Non-invasive prenatal paternity testing method

Abstract

To provide a novel technology to verify erroneous determination due to sample mix-up in a blood relationship test.SOLUTION: Provided is a method for verifying erroneous determination due to sample mix-up in a blood relationship test between a specific person and a pseudo-blood relative based on samples taken from a pseudo-blood relative of a specific person to be tested and a set of specific target families including the specific person, the method including verification steps defined below. (Verification pattern 1) Based on the result of genotyping a specific person belonging to a specific target family and the result of genotyping a pseudo-blood relative belonging to another target family, blood relationship testing is performed for the combination of this pseudo-blood relative and the specific person. (Verification pattern 2) Based on the result of genotyping a specific person belonging to another target family and the result of genotyping a pseudo-blood relative belonging to the specific target family, blood relationship testing is performed for the combination of this pseudo-blood relative and the specific person.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to non-invasive prenatal paternity testing techniques. [Background technology]

[0002] There is a known technology for prenatal paternity testing between a fetus and a putative father based on the analysis of circulating cell-free DNA (cfDNA) in the blood. By analyzing cell-free fetal DNA (cffDNA), which is genetic material derived from the fetus that is mixed into the mother's blood circulation, it is possible to perform a non-invasive prenatal paternity test (NIPPT) (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2014-502845 Summary of the Invention [Problem to be solved by the invention]

[0004] In non-invasive prenatal paternity testing, the genotype of a fetus is identified based on the analysis results of a cfDNA sample. However, since cfDNA contains a mixture of DNA derived from the mother and DNA derived from the fetus, a signal measured from one polymorphic locus contains a mixture of signals caused by alleles derived from the mother and signals caused by alleles derived from the fetus. In order to perform genotyping of a fetus, it is necessary to estimate the signal of the allele derived from the fetus among the mixed signals. Therefore, the first object of the present invention is to provide a novel non-invasive prenatal paternity testing method that can perform genotyping of a fetus with a simple standard. [Means for solving the problem]

[0005] The present invention for solving the above-described first problem is as follows. [1] A non-invasive prenatal paternity testing method, A non-invasive prenatal paternity testing method including a two-sample testing step and / or a three-sample testing step defined below.

[0006] <Two-sample testing step> A step of determining the existence or non-existence of a biological paternity relationship between a putative father and a fetus based on the results of genotyping of the putative father and the fetus at a plurality of polymorphic loci. However, the genotyping of the fetus is performed according to a reference a determination method defined below.

[0007] (Reference a determination method) Based on data obtained by analyzing a cell-free nucleic acid sample collected from a pregnant mother, derived from a specific polymorphic locus, The allele detected with the strongest signal intensity is designated as allele A, The allele detected with the same signal intensity as allele A or the second strongest signal intensity is designated as allele B, When so designated, using the ratio (Fa) of the signal intensity of allele A and the ratio (Fb) of the signal intensity of allele B to the total signal intensity derived from the specific polymorphic locus as indices, the following reference a is used.

[0008] <Reference a> · When 0% ≦ Fb {≦, <} L% is satisfied, it is determined that the genotype of the fetus at the specific polymorphic locus is homozygous AA · When M% {≦, <} Fb {≦, <} N% is satisfied, it is determined that the genotype of the fetus at the specific polymorphic locus is heterozygous AB · When Fa = Fb is satisfied, skip without performing genotyping of the specific polymorphic locus · When Fb does not satisfy any of the above conditions, skip without performing genotyping of the specific polymorphic locus (N is a numerical value less than 50, and satisfies the relational expression 0 {≦, <} L < M {≦, <} N. Note that the notation {≦, <} indicates that either the inequality sign ≦ or < can be arbitrarily selected.)

[0009] <3-Sample Genotyping Process> A process of determining the existence or non-existence of the biological parent-child relationship between the alleged father and the fetus based on the genotyping results of the biological mother, the alleged father, and the fetus at multiple polymorphic loci. However, the genotyping of the fetus is performed according to the above-mentioned criterion a determination method and / or the criterion b determination method defined below.

[0010] (Criterion b determination method) Deriving from a specific polymorphic locus determined to be homozygous in the mother, included in the data obtained by analyzing the circulating cell-free nucleic acid sample, The allele detected with the strongest signal intensity is designated as allele A, The allele detected with the same signal intensity as allele A or the second strongest signal intensity is designated as allele B, When expressed as such, using the ratio (Fa) of the signal intensity of allele A and the ratio (Fb) of the signal intensity of allele B to the total signal intensity derived from the specific polymorphic locus as indicators, the following criterion b is used.

[0011] <Criterion b> ·When 0% ≤ Fb {≤, <} L% is satisfied, it is determined that the genotype of the fetus at the specific polymorphic locus is homozygous AA ·When M% {≤, <} Fb is satisfied, it is determined that the genotype of the fetus at the specific polymorphic locus is heterozygous AB ·When Fa = Fb is satisfied, skip without performing genotyping of the specific polymorphic locus ·When Fb does not satisfy any of the above conditions, skip without performing genotyping of the specific polymorphic locus (Satisfies the relational expression of 0 {≤, <} L < M. Note that the notation {≤, <} indicates that either the inequality sign ≤ or < can be arbitrarily selected.)

[0012] [2] The determination of the existence or non-existence of the biological parent-child relationship is By multiplying the paternity index (PI) obtained for each polymorphic locus, the combined paternity index (CPI) is determined, and a determination of either "affirmative", "negative", or "indeterminate" is made according to a predetermined criterion based on the value of the CPI. In the two-sample identification process, the PI is determined based on Table 1 below. In the three-sample identification process, the PI is determined based on Table 1 or Table 2 below. The non-invasive prenatal paternity testing method described in [1].

Table 1

Table 2

[0013] [3] The non-invasive prenatal paternity testing method described in [1] or [2], including the three-sample identification process and the MC filter process defined below.

[0014] <MC filter process> Genotyping of the mother is performed based on a sample that contains nucleic acid containing the genetic information of the mother collected from the mother and does not substantially contain nucleic acid containing the genetic information of the fetus. (i) A polymorphic locus that is homozygous for an allele whose genotype in the mother and the fetus or cell-free nucleic acid is different. And / or (ii) A polymorphic locus in which the genotype of the mother is heterozygous and the genotype of the cell-free nucleic acid is homozygous. However, if a preset number of times or more is observed, paternity testing based on the collected sample is not performed and the process is stopped.

[0015] [4] The non-invasive extraction method according to any one of [1] to [3], characterized in that a verification step defined below is carried out based on a sample collected from a specific subject family. Prenatal paternity testing method.

[0016] <Verification process> In order to verify erroneous determination due to misidentification of a sample taken from a family to be identified other than the specific family to be identified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below.

[0017] (Verification pattern 1) The genotyping results of the fetus belonging to the particular subject family; and Optionally, the genotyping results of the mother belonging to the particular subject family; The genotyping results of a putative father belonging to the other family to be tested; Based on this, a paternity test is performed for this combination of putative father and fetus.

[0018] (Verification pattern 2) The genotyping results of a fetus belonging to the other subject family; and Optionally, the genotyping results of the biological mother of the fetus belonging to the other subject family; The genotyping results of the putative father belonging to the specific subject family; Based on this, a paternity test is performed for this combination of putative father and fetus.

[0019] [5] In the verification step, The non-invasive prenatal parent-child testing method described in [4], wherein verification is performed according to verification pattern 1 and / or verification pattern 2 by referring to a database storing genotyping results of a putative father, a fetus, and optionally the biological mother of the fetus, who belong to one or more of the other families to be tested.

[0020] [6] After completing genotyping of the putative father, the fetus, and optionally the mother belonging to the specific subject family, storing and updating the results in the database; The verification step includes executing the verification step by referring to the updated database, The non-invasive prenatal parent-child testing method described in [5], in which, in the verification step, the genotyping results of the specific family to be tested reflected in the database are regarded as the genotyping results of another family to be tested, and verification is performed according to verification pattern 1 and / or verification pattern 2.

[0021] [7] Genotyping the putative father and genotyping the fetus, each performed at different locations and / or at different times; and / or A non-invasive prenatal paternity test described in any one of [4] to [6], each of which is performed by a different operator.

[0022] In addition, in the field of blood relationship testing, one person tests a sample of a pseudo-blood relative taken from a specific family to be tested and a sample of a specific person. In this case, a mix-up of samples may occur, resulting in an incorrect blood relationship test result. To prevent such a mix-up of samples, the same test is sometimes performed twice to double check, but this has the problem of doubling the cost of the test. In view of these problems, a second objective of the present invention is to provide a new technology for verifying erroneous determinations due to sample mix-ups in blood relationship testing. The present invention for solving the second problem is as follows.

[0023] [1] A method for verifying an erroneous determination due to a mix-up of samples in a blood relationship test between a specific person and a potential blood relative, based on samples taken from a set of specific subject families including the specific person and, optionally, a biological blood relative of the specific person, the specific person being the subject of the test to determine whether or not there is a biological blood relationship between the specific person and the potential blood relative, the method comprising the steps of:

[0024] <Verification process> In order to verify erroneous determination due to misidentification of a sample taken from a family to be identified other than the specific family to be identified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below.

[0025] (Verification pattern 1) The result of genotyping the specific person belonging to the specific subject family; and Optionally, the results of genotyping biological relatives of said particular person belonging to said particular subject family; the genotyping results of the relatives belonging to the other subject family; Based on this, a blood relationship test is performed between this pseudo-blood relative and a specific person.

[0026] (Verification pattern 2) The genotyping results of a specific person belonging to the other subject family; and Optionally, the results of genotyping biological relatives of said particular person who belong to said other subject family; the genotyping results of the relatives belonging to the particular subject family; and Based on this, a blood relationship test is performed between this pseudo-blood relative and a specific person.

[0027] [2] The pseudo-blood relative is a pseudo-parent, The specific person is a child, A biological relative of the particular person is a biological parent of the child who is of a different sex than the assumed parent; The blood relationship test is a parent-child test, The verification method described in [1] is a method for verifying a misjudgment due to a mix-up of samples in a parent-child test of the pseudo parent and the child based on samples taken from a specific set of subject families including the pseudo parent, the child, and optionally the biological parent, which are the subjects of the test for the existence or absence of a biological parent-child relationship, and is characterized in that it includes a verification step defined below.

[0028] <Verification process> In order to verify erroneous determination due to misidentification of a sample taken from a family to be identified other than the specific family to be identified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below.

[0029] (Verification pattern 1) The genotyping results of the child belonging to the particular subject family; and Optionally, the genotyping results of the biological parents belonging to the particular subject family; and The genotyping results of a pseudo-parent belonging to the other family to be verified; Based on this, parentage testing is performed for this combination of pretend parent and child.

[0030] (Verification pattern 2) The genotyping results of a child belonging to the other subject family; and Optionally, the genotyping results of the child's biological parents who belong to the other subject family; The genotyping results of the pseudo-parents belonging to the specific subject family; and Based on this, parentage testing is performed for this combination of pretend parent and child.

[0031] [3] The verification method described in [1] or [2], wherein the blood relationship testing is based on the results of genotyping two individuals, the related relative and the specific individual, who are the subjects of the testing to determine whether or not there is a biological blood relationship.

[0032] [4] The verification method described in any one of [1] to [3], wherein the blood relationship testing is performed based on the results of genotyping three people: the pseudo-blood relative who is the subject of the testing for the existence of a biological parent-child relationship, the specific person, and the biological blood relative.

[0033] [5] In the verification step, The verification method according to any one of [1] to [4], wherein verification is performed according to the verification pattern 1 and / or the verification pattern 2 by referring to a database storing the genotyping results of a specific individual and a related relative belonging to one or more of the other families to be identified. [6] In the verification step, The verification method described in [2], wherein verification is performed according to verification pattern 1 and / or verification pattern 2 by referring to a database storing the genotyping results of a pseudo-parent, a child, and optionally the biological parent of the child, who belong to one or more of the other families to be verified.

[0034] [7] The verification method described in [5], which includes storing and updating the results in the database after genotyping of the pseudo-blood relatives belonging to the specific family to be verified and the specific person is completed.

[0035] [8] The verification method described in [6], which includes storing and updating the results in the database after genotyping of the pseudo parent, the child, and optionally the biological parent belonging to the specific family to be verified is completed.

[0036] [9] The verification step includes: The verification method described in [7] or [8] includes performing the verification step by referring to the updated database, and performing verification according to verification pattern 1 and / or verification pattern 2 by regarding the genotyping result of the specific family to be verified reflected in the database as the genotyping result of another family to be verified.

[0037]

[10] Genotyping the kin and genotyping the particular person, each performed at different locations and / or at different times; and / or The verification method according to any one of [1] to [9], wherein each of the verification methods is performed by a different worker. Effect of the Invention

[0038] According to the present invention which solves the first problem, non-invasive prenatal parent-child testing can be carried out easily and with clear criteria, Criterion A or Criterion B, based on the genotyping results of both the putative father and the fetus.

[0039] Moreover, according to the present invention which solves the second problem, erroneous determination due to sample mix-up can be verified with high accuracy. [Brief description of the drawings]

[0040] [Figure 1] 2 shows a flowchart of an embodiment including a sample authentication process and a verification process. [Diagram 2] 1 shows a flowchart of an embodiment including an MC filter step, a three-sample authentication step, and a verification step. [Diagram 3] FIG. 1 is a schematic diagram of verification pattern 1 in which two-sample authentication is performed by referring to a database. [Figure 4] FIG. 13 is a schematic diagram of verification pattern 2 in which two-sample authentication is performed by referring to a database. [Diagram 5] FIG. 1 is a schematic diagram of verification pattern 1 in which three-sample authentication is performed by referring to a database. [Figure 6] FIG. 13 is a schematic diagram of verification pattern 2 in which three-sample authentication is performed by referring to a database. [Figure 7] Circulating cell-free nucleic acid samples were analyzed, and the signal intensities of alleles at each polymorphic locus were blotted. The vertical axis represents the ratio of the signal indicating the presence of a particular allele to the sum of the signal intensities derived from the particular polymorphic locus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] <1> Non-invasive prenatal paternity testing method The present invention, which solves the first problem, is a non-invasive prenatal paternity testing method using maternal blood (circulating cell-free nucleic acid sample). An embodiment of the non-invasive prenatal paternity testing method of the present invention will be described with appropriate reference to Figures 1 and 2. Note that the present invention is not limited to the embodiment described in Figures 1 and 2.

[0042] The present invention includes a two-sample authentication process and / or a three-sample authentication process as defined below.

[0043] <2 Sample authentication process> The two-sample identification step is a step of identifying the presence or absence of a biological parent-child relationship between the putative father and the fetus based on the results of genotyping the putative father and the fetus at multiple polymorphic loci. In the two-sample identification step, the fetus is genotyped according to the Criterion A determination method defined below.

[0044] (Standard a judgment method) derived from specific polymorphic loci contained in data obtained by analysis of circulating cell-free nucleic acid samples obtained from pregnant mothers, The allele detected with the strongest signal intensity is called allele A. The allele detected with the same or second strongest signal intensity as allele A is allele B. When the above expression is used, the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensities derived from the specific polymorphic locus are used as indicators, and the following criterion a is used.

[0045] <Standard a> If 0%≦Fb{≦,<}L%, the genotype of the fetus at the specific polymorphic locus is determined to be AA homozygous. If M%{≦,<}Fb{≦,<}N%, the genotype of the fetus at the specific polymorphic locus is determined to be heterozygous for AB. ·When Fa = Fb is satisfied, skip without performing genotyping of the specific polymorphic locus. ·When Fb does not satisfy any of the above conditions, skip without performing genotyping of the specific polymorphic locus.

[0046] N is a numerical value less than 50. The specific numerical values of L, M, and N can be appropriately set within the range that satisfies the relational expression 0 {≦, <} L < M {≦, <} N. Note that the notation {≦, <} indicates that either the inequality sign ≦ or < can be arbitrarily selected. For example, the lower limit value of L can be set to 0.1 or more. Also, the upper limit value of L can be set to less than 1, or 0.8 or less, or 0.5 or less. Also, the lower limit value of M can be set to 0.5 or more, or 0.8 or more, or 1 or more. The upper limit value of M can be set to 3 or less, or 2 or less.

[0047] From the perspective of preventing misidentification of the signal of the allele derived from the mother as the allele derived from the fetus, N is preferably set to 40 or less, more preferably 30 or less, and even more preferably 25 or less. Thereby, polymorphic loci with a high possibility of misidentifying the signal of the allele derived from the mother as the allele derived from the fetus can be excluded from the basis of paternity testing so that the possibility of occurrence of misjudgment can be suppressed.

[0048] Also, a condition of skipping without performing genotyping of the specific polymorphic locus when the noise derived from the specific polymorphic locus exceeds a certain standard may be added to the above-mentioned standard a. That is, when the alleles detected with the third and fourth strongest signal intensities, which are derived from a specific polymorphic locus and included in the data obtained by analyzing the circulating cell-free nucleic acid sample, are denoted as allele C and allele D, respectively, the ratio (Fc) of the signal intensity of allele C and the ratio (Fd) of the signal intensity of allele D to the total signal intensity derived from the specific polymorphic locus may be used as an index, and the following skip condition may be added to the above-mentioned standard a. If K{≦,<}Fc+Fd is satisfied, skip the genotyping of the specific polymorphic locus.

[0049] The value of K can be set arbitrarily, for example, to 10 or less, preferably 5 or less.

[0050] <3 Sample authentication process> The three-sample identification step is a step of identifying the presence or absence of a biological parent-child relationship between the putative father and the fetus based on the results of genotyping the biological mother, the putative father, and the fetus at multiple polymorphic loci. In the three-sample identification step, the fetus is genotyped according to the Criterion A determination method and / or the Criterion B determination method defined below.

[0051] (Standard b judgment method) from a particular polymorphic locus determined to be homozygous in the mother, contained in data obtained by analysis of the circulating cell-free nucleic acid sample; The allele detected with the strongest signal intensity is called allele A. The allele detected with the same or second strongest signal intensity as allele A is allele B. When the above expression is used, the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensities derived from the specific polymorphic locus are used as indicators, and the following criterion b is applied.

[0052] <Criteria b> If 0%≦Fb{≦,<}L%, the genotype of the fetus at the specific polymorphic locus is determined to be AA homozygous. If M%{≦,<}Fb is satisfied, the genotype of the fetus at the specific polymorphic locus is determined to be heterozygous for AB. If Fa=Fb, skip the genotyping of the specific polymorphic locus. If Fb does not meet any of the above conditions, skip without genotyping the specific polymorphic locus.

[0053] (satisfies the condition.) Specific numerical values of L and M can be appropriately set within the range that satisfies the relational expression 0{≦,<}L<M. Note that the notation {≦,<} indicates that either the inequality sign ≦ or < can be arbitrarily selected. For example, the lower limit value of L can be set to 0.1 or more, for example. Also, the upper limit value of L can be set to less than 1, or 0.8 or less, or 0.5 or less, for example. Also, the lower limit value of M can be set to 0.5 or more, or 0.8 or more, or 1 or more, for example. There is no limitation on the upper limit value of M.

[0054] Further, when the noise derived from a specific polymorphic locus exceeds a certain standard, the following condition of skipping without performing genotyping of the specific polymorphic locus may be added to the above-mentioned standard b. That is, when alleles detected with the third and fourth strongest signal intensities derived from a specific polymorphic locus included in the data obtained by analyzing a circulating cell-free nucleic acid sample are denoted as allele C and allele D, respectively, the following skip condition may be added to the above-mentioned standard b using, as indices, the ratio (Fc) of the signal intensity of allele C to the total signal intensity derived from the specific polymorphic locus and the ratio (Fd) of the signal intensity of allele D. ·Skip without performing genotyping of the specific polymorphic locus when K{≦,<}Fc + Fd is satisfied

[0055] The numerical value of K can be arbitrarily set, but can be an arbitrary numerical value of 10 or less, preferably 5 or less, for example.

[0056] In the present invention, the analysis means of the nucleic acid sample for genotyping is not particularly limited. Analytical methods capable of distinguishing and detecting single nucleotide substitutions (SNVs) at polymorphic loci can be preferably exemplified, and specifically, nucleotide sequence analysis, mass spectrometry, digital PCR, SNV microarray, real-time PCR, etc. can be mentioned. As a specific means of nucleotide sequence analysis, a next-generation sequencer (NGS) can be mentioned. Also, as the polymorphic locus to be analyzed, a single nucleotide polymorphic locus can be preferably exemplified.

[0057] Genotyping of the putative father, and also of the mother when a three-sample identification process is performed, can be easily performed by a conventional method, for example, by analyzing a sample commonly used in genetic testing, such as an oral mucosal cell sample taken from the oral cavity.

[0058] When the two-sample identification process is performed, the existence of a biological parent-child relationship between the fetus and the putative father is identified based on the genotyping results of the fetus determined by the above-mentioned method and the genotyping results of the putative father (Figure 1). In addition, when a three-sample identification process is performed, the existence of a biological parent-child relationship between the fetus and the pretended father is identified based on the genotyping results of the fetus determined by the above-mentioned method, the genotyping results of the pretended father, and the genotyping results of the fetus's biological mother (Figures 1 and 2).

[0059] The method of parentage testing in the two-sample identification process and the three-sample identification process is not particularly limited. In the case of performing genotyping of single nucleotide polymorphism loci, a paternity index (PI) can be calculated for each single nucleotide polymorphism locus based on the genotyping results of the putative father and the fetus. The method of calculating the paternity index is not particularly limited, and in the case of the two-sample identification process, it can be calculated according to, for example, Table 1 below, and in the case of the three-sample identification process, it can be calculated according to, for example, Table 1 or Table 2 below.

[0060] [Table 1]

[0061] [Table 2]

[0062] In Tables 1 and 2, an allele with a relatively high signal intensity in the analysis of a circulating cell-free nucleic acid sample is denoted as A, and an allele with a relatively low signal intensity is denoted as B. In Table 1, k0 is the false negative rate, kb is the false positive rate, a is the frequency of occurrence of A, and b is the frequency of occurrence of B. Note that k0 and kb can be set appropriately depending on the accuracy of the inspection system, and can be set to, for example, 0.1 or less, preferably 0.01 or less, and more preferably 0.001 or less.

[0063] After the PI is calculated for each single nucleotide polymorphism locus, a combined paternity index (CPI) may be calculated. The CPI is calculated as the sum of the PIs.

[0064] In the present invention, a preferred embodiment is one in which a judgment is made as to whether the result is "positive," "negative," or "undecidable" according to a predetermined criterion corresponding to the CPI value, for example as shown below. 10 4 ≦CPI Result: “affirmative” 10 -6 ≦CPI<10 4 ...Result: "Unable to judge" CPI<10 -6 ...Result: "Negative"

[0065] The above criteria are merely examples, and the specific CPI criteria for judgment can be set as appropriate.

[0066] After calculating the CPI, the Probability of Paternity (POP) may be calculated. POP can be calculated using the following formula. Note that P in the formula represents the prior probability of paternity.

number

[0067] In an embodiment including a 3-sample identification step, it is preferable to include an MC filter step defined below (FIG. 2).

[0068] <MC filter step> Based on a sample that contains nucleic acid including the genetic information of the mother collected from the mother and substantially does not contain nucleic acid including the genetic information of the fetus, perform genotyping of the mother, and when the following (i) and / or (ii) are observed to be equal to or more than a preset specified number, stop without performing paternity testing based on the collected sample. (i) A polymorphic locus that is a homozygote of an allele whose genotype in the mother and the fetus or cell-free circulating nucleic acid is different from each other (ii) A polymorphic locus in which the genotype of the mother is heterozygous and the genotype of the cell-free circulating nucleic acid is homozygous

[0069] The cell-free circulating nucleic acid sample mainly contains nucleic acid derived from the mother. Therefore, as a result of analyzing the cell-free circulating nucleic acid sample to perform genotyping of the fetus or cell-free circulating nucleic acid, it is usually considered that the genotypes of the mother and the fetus or cell-free circulating nucleic acid do not meet the conditions described in the above (i) and / or (ii). In such a case, mislabeling of the sample is suspected.

[0070] The MC filter step is performed to prevent incorrect determination of the identification result and wasted work by stopping the identification without performing the 3-sample identification step when such cases where mislabeling of the sample is suspected are observed to be equal to or more than a predetermined specified number (FIG. 2). The "preset specified number" can be arbitrarily set.

[0071] The specified number can be set to less than 5% of the total number of polymorphic loci observed in genotyping, preferably less than 3%, more preferably less than 1%.

[0072] ​The specific number of the specified number is not particularly limited, but can be set to 5 or less, more preferably 3 or less, and further preferably 1.

[0073] Genotyping of circulating cell-free nucleic acid to identify the genotype of the circulating cell-free nucleic acid will now be described with reference to FIG. Circulating cell-free nucleic acid samples contain primary nucleic acids derived from the mother and secondary nucleic acids derived from the fetus. Therefore, when circulating cell-free nucleic acid samples are analyzed and the ratio of signals indicating the presence of specific alleles to the total signal intensity derived from specific polymorphic loci is blotted, the ratios are distributed between 0-25%, 35-65%, and 75-100%, as shown in Figure 7. Assuming that there is no external contamination with DNA from other people, the above ratios at each polymorphic locus are treated as real signals when they are 5% or more.

[0074] In light of this, the present invention provides a method for determining whether a specific polymorphic locus is included in the data obtained by analyzing the circulating cell-free nucleic acid sample. The allele detected with the strongest signal intensity is called allele A. The allele detected with the same or second strongest signal intensity as allele A is allele B. When the above expression is used, the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the sum of the signal intensities derived from the specific polymorphic locus are used as indices, and genotyping of the circulating cell-free nucleic acid is performed according to the following criterion c.

[0075] <Criteria c> If X{≦,<}Fa{≦,<}Y%, the genotype at the specific polymorphic locus in the circulating cell-free nucleic acid is determined to be heterozygous for AB. If Z%{≦,<}Fa≦100 is satisfied, the genotype at the specific polymorphic locus in the cell-free circulating nucleic acid is determined to be homozygous AA. When Fa=Fb is satisfied, the genotype at the specific polymorphic locus is determined to be heterozygous for AB.

[0076] The specific numerical values of X, Y, and Z can be appropriately set within the range that satisfies the relational expression of X {≦, <} Y < Z. Note that the notation {≦, <} means that either the inequality sign ≦ or < can be arbitrarily selected. This indicates.

[0077] In addition, when the ratio does not correspond to any of the above, it may be an embodiment to skip without performing the genotyping of the specific allele.

[0078] The numerical value of X can preferably be set to any value of 40 or more, more preferably 45 or more. The numerical value of Y can preferably be set to any value of 65 or less, more preferably 60 or less. The numerical value of Z can preferably be set to any value of 75 or more, more preferably 95 or more.

[0079] In one embodiment of the present invention, after performing the above-mentioned "genotyping of the mother based on a sample containing nucleic acid containing the genetic information of the mother collected from the mother and substantially not containing nucleic acid containing the genetic information of the fetus" and "genotyping of cell-free circulating nucleic acid based on a cell-free circulating nucleic acid sample collected from the mother", an MC filter step is executed.

[0080] The stop condition employed in the MC filter step may be either one or both of the above-mentioned (i) and (ii).

[0081] Even when performing the three-sample identification step, the implementation of the MC filter step is not essential. The MC filter step is an optional step and may or may not be performed.

[0082] In one embodiment of the present invention, when no stop determination is made in the MC filter step, the identification based on the collected sample is continued (Figure 2). On the other hand, when a stop determination is made in the MC filter step, it stops without performing the three-sample identification step based on the collected sample (Figure 2).

[0083] In normal legal cases, a witness is required to be present when all samples are collected. However, when the MC filter process is implemented, it is possible to determine whether the maternal nucleic acid sample and the circulating cell-free nucleic acid sample are mixed up, so there is no need to be present when the maternal blood sample is collected, which is necessary for legal testing. This has the advantage that legal testing can be performed with only the presence of the mother's genomic DNA sample.

[0084] The present invention may be implemented in a form including either one of a two-sample authentication process and a three-sample authentication process, or both. In the case of a form including both a two-sample authentication process and a three-sample authentication process, the authentication result of either one of the two may be output as the final authentication result, or a process of comparing the authentication results from these two authentication processes may be included. Then, an embodiment may be implemented in which the final authentication result is output according to a predetermined criterion based on the pattern of agreement or disagreement between the two authentication results.

[0085] The above-mentioned predetermined criteria can be set arbitrarily. For example, the criteria shown in the following Table 3 according to combination patterns 1 to 9 of the verification results by the two-sample verification process and the three-sample verification process may be adopted.

[0086] [Table 3]

[0087] The non-invasive prenatal paternity testing method of the present invention may further include a verification step for verifying erroneous determination due to mix-up of samples (FIGS. 1 and 2).

[0088] 1 and 2 show an embodiment in which a verification step is performed regardless of the result of the two-sample authentication step or the three-sample step. In such an embodiment, it is possible to verify whether the result of denying or affirming the biological parent-child relationship by the two-sample authentication step or the three-sample step includes any erroneous determination due to mixing up samples. If it is determined that a mix-up of samples occurred in the verification process, it can be determined that the results of the two-sample authentication process or the three-sample process are unreliable.

[0089] 1 and 2 are merely one embodiment, and an embodiment in which the verification step is performed depending on the results of the two-sample authentication step or the three-sample step may also be used.

[0090] When two or more workers perform inspections independently, or when one worker performs multiple inspections at different locations and times, the possibility of the same sample being mixed up in all inspections is extremely low. Under such a double-check system, the verification step may be carried out if a negative determination result is obtained in each test. Conversely, under the above double-check system, if a "positive" judgment result is obtained in each test, the judgment result may be regarded as correct and the verification step may not be carried out. Of course, a triple-check system may be established by carrying out an additional verification step.

[0091] A specific embodiment of the verification step will be described in detail in the following description of the verification method invention.

[0092] <2> How to Test The present invention is a method for verifying erroneous determinations in blood relationship testing due to sample mix-ups based on samples collected from a set of specific test subjects.

[0093] The verification method of the present invention can be applied to methods for verifying biological parent-child relationships, biological sibling relationships, relationships between biological grandparents and grandchildren, and any other biological blood relationships.

[0094] The subject family as used herein refers to a group including at least a specific person to be examined for the presence or absence of a biological blood relationship and his / her blood relatives. The subject family may optionally include one or more biological blood relatives of the specific person. In other words, the family to be verified may include only two members, a pseudo-blood relative and a specific person, or may include three members, a pseudo-blood relative, a specific person, and a biological blood relative of the specific person. In other words, the verification method of the present invention is applicable to both the two-sample verification process and the three-sample verification process described above.

[0095] For example, in the case of biological sibling relationships, a specific person and his / her pseudo-siblings constitute the family to be verified. In this case, the family to be verified may optionally include biological relatives such as the specific person's biological parents and siblings. In the case of the appraisal of the relationship between biological grandparents and grandchildren, the family to be appraised is composed of the biological grandfather or grandmother and the grandchild. In this case, the family to be appraised may optionally include biological blood relatives such as the biological parents of the grandchild or a biological grandmother or grandfather of a different sex from the biological blood relatives, the biological grandfather or grandmother.

[0096] In addition, when parentage testing is performed, the family to be tested includes a pseudo parent and a child to be tested for the presence or absence of a biological parent-child relationship, and may optionally include a biological parent of the child who is of a different sex from the pseudo parent. In other words, the family to be verified may include only two people, the pseudo parent and the child, or it may include three people, the pseudo parent, the child, and the biological parent. In other words, the verification method of the present invention is applicable to both the two-sample verification process and the three-sample verification process described above.

[0097] "The biological parent of the child, who is of a different sex from the imitation parent," is the child's biological parent who is the imitation father. In this case, the biological mother of the child is the biological mother of the child, and in this case, the pseudo-parent is the biological father of the child. In other words, the verification method of the present invention is applicable to both paternity testing and maternity testing.

[0098] The following description focuses on an embodiment in which the verification method of the present invention is applied to a parent-child test including a "child," a "false father," and optionally a "biological mother" as family members to be tested. It should be noted that the present invention is not limited to the parent-child test described below, and can be applied to the verification of any kind of blood relationship test.

[0099] The verification method of the present invention can also be applied to parentage testing carried out between a post-born child and a putative parent, and can also be naturally applied to parentage testing carried out between a pre-born child (fetus) and a putative parent (putative father), such as the non-invasive pre-natal parentage testing method of the present invention described above.

[0100] When the verification method of the present invention is applied to paternity testing, the specific form of the paternity testing is not limited. For example, PI calculation may be performed based on the above Table 1 or Table 2. In addition, in the case of a child after birth, the "fetus" in the above Table 1 or Table 2 may be read as "child after birth" and applied mutatis mutandis. Furthermore, CPI may be calculated based on the calculated PI, and POP may be obtained based on the CPI.

[0101] When the verification method of the present invention is applied to a paternity test, the specific form of the paternity test is not limited. For example, a maternity index (MI) can be calculated according to the PI calculation in a paternity test. Furthermore, a combined maternity index (CMI) may be calculated according to the CPI calculation method based on the calculated MI. Furthermore, a probabilistic probability of maternity (POM) may be calculated according to the POP calculation method based on the calculated CMI.

[0102] Naturally, the verification method of the present invention can also be applied to paternity testing that does not use the PI or MI calculation formulas shown in Table 1 or Table 2. For example, in addition to methods that use the calculation formulas in Table 1 or Table 2, the verification method of the present invention can also be applied to methods that utilize a likelihood ratio that represents the probability of a parent-child relationship.

[0103] When genotyping of a pseudo-family member and genotyping of a specific individual are performed by a single operator at the same place and at the same time, there is a possibility that the samples of the entire family to be identified, rather than just a portion of the family to be identified, may be mixed up. If a sample mix-up occurs for each family to be identified in this way, it may not be possible to definitively detect the occurrence of a sample mix-up in the verification process described below.

[0104] To address this issue, in a preferred embodiment of the verification method of the present invention, the genotyping of the kin and the genotyping of the specific individual are performed at different locations and / or at different times and / or by different operators. In the case of parentage testing, genotyping of the putative parent and genotyping of the child may be performed at different locations and / or at different times and / or by different operators.

[0105] In this way, when genotyping of the similes and the specific individual is performed at different places, at different times, and by different operators, the possibility of the same sample being mixed up in each genotyping is reduced. By implementing such a genotyping embodiment and then performing the verification step described below, it is possible to obtain the extremely advantageous effect of significantly improving the verification accuracy.

[0106] The verification method of the present invention is characterized by including a verification step defined below.

[0107] <Verification process> In order to verify erroneous determination due to misidentification of a sample taken from a family to be identified other than the specific family to be identified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below.

[0108] (Verification pattern 1) The genotyping results of the child belonging to the particular subject family; and Optionally, the genotyping results of the biological parents belonging to the particular subject family; and The genotyping results of a pseudo-parent belonging to the other family to be verified; Based on this, parentage testing is performed for this combination of pretend parent and child. (Verification pattern 2) The genotyping results of a child belonging to the other subject family; and Optionally, the genotyping results of the child's biological parents who belong to the other subject family; The genotyping results of the pseudo-parents belonging to the specific subject family; and Based on this, parentage testing is performed for this combination of pretend parent and child.

[0109] Verification pattern 1 is a pattern in which parent-child testing is performed for a combination of a child belonging to a specific family to be verified and a pseudo-parent belonging to another family to be verified. The parentage testing carried out here may be a two-sample test or a three-sample test.

[0110] In the two-sample testing in verification pattern 1, parent-child testing is performed based on the genotyping results of two individuals, a child belonging to a specific family to be tested and a putative parent belonging to another family to be tested. The specific aspects of this method can be applied as is to the above description of the two-sample testing process.

[0111] In the three-sample testing in verification pattern 1, parent-child testing is performed based on the genotyping results of three people: a child who belongs to a specific family to be tested, the child's biological parent who belongs to the specific family to be tested, and a pseudo-parent who belongs to another family to be tested. The specific aspects of this method can be applied directly to the explanation of the three-sample testing process described above.

[0112] Verification pattern 2 is a pattern in which parent-child testing is performed for a combination of a child belonging to another family to be verified and a pseudo-parent belonging to a specific family to be verified. The parentage testing carried out here may be a two-sample test or a three-sample test.

[0113] In the two-sample testing in verification pattern 2, parent-child testing is performed based on the genotyping results of two individuals, a child belonging to another family to be tested and a putative parent belonging to a specific family to be tested. The specific aspects of this method can be applied as is to the above description of the two-sample testing process.

[0114] In the three-sample testing in verification pattern 2, parent-child testing is performed based on the genotyping results of three people: a child belonging to another family to be tested, the child's biological parent belonging to another family to be tested, and a pseudo-parent belonging to a specific family to be tested. The specific embodiment can be directly applied to the above description of the three-sample testing process.

[0115] In addition, if the parentage testing to be verified for sample mix-up is a two-sample testing, the parentage testing performed in the verification process is not limited to a two-sample testing, and there is no problem in conducting a three-sample testing in the verification process.

[0116] On the other hand, if the parentage testing to be verified to determine whether or not there was a mix-up in samples is a three-sample testing, the parentage testing performed in the verification process is not limited to a three-sample testing, and there is no problem in conducting a two-sample testing in the verification process.

[0117] In the case where a test result that confirms a biological parent-child relationship is obtained in Verification Pattern 1 and / or Verification Pattern 2, it can be determined that there is a high possibility that a sample from a specific family to be tested has been mixed up with a sample from another family to be tested. In other words, there is a high possibility that a sample from a subject who belongs to a specific family to be tested and who has a biological parent-child relationship has been treated as belonging to a different family to be tested, and it can be determined that the test result of the parent-child test that is the subject of the verification contains a misjudgment.

[0118] In addition, if the "negative" result of the paternity test being verified is the correct result without any mix-up of samples, it can be confirmed that there was no error in the result in the verification process. Furthermore, even if the "negative" result of the paternity test being verified is due to a mix-up of samples, if the verification process does not obtain a result that affirms the biological parent-child relationship, the result of the paternity test being verified will be "negative." In other words, if the parent-child testing being verified produces results that deny the biological parent-child relationship, and if the verification process does not produce results that affirm the biological parent-child relationship, the existence of a biological parent-child relationship between the putative parent and child being verified can be denied.

[0119] If, through the verification process, it is determined that the results of the parent-child testing being verified contain a misjudgment due to mixing up a sample taken from a specific family to be tested with a sample taken from a different family to be tested, a final conclusion of "inconclusive" may be reached, or a sample may be re-obtained from a subject belonging to the specific family to be tested and a re-test may be conducted.

[0120] Next, an embodiment in which the verification step is performed by referring to a database in which the genotyping results of another family to be identified are stored will be described with reference to FIGS.

[0121] First, an embodiment for executing the verification pattern 1 is shown in Fig. 3. In this embodiment, parent-child tests (paternity tests) performed on two target families, a specific target family Y including two members, a child CY and a putative father AFY, and a specific target family Z including two members, a child CZ and a putative father AFZ, are the targets of verification in the verification process.

[0122] Naturally, the verification process may be performed on a single family. Also, as shown in FIG. 3, the verification process may be performed on two or more families.

[0123] The database pre-stores the genotyping results of offspring CA to CX and putative fathers AFA to AFX, who belong to multiple sets of different target families A to X, which are different from the specific target families Y and Z (Figure 3). In addition, after genotyping of child CY and putative father AY belonging to family Y to be examined, and child CZ and putative father AZ belonging to family Z to be examined, is completed, the database is updated before the verification process is performed, and the genotyping results of child CY and putative father AFY, and child CZ and putative father AFZ are stored in the database (Figure 3). That is, the verification step is carried out by referring to a database in which the genotyping results of the offspring CA to CZ and the putative fathers AFA to AFZ are stored.

[0124] 3, parentage testing is performed based on the genotyping results of child CY and the genotyping results of pseudo-fathers AFA to AFZ stored in the database. That is, parentage testing is performed for each of the combinations of child CY and pseudo-father AFA, child CY and pseudo-father AFB, child CY and pseudo-father AFX, child CY and pseudo-father AFY, and child CY and pseudo-father AFZ. In addition, in the embodiment of Figure 3, parent-child testing is similarly performed independently based on the genotyping results of the child CZ and the genotyping results of the putative fathers AFA to AFZ stored in the database.

[0125] In the following, to avoid duplication, we will focus on cases where parentage testing is performed based on the genotyping results of the child CZ and the putative father AFA-AFZ separately. Needless to say, the following explanation also applies when parentage testing is performed based on the genotyping results of the child CY and the putative father AFA-AFZ separately.

[0126] If the existence of a biological parent-child relationship between child CZ and any of the putative fathers AFA-AFY is confirmed, it can be determined that the combination of samples from child CZ and putative father AFZ, which had been treated as having been obtained from a specific set of family Z being tested, was mistaken for a sample obtained from a member of another family being tested. In other words, it can be determined that the results of the paternity test performed by analyzing samples from child CZ and putative father AFZ belonging to the subject family Z contain an error.

[0127] Furthermore, if a "positive" verdict is obtained in the paternity test that is the subject of the verification, the verification of the putative father AFZ stored in the database through the update should also give a "positive" result. On the other hand, unless there are special circumstances such as the putative father AFZ having been registered in the database as one of the putative fathers AFA-AFY because he was the subject of a paternity test in another case, the verification of the putative fathers AFA-AFY stored in the database should give a "negative" result. In other words, if a ``positive'' verdict is obtained in the parent-child test that is the subject of verification, the putative father AFZ stored in the database by update in the verification process will act as a positive control, while the putative fathers AFA to AFY will act as negative controls unless there are special circumstances as mentioned above, so it can be confirmed that the verification process itself is being carried out normally.

[0128] Conversely, if a “negative” verdict is obtained in the paternity test that is the subject of the verification, the verification of the putative father AFZ stored in the database through the update should also yield a “negative” result. In other words, in the verification process, the pseudo-father AFZ stored in the database by updating serves as a negative control, so that it is possible to confirm that the verification process itself is being carried out normally.

[0129] 3 shows an embodiment in which the database is updated with the genotyping results of child CY and putative father AFY belonging to family Y to be examined, and child CZ and putative father AFZ belonging to family Z to be examined, prior to verification. However, it goes without saying that valid verification can be performed even if the database is not updated and a database in which only the genotyping results of children CA-CX and putative fathers AFA-AFX are stored in advance is used.

[0130] Next, an embodiment for executing the verification pattern 2 is shown in FIG.

[0131] The database configuration of the embodiment in Fig. 4 is the same as that in Fig. 3. In the embodiment in Fig. 4, parentage testing is performed based on the genotyping results of the pseudo-father AFY and the genotyping results of the children CA to CZ stored in the database. That is, parentage testing is performed for each of the combinations of pseudo-father AFY and child CA, pseudo-father AFY and child CB, ... pseudo-father AFY and child CX, pseudo-father AFY and child CY, and pseudo-father AFY and child CZ. In addition, in the embodiment of FIG. 4, parent-child testing is similarly performed independently based on the genotyping results of the putative father AFZ and the genotyping results of the children CA to CZ stored in the database.

[0132] In the following, to avoid duplication, we will focus on cases where parentage testing is performed based on the genotyping results of the putative father AFZ and the children CA to CZ. Needless to say, the following explanation also applies when parentage testing is performed based on the genotyping results of the putative father AFY and the children CA to CZ.

[0133] If the existence of a biological parent-child relationship between the putative father AFZ and any of the children CA through CY is confirmed, it can be determined that the combination of samples from child CZ and putative father AFZ, which had been treated as having been obtained from a specific subject family Z, was mistaken for a sample obtained from a member of another subject family. In other words, it can be determined that the results of the paternity test performed by analyzing samples from child CZ and putative father AFZ belonging to the subject family Z contain an error.

[0134] Furthermore, if a "positive" verdict is obtained in the parentage test that is the subject of the verification, the verification of the child CZ stored in the database through the update should also give a "positive" result. On the other hand, unless there are special circumstances such as the child CZ having been registered in the database as one of the children CA-CY because it was the subject of a parentage test in another case, the verification of the children CA-CY stored in the database should give a "negative" result. In other words, if a ``positive'' verdict is obtained in the parent-child test that is the subject of verification, then in the verification process, child CZ stored in the database by update acts as a positive control, while children CA to CY act as negative controls unless there are special circumstances as mentioned above, so it can be confirmed that the verification process itself is being carried out normally.

[0135] Conversely, if a “negative” verdict is obtained in the parent-child test that is the subject of the verification, a “negative” verdict should also be obtained in the verification of the child CZ stored in the database through an update. In other words, in the verification process, the child CZ stored in the database by updating acts as a negative control, so that it is possible to confirm that the verification process itself is being executed normally.

[0136] Next, an embodiment for executing the verification pattern 1 is shown in Fig. 5. In this embodiment, the parent-child tests (paternity tests) performed on two target families, a specific target family Y including three members, a child CY, the child CY's biological mother MY, and the pretended father AFY, and a specific target family Z including three members, a child CZ, the child CZ's biological mother MZ, and the pretended father AFZ, are the targets of verification in the verification process.

[0137] Naturally, the verification process may be performed on a single family. Also, as shown in FIG. 5, the verification process may be performed on two or more families.

[0138] The database pre-stores the genotyping results of offspring CA-CX, their respective biological mothers MA-MX, and putative fathers AFA-AFX, who belong to multiple pairs of families A-X that are different from the specific families Y and Z that are being examined (Figure 5). In addition, after genotyping is completed for child CY, putative father AFY, and mother MZ belonging to family Y to be examined, as well as child CZ, putative father AFZ, and mother MZ belonging to family Z to be examined, the database is updated before the verification process is performed, and the genotyping results for child CY, putative father AFY, and mother MY, as well as child CZ, putative father AFZ, and mother MZ are stored in the database (Figure 5). That is, the verification step is carried out by referring to a database in which the genotyping results of the offspring CA to CZ, putative fathers AFA to AFZ, and mothers MA to MZ are stored.

[0139] 5, parentage testing is performed based on the genotyping results of child CY and mother MY and the genotyping results of pseudo-fathers AFA to AFZ stored in the database. That is, parentage testing (three-sample testing) is performed for each of the combinations of child CY, mother MY, and pseudo-father AFA; child CY, mother MY, and pseudo-father AFB; child CY, mother MY, and pseudo-father AFX; child CY, mother MY, and pseudo-father AFY; child CY, mother MY, and pseudo-father AFZ. In addition, in the embodiment of Figure 5, parentage testing is similarly performed independently based on the genotyping results of the child CZ and mother MZ, and the genotyping results of the putative fathers AFA to AFZ stored in the database.

[0140] In the following, to avoid duplication, the explanation will be limited to cases where parentage testing is performed based on the genotyping results of the child CZ, the mother MZ, and the putative father AFA-AFZ. Needless to say, the following explanation also applies when parentage testing is performed based on the genotyping results of the child CY, the mother MY, and the putative father AFA-AFZ.

[0141] If the existence of a biological parent-child relationship between child CZ and any of the putative fathers AFA-AFY is confirmed, it can be determined that the combination of samples from child CZ, mother MZ, and putative father AFZ, which were treated as being obtained from a specific set of family Z to be tested, was mistaken for a sample obtained from a member of another family to be tested. In other words, it can be determined that the results of the paternity test performed by analyzing samples from child CZ, mother MZ, and putative father AFZ belonging to the subject family Z contain an error. In addition, if the above-mentioned MC filter process has been passed, there is no need to take into consideration the mix-up of samples of the child CZ and the mother MZ.

[0142] Furthermore, if a "positive" verdict is obtained in the paternity test that is the subject of the verification, the verification of the putative father AFZ stored in the database through the update should also give a "positive" result. On the other hand, unless there are special circumstances such as a person (including ascendants and descendants) who has a biological parent-child relationship with child CZ being registered in the database as one of the putative fathers AFA-AFY because he was the subject of a paternity test in another case, the verification of the putative fathers AFA-AFY stored in the database should give a "negative" result. In other words, if a ``positive'' verdict is obtained in the parent-child test that is the subject of verification, the putative father AFZ stored in the database by update in the verification process will act as a positive control, while the putative fathers AFA to AFY will act as negative controls unless there are special circumstances as mentioned above, so it can be confirmed that the verification process itself is being carried out normally.

[0143] Conversely, if the paternity test being verified yields a negative verdict, the verification of the putative father AFZ stored in the database through the update should also yield a negative verdict. In other words, in the verification process, the pseudo-father AFZ stored in the database by updating serves as a negative control, so that it is possible to confirm that the verification process itself is being carried out normally.

[0144] Next, an embodiment for executing the verification pattern 2 is shown in Fig. 6. In this embodiment, the configurations of the families Y and Z to be verified and the data configuration stored in the database are the same as those in the embodiment in Fig. 5.

[0145] 6, parentage testing is performed based on the genotyping results of pseudo-father AFY and the genotyping results of offspring CA-CZ and mothers MA-MZ stored in the database. That is, parentage testing (three-sample testing) is performed for each of the combinations of pseudo-father AFY, offspring CA, and mother MA; pseudo-father AFY, offspring CB, and mother MB, ... pseudo-father AFY, offspring CX, and mother MX; pseudo-father AFY, offspring CY, and mother MY; pseudo-father AFY, offspring CZ, and mother MZ. In addition, in the embodiment of Figure 6, parentage testing is similarly performed independently based on the genotyping results of the putative father AFZ and the genotyping results of the children CA to CZ and the mothers MA to MZ stored in the database.

[0146] In the following, to avoid duplication, the explanation will be limited to the case where parentage testing is performed based on the genotyping results of the putative father AFZ, the offspring CA-CZ, and the mother MA-MZ. Needless to say, the following explanation also applies to the case where parentage testing is performed based on the genotyping results of the putative father AFY, the offspring CA-CZ, and the mother MA-MZ.

[0147] If the existence of a biological parent-child relationship between the putative father AFZ and any of the children CA through CY is confirmed, it can be determined that the combination of samples from child CZ, mother MZ, and putative father AFZ, which were treated as having been obtained from a specific set of family Z to be tested, was mistaken for a sample obtained from a member of another family to be tested. In other words, it can be determined that the results of the paternity test performed by analyzing samples from child CZ, mother MZ, and putative father AFZ belonging to the subject family Z contain an error. In addition, if the above-mentioned MC filter process has been passed, there is no need to take into consideration the mix-up of samples of the child CZ and the mother MZ.

[0148] Furthermore, if a "positive" verdict is obtained in the paternity test that is the subject of the verification, the verification of child CZ and mother MZ stored in the database through the update should also give a "positive" result. On the other hand, unless there are special circumstances such as child CZ and mother MZ having been registered in the database as children CA-CY and mothers MA-MY because they were the subject of a paternity test in another case, the verification of children CA-CY and mothers MA-MY stored in the database should give a "negative" result. In other words, if a ``positive'' verdict is obtained in the parent-child testing that is the subject of verification, then in the verification process, child CZ and mother MZ stored in the database by update act as positive controls, while children CA-CY and mothers MA-MY act as negative controls unless there are special circumstances as mentioned above, so it can be confirmed that the verification process itself is being carried out normally.

[0149] Conversely, if a “negative” verdict is obtained in the parent-child test that is the subject of the verification, a “negative” verdict should also be obtained in the verification of the child CZ and mother MZ that have been stored in the database through the update. In other words, in the verification process, the child CZ and mother MZ stored in the database by updating act as negative controls, so that it is possible to confirm that the verification process itself is being carried out normally.

[0150] Next, the effectiveness of the verification common to the embodiments shown in FIGS. 3 to 6 will be described. 3 to 6, the subject of verification is parent-child testing for families Y and Z to be tested. Here, it is assumed that a mix-up of samples has occurred between families Y and Z to be tested. In this case, the following four cases are possible as the results of paternity testing for each of the subject families X and Y.

[0151] Case 1: Paternity tests conducted on both families Y and Z confirmed the biological parent-child relationship between the child and the putative father. Case 2: A paternity test conducted on family Y obtained results that confirmed the biological parent-child relationship, while a paternity test conducted on family Z obtained results that denied the biological parent-child relationship. Case 3: A paternity test conducted on family Y denied the biological parent-child relationship, while a paternity test conducted on family Z confirmed the biological parent-child relationship. Case 4: Paternity tests conducted on both families Y and Z denied the biological parent-child relationship.

[0152] In the above-mentioned cases 1 to 3, the occurrence of a sample mix-up can be discovered by going through a verification process. However, in the case of Case 4, if there is in fact no biological parent-child relationship between child CY and putative father AFY, and between child CZ and putative father AFZ, the fact that samples were mixed up cannot be discovered even after the verification process. However, in this case, although there is an error in the DNA profile of the subject due to the mix-up of samples, the final result of the determination denying the biological parent-child relationship remains unchanged.

[0153] The embodiment shown in Figures 3 to 6 is an embodiment related to paternity testing. However, the present invention is not limited to paternity testing, and can naturally be applied to mother-child testing as well. When applied to mother-child testing, the "pseudo father" in the embodiment shown in Figures 3 to 6 can be replaced with the "pseudo mother." Also, in the embodiment shown in Figures 5 and 6, the "biological mother" can be replaced with the "biological father."

[0154] The verification method of the present invention can be applied to both parentage testing carried out before the child is born and parentage testing carried out after the child is born. When the parentage testing to which the verification method of the present invention is applied is carried out before the birth of the child, an MC filter step may be included as described above. In addition, even when the parent-child test to which the verification method of the present invention is applied is carried out after the birth of the child, a filter similar to the MC filter described above can be adopted. Below, two cases, paternity testing and mother-child testing, will be described respectively.

[0155] In a paternity test performed by analyzing samples taken from three people after birth: the child, the child's biological mother, and the putative father, it is preferable to include a post-natal MC filter step as defined below.

[0156] <postnatal MCフィルター> Genotyping the child based on a sample containing nucleic acid comprising genetic information of the child collected from the child after birth; Genotyping the biological mother of the child based on a sample containing nucleic acid comprising genetic information of the mother, the sample being collected from the biological mother of the child; If the following (i) is observed more than a predetermined number of times, the paternity test based on the collected sample is not performed and the process is stopped. (i) a polymorphic locus in which the mother and the child have different genotypes and are homozygous for an allele different from each other;

[0157] The postnatal MC filter eliminates the need for a witness to be present when a sample is taken from the biological mother, even in legal cases.

[0158] In addition, in paternity testing (maternity testing) that is conducted by analyzing samples taken from three people after birth: the child, the child's biological father, and the adoptive mother, the following post natal It is preferred to include a FC filter step.

[0159] <postnatal FCフィルター> Genotyping the child based on a sample containing nucleic acid comprising genetic information of the child collected from the child after birth; Genotyping the father of the child based on a sample containing nucleic acid comprising genetic information of the father taken from the biological father of the child; If the following (i) is observed more than a predetermined number of times, the paternity test based on the collected sample is not performed and the process is stopped. (i) a polymorphic locus in which the genotypes of the father and the child are different from each other in the homozygote allele;

[0160] The postnatal MC filter eliminates the need for a witness to be present when a sample is taken from the biological mother, even in legal cases.

[0161] As described above, the verification method of the present invention is not limited to parent-child testing, but can be applied to the verification of any kind of blood relationship testing. Depending on the composition of the family to be verified in the blood relationship testing to be verified, the "pseudo-father" and "child" in Figures 3 and 4 can be replaced with any "pseudo-relative" and "specific person," respectively. In addition, depending on the composition of the family in the blood relationship testing being verified, the "pseudo father," "child," and "biological mother" in Figures 5 and 6 can be replaced with any "pseudo blood relative," "specific person," and "biological blood relative," respectively.

[0162] <3> Specific embodiments of parentage testing The parent-child testing method employing the detection method of the present invention will be described with reference to FIGS.

[0163] The embodiment shown in Fig. 1 includes a two-sample identification process based on two samples obtained from a specific family to be identified, including the putative father and the child. In this embodiment, first, the two samples of the putative father and the child are analyzed to obtain the genotyping results of the putative father and the child.

[0164] The database to be referred to in the verification process stores the genotyping results of another family to be identified in advance. In addition, this embodiment includes storing the results of genotyping of two members of a specific family to be identified in the database after the genotyping is completed, and updating the database (FIG. 1). If there are two or more families to be verified in the verification process, the database may be updated all at once by registering them at once. In this case, the updated database will contain the genotyping results of the children CA to CZ and the putative fathers AFA to AFZ, as shown in Figures 3 to 6.

[0165] After genotyping of two members of a particular family to be identified is completed, a two-sample identification process is performed based on the results (FIG. 1). In this embodiment, a verification process is performed regardless of the results of the two-sample identification process.

[0166] When the verification step is executed, the updated database is referenced and verification is performed according to verification pattern 1 and / or verification pattern 2. If we explain this in detail according to an embodiment in which there are two authenticated families to be verified, as shown in Figures 3 to 6, in the verification process, verification is performed according to the following verification pattern 1-1 and / or verification pattern 2-1, and verification pattern 1-2 and / or verification pattern 2-2.

[0167] (Verification pattern 1-1) Genotyping results of child CY and The genotyping results for each of the putative fathers AFA~AFZ stored in the updated database, Based on this, parentage testing will be conducted for each combination of child CY and putative fathers AFA to AFZ.

[0168] (Verification pattern 2-1) Genotyping results of putative father AFY and The genotyping results for each of the children CA~CZ stored in the updated database, Based on this, parentage testing will be conducted for each combination of putative father AFY and children CA~CZ.

[0169] (Verification pattern 1-2) Genotyping results of child CZ and The genotyping results for each of the putative fathers AFA~AFZ stored in the updated database, Based on this, parentage testing will be performed for each combination of child CZ and putative father AFA~AFZ.

[0170] (Verification pattern 2-2) Genotyping results of the pseudofather AFZ and The genotyping results for each of the children CA~CZ stored in the updated database, Based on this, parentage testing will be performed for each combination of putative father AFZ and children CA~CZ.

[0171] In other words, in this embodiment, the genotyping results of the specific family to be identified reflected in the updated database are regarded as the genotyping results of another family to be identified, and verification is performed according to verification pattern 1 and / or verification pattern 2.

[0172] In the following, in order to avoid duplication of explanation, the following description will be given based on an embodiment in which verification patterns 1-2 and / or 2-2 are executed. Note that the following description also naturally applies to an embodiment in which verification patterns 1-1 and / or 2-1 are executed.

[0173] If the biological parent-child relationship is confirmed by the two-sample testing process, the verification process should produce positive results in paternity testing based on the genotyping results of the child CZ and the putative father AFZ stored in the updated database (verification pattern 1-2), and in paternity testing based on the genotyping results of the putative father AFZ and the child CZ stored in the updated database (verification pattern 2-2).

[0174] Furthermore, if the biological parent-child relationship is denied by the two-sample testing process, the verification process should produce a ``negative'' result in a paternity test based on the genotyping results of the child CZ and the putative father AFZ stored in the updated database (verification pattern 1-2), and in a paternity test based on the genotyping results of the putative father AFZ and the child CZ stored in the updated database (verification pattern 2-2).

[0175] In other words, the updated database will always contain positive controls that will produce a "positive" result or negative controls that will produce a "negative" result, making it possible to check whether or not any errors have occurred in the verification process itself.

[0176] The embodiment shown in Figure 2 includes a three-sample identification process based on three samples obtained from a specific family to be identified, including a putative father, a child, and the child's biological mother. This embodiment also includes an MC filter process (Figure 2), and after genotyping of the three members of the specific family to be identified is completed, the results are stored in a database and updated (Figure 2). If there are two or more families to be verified in the verification process, the database may be updated all at once by registering them at once. In this case, the updated database will contain the genotyping results of the children CA to CZ and the putative fathers AFA to AFZ, as shown in Figures 3 to 6.

[0177] In this embodiment, the updated database is referenced, and verification is performed according to verification pattern 1 and / or verification pattern 2 in the verification process. If we explain this in detail according to an embodiment in which there are two authenticated families to be verified, as shown in Figures 3 to 6, in the verification process, verification is performed according to the following verification pattern 1-1 and / or verification pattern 2-1, and verification pattern 1-2 and / or verification pattern 2-2.

[0178] (Verification pattern 1-1) Genotyping results of child CY and Optionally, maternal MY genotyping results and The genotyping results for each of the putative fathers AFA~AFZ stored in the updated database, Based on this, parentage testing will be conducted for each combination of child CY and putative fathers AFA to AFZ.

[0179] (Verification pattern 2-1) Genotyping results of putative father AFY and Optionally, the genotyping results for each of the mothers MA to MZ stored in the updated database, The genotyping results for each of the children CA~CZ stored in the updated database, Based on this, parentage testing will be conducted for each combination of putative father AFY and children CA~CZ.

[0180] (Verification pattern 1-2) Genotyping results of child CZ and Optionally, maternal MZ genotyping results and The genotyping results for each of the putative fathers AFA~AFZ stored in the updated database, Based on this, parentage testing will be performed for each combination of child CZ and putative father AFA~AFZ.

[0181] (Verification pattern 2-2) Genotyping results of the pseudofather AFZ and Optionally, the genotyping results for each of the mothers MA to MZ stored in the updated database, The genotyping results for each of the children CA~CZ stored in the updated database, Based on this, parentage testing will be performed for each combination of putative father AFZ and children CA~CZ.

[0182] As in the embodiment of Figure 1, in the embodiment of Figure 2, the genotyping results of the specific family to be identified reflected in the updated database are treated as the genotyping results of another family to be identified, and verification is performed according to verification pattern 1 and / or verification pattern 2.

[0183] In the embodiment of FIG. 2, like the embodiment of FIG. 1, the updated database will always contain a positive control that will give a "positive" result or a negative control that will give a "negative" result if there is a corresponding authentication family with a positive result, so it is possible to check whether or not an error has occurred in the verification process itself. [Industrial Applicability]

[0184] The present invention can be applied to parentage testing.

Claims

1. A method for verifying erroneous determination due to sample mix-up in blood relationship testing between a specific person and a potential blood relative, based on samples taken from a set of specific subject families including a potential blood relative of the specific person who is the subject of the biological blood relationship testing, the specific person, and optionally a biological blood relative of the specific person, the method comprising the steps of: <Verification process> In order to verify erroneous determination due to misidentification of a sample taken from a family to be verified other than the specific family to be verified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below. (Verification pattern 1) The result of genotyping the specific person belonging to the specific subject family; and Optionally, the results of genotyping biological relatives of said particular person belonging to said particular subject family; the genotyping results of the relatives belonging to the other subject family; Based on this, a blood relationship test is performed between this pseudo-blood relative and a specific person. (Verification pattern 2) The genotyping results of a specific person belonging to the other subject family; Optionally, the genotyping results of biological relatives of said particular person who belong to said other subject family; the genotyping results of the relatives belonging to the particular subject family; and Based on this, a blood relationship test is performed between this pseudo-blood relative and a specific person.

2. The pseudo-kin is a pseudo-parent, The specific person is a child, A biological relative of said particular person is a biological parent of said child who is of a different sex than said assumed parent; The blood relationship test is a parent-child test, 2. The method of claim 1, which verifies erroneous determination due to mixing up samples in parent-child testing of the pseudo parent and the child based on samples taken from a set of a specific subject family including the pseudo parent, the child, and optionally the biological parent, which is the subject of testing for the existence or nonexistence of a biological parent-child relationship, comprises the verification step defined below. <Verification process> In order to verify erroneous determination due to misidentification of a sample taken from a family to be verified other than the specific family to be verified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below. (Verification pattern 1) The genotyping results of the child belonging to the particular subject family; and Optionally, the genotyping results of the biological parents belonging to the particular subject family; and The genotyping results of a pseudo-parent belonging to the other family to be verified; Based on this, parentage testing is performed for this combination of pretend parent and child. (Verification pattern 2) The genotyping results of a child belonging to the other subject family; and Optionally, the genotyping results of the child's biological parents who belong to the other subject family; The genotyping results of the pseudo-parents belonging to the specific subject family; and Based on this, parentage testing is performed for this combination of pretend parent and child.

3. The blood relationship test is An appraisal based on the results of genotyping of the two subjects, the said related relative and the said specific person, to determine whether or not there is a biological blood relationship; or An appraisal based on the results of genotyping three people, the said pseudo-blood relative who is the subject of the appraisal of the existence of a biological parent-child relationship, the said specific person, and the said biological blood relative. The method of claim 1 , comprising:

4. In the verification step, The verification method according to claim 1, wherein verification is performed according to the verification pattern 1 and / or the verification pattern 2 by referring to a database storing genotyping results between related relatives and specific individuals belonging to one or more of the other families to be verified.

5. In the verification step, The verification method according to claim 2, wherein verification according to the verification pattern 1 and / or the verification pattern 2 is performed by referring to a database storing genotyping results of a pseudo-parent, a child, and optionally the biological parent of the child, belonging to one or more of the other families to be verified.

6. The method of claim 4 or 5, further comprising, after genotyping of the specific person and the related relatives belonging to the specific family to be identified is completed, storing the result in the database and updating it.

7. The verification step includes: executing the verification step by referring to the updated database; and The verification method according to claim 6, further comprising: regarding the genotyping result of the specific family to be verified reflected in the database as the genotyping result of the other family to be verified, and performing verification according to the verification pattern 1 and / or the verification pattern 2.

8. genotyping the kin and genotyping the particular individual; each performed at different locations and / or at different times; and / or The verification method according to any one of claims 1 to 5, wherein each of the verification steps is performed by a different operator.

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