 0.114 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present.
(10) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4. When the abundance ratio of fragments having a base length of 265 bp is 38.276 or more and the abundance ratio of fragments having a base length of 45 bp is 1.258 or more, it is 2 in type 1 diabetes. It is determined that it is not type 2 diabetes.
(11) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is 4. If the abundance ratio of fragments having a base length of 265 bp is less than 38.276 and the abundance ratio of fragments having a base length of 45 bp is less than 1.328, it is 2 for type 1 diabetes. It is determined that it is not type 2 diabetes. The method for examining diabetes according to claim 10, comprising at least one of the following (2), (6) or (8).
(2) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of less than 4.140 and a base length of 203 bp is less than 2.636, it is determined as having type 1 diabetes.
(6) The abundance ratio of fragments having a base length of 85 bp is less than 0.392, the abundance ratio of fragments having a base length of 177 bp is 4.067 or more, and the abundance ratio of fragments having a base length of 236 bp is When the abundance ratio of fragments having a base length of 265 bp is 38.276 or more and the abundance ratio of fragments having a base length of 45 bp is less than 1.258, it is type 2 diabetes Is determined.
(8) The abundance ratio of fragments having a base length of 85 bp is 0.392 or more, the abundance ratio of fragments having a base length of 315 bp is 7.941 or more, and the abundance ratio of fragments having a base length of 79 bp is When it is 0.856 or more, it is determined that neither type 1 diabetes nor type 2 diabetes is present. The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 3, and cleaving the obtained amplification product with MspI. 6. The method according to claim 1, comprising at least one of the conditions (1) to (12) of the abundance ratio of the end-labeled restriction enzyme fragment having a specific base length with respect to the total amount of the labeled restriction enzyme fragment. 2. Diabetes testing method described in 1.
(1) Fragment having an abundance ratio of fragments having a base length of 293 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 457 bp being equal to or greater than a predetermined threshold, and a base length being 120 bp When the abundance ratio is equal to or greater than a predetermined threshold, it is determined that the patient has type 1 diabetes.
(2) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp, a ratio of fragments having a base length of 150 bp and a base length of 194 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 88 bp is less than a predetermined threshold value, it is determined as having type 1 diabetes.
(3) Fragment having a base length of 293 bp that is less than a predetermined threshold, a base length of 457 bp being greater than or equal to a predetermined threshold, and a base length of 120 bp Is less than a predetermined threshold value, and a fragment having a base length of 81 bp is less than the predetermined threshold value, it is determined as having type 1 diabetes.
(4) A fragment whose base length is 293 bp or more is a predetermined threshold or more, a fragment whose base length is 150 bp or more is a predetermined threshold or more and a base length is 194 bp The abundance ratio of fragments having a base length of 390 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 200 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(5) Fragment having an abundance ratio of fragments having a base length of 293 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 150 bp being not less than a predetermined threshold, and a base length of 194 bp The abundance ratio of fragments whose base length is 390 bp is less than the predetermined threshold, and the abundance ratio of fragments whose base length is 45 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(6) Type 2 diabetes when the abundance ratio of fragments having a base length of 293 bp is equal to or greater than a predetermined threshold and the abundance ratio of fragments having a base length of 150 bp is less than the predetermined threshold Is determined.
(7) Fragment having an abundance ratio of fragments having a base length of 293 bp less than a predetermined threshold, an abundance ratio of fragments having a base length of 457 bp being equal to or greater than a predetermined threshold, and a base length being 120 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 81 bp is equal to or greater than the predetermined threshold value, it is determined as having type 2 diabetes.
(8) Fragment having an abundance ratio of fragments having a base length of 293 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 150 bp being not less than a predetermined threshold, and having a base length of 194 bp The abundance ratio of fragments having a base length of 390 bp is less than a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is not less than a predetermined threshold. Is determined to be type 2 diabetes.
(9) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp and a base ratio of 150 bp and a fragment having a base length of 194 bp The abundance ratio of fragments having a base length of 88 bp is not less than a predetermined threshold, and the abundance ratio of fragments having a base length of 45 bp is not less than a predetermined threshold. Is determined to be type 2 diabetes.
(10) When the abundance ratio of fragments having a base length of 293 bp is less than a predetermined threshold value and the abundance ratio of fragments having a base length of 457 bp is less than a predetermined threshold value, 2 even in type 1 diabetes It is determined that it is not type 2 diabetes.
(11) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp, a ratio of fragments having a base length of 150 bp and a base length of 194 bp The abundance ratio of fragments having a base length of 390 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 200 bp is greater than or equal to a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes.
(12) A fragment having a base length of 293 bp and a fragment having a base length of 194 bp, a ratio of fragments having a base length of 150 bp and a base length of 194 bp The abundance ratio of fragments having a base length of 88 bp is greater than or equal to a predetermined threshold and the abundance ratio of fragments having a base length of 45 bp is less than a predetermined threshold. If it is, it is determined that it is neither type 1 diabetes nor type 2 diabetes. The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined threshold values of the conditions (1) to (12) according to claim 12 are the following (1) The method for examining diabetes according to claim 12, which is defined as shown in (12).
(1) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When it is 0.312 or more, it is determined that it is type 1 diabetes.
(2) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is When the abundance ratio of fragments having a length of less than 2.617 and a base length of 88 bp is less than 1.179, it is determined as having type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When the abundance ratio of a fragment having a base length of less than 0.312 and a base length of 81 bp is less than 7.009, it is determined as having type 1 diabetes.
(4) The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2. Type 1 diabetes when the abundance ratio of fragments having a base length of 390 bp is 0.750 or more and the abundance ratio of fragments having a base length of 200 bp is less than 0.542 Is determined.
(5) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is If the abundance ratio of fragments having a base length of 390 bp is less than 0.750 and the abundance ratio of fragments having a base length of 45 bp is less than 1.263, it is type 1 diabetes Is determined.
(6) When the abundance ratio of a fragment having a base length of 293 bp is 9.740 or more and the abundance ratio of a fragment having a base length of 150 bp is less than 3.879, it is determined as having type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When the abundance ratio of a fragment having a base length of less than 0.312 and a base length of 81 bp is 7.009 or more, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is 2. Type 2 diabetes when the abundance ratio of fragments having a base length of 390 bp is less than 0.750 and the abundance ratio of fragments having a base length of 45 bp is 1.263 or more Is determined.
(9) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is It is type 2 diabetes when the abundance ratio of fragments having a base length of less than 2.617, a base length of 88 bp is 1.179 or more, and the abundance ratio of fragments having a base length of 45 bp is 1.688 or more Is determined.
(10) When the abundance ratio of fragments having a base length of 293 bp is less than 9.740 and the abundance ratio of fragments having a base length of 457 bp is less than 1.620, neither type 1 diabetes nor type 2 diabetes Is determined.
(11) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is 2. If the abundance ratio of fragments having a base length of 390 bp is 0.750 or more and the abundance ratio of fragments having a base length of 200 bp is 0.542 or more, it is 2 even in type 1 diabetes. It is determined that it is not type 2 diabetes.
(12) The abundance ratio of fragments having a base length of 293 bp is 9.740 or more, the abundance ratio of fragments having a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments having a base length of 194 bp is If the abundance ratio of fragments having a base length of less than 2.617, a base length of 88 bp is 1.179 or more, and the abundance ratio of fragments having a base length of 45 bp is less than 1.688, even in type 1 diabetes, 2 It is determined that it is not type 2 diabetes. The method for testing diabetes according to claim 13, comprising at least one of the following (1), (6), (7) or (11).
(1) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When it is 0.312 or more, it is determined that it is type 1 diabetes.
(6) When the abundance ratio of a fragment having a base length of 293 bp is 9.740 or more and the abundance ratio of a fragment having a base length of 150 bp is less than 3.879, it is determined as having type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 293 bp is less than 9.740, the abundance ratio of fragments having a base length of 457 bp is 1.620 or more, and the abundance ratio of fragments having a base length of 120 bp is When the abundance ratio of a fragment having a base length of less than 0.312 and a base length of 81 bp is 7.009 or more, it is determined as having type 2 diabetes.
(11) The abundance ratio of fragments with a base length of 293 bp is 9.740 or more, the abundance ratio of fragments with a base length of 150 bp is 3.879 or more, and the abundance ratio of fragments with a base length of 194 bp is 2. If the abundance ratio of fragments having a base length of 390 bp is 0.750 or more and the abundance ratio of fragments having a base length of 200 bp is 0.542 or more, it is 2 even in type 1 diabetes. It is determined that it is not type 2 diabetes. The T-RFLP method includes performing nucleic acid amplification using a forward primer represented by SEQ ID NO: 3, and cleaving the obtained amplification product with AluI. 6. The method according to claim 1, comprising at least one of the conditions (1) to (10) of the abundance ratio of the end-labeled restriction enzyme fragments having a specific base length with respect to the total amount of the labeled restriction enzyme fragments. 2. Diabetes testing method described in 1.
(1) Type 1 diabetes when the abundance ratio of fragments having a base length of 44 bp is less than a predetermined threshold and the abundance ratio of fragments having a base length of 58 bp is less than a predetermined threshold Is determined.
(2) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is not less than a predetermined threshold, and the base length is 76 bp If the abundance ratio is less than a predetermined threshold value, it is determined that the patient has type 1 diabetes.
(3) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is less than the predetermined threshold, and the base length is 487 bp Is less than a predetermined threshold value, and when the abundance ratio of a fragment having a base length of 83 bp is less than a predetermined threshold value, it is determined as having type 1 diabetes.
(4) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is less than the predetermined threshold, and the base length is 487 bp The abundance ratio of fragments having a base length of 203 bp is greater than or equal to a predetermined threshold and the abundance ratio of fragments having a base length of 830 bp is less than the predetermined threshold. Is determined to be type 1 diabetes.
(5) Type 2 diabetes when the abundance ratio of fragments having a base length of 44 bp is less than a predetermined threshold and the abundance ratio of fragments having a base length of 58 bp is not less than a predetermined threshold Is determined.
(6) Fragment having an abundance ratio of fragments having a base length of 44 bp is equal to or greater than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp is not less than a predetermined threshold, and a base length is 76 bp If the abundance ratio is equal to or greater than a predetermined threshold value, it is determined that the patient has type 2 diabetes.
(7) Fragment having an abundance ratio of fragments having a base length of 44 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp being less than the predetermined threshold, and a base length of 487 bp Is greater than or equal to a predetermined threshold value, and a fragment having a base length of 203 bp is less than the predetermined threshold value, it is determined that the subject has type 2 diabetes.
(8) Fragment having an abundance ratio of fragments having a base length of 44 bp is equal to or greater than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp is equal to or greater than a predetermined threshold, and a base length is 76 bp When the abundance ratio is equal to or greater than a predetermined threshold, it is determined that the person is a healthy person.
(9) Fragment in which the abundance ratio of fragments having a base length of 44 bp is not less than a predetermined threshold, the abundance ratio of fragments having a base length of 216 bp is less than the predetermined threshold, and the base length is 487 bp If the abundance ratio of the fragment is less than a predetermined threshold and the abundance ratio of the fragment having a base length of 83 bp is equal to or greater than the predetermined threshold, it is determined that the person is a healthy person.
(10) Fragment having an abundance ratio of fragments having a base length of 44 bp that is not less than a predetermined threshold, an abundance ratio of fragments having a base length of 216 bp being less than the predetermined threshold, and a base length of 487 bp The abundance ratio of fragments having a base length of 203 bp is greater than or equal to a predetermined threshold, and the abundance ratio of fragments having a base length of 830 bp is greater than or equal to a predetermined threshold. When it is, it determines with it being a healthy person. The T-RFLP method uses a reverse primer represented by SEQ ID NO: 2, and the predetermined threshold values of the conditions (1) to (10) according to claim 15 are the following (1) The method for examining diabetes according to claim 15, which is defined as shown in (10).
(1) When the abundance ratio of a fragment having a base length of 44 bp is less than 9.028 and the abundance ratio of a fragment having a base length of 58 bp is less than 1.062, it is determined as having type 1 diabetes.
(2) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is If it is less than 4.11, it is determined that the patient has type 1 diabetes.
(3) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of a fragment having a base length of less than 0.762 and a base length of 83 bp is less than 9.744, it is determined as having type 1 diabetes.
(4) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp Is 0.762 or more, the abundance ratio of fragments having a base length of 203 bp is 0.749 or more, and the abundance ratio of fragments having a base length of 830 bp is less than 0.317, Judge that there is.
(5) When the abundance ratio of a fragment having a base length of 44 bp is less than 9.028 and the abundance ratio of a fragment having a base length of 58 bp is 1.062 or more, it is determined as having type 2 diabetes.
(6) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is When it is 0.405 or more, it is determined that it is type 2 diabetes.
(7) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of fragments having a length of 0.762 or more and a base length of 203 bp is less than 0.749, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is 4. If it is 121 or more, it is determined that the person is healthy.
(9) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is If the abundance ratio of fragments having a length of less than 0.762 and a base length of 83 bp is 9.744 or more, it is determined that the subject is a healthy person.
(10) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of fragments having a base length of 0.762 or more is 0.749 or more and the abundance ratio of fragments having a base length of 830 bp is 0.317 or more, judge. The method for examining diabetes according to claim 16, comprising at least one of the following (3), (5), and (8).
(3) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is less than 0.861, and the abundance ratio of fragments having a base length of 487 bp is When the abundance ratio of a fragment having a base length of less than 0.762 and a base length of 83 bp is less than 9.744, it is determined as having type 1 diabetes.
(5) When the abundance ratio of a fragment having a base length of 44 bp is less than 9.028 and the abundance ratio of a fragment having a base length of 58 bp is 1.062 or more, it is determined as having type 2 diabetes.
(8) The abundance ratio of fragments having a base length of 44 bp is 9.028 or more, the abundance ratio of fragments having a base length of 216 bp is 0.861 or more, and the abundance ratio of fragments having a base length of 76 bp is 4. If it is 121 or more, it is determined that the person is healthy.