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 a non-invasive prenatal paternity testing technique.

Background Art

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

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0005] The present invention for solving the above 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 the reference a determination method defined below.

[0007] (Reference a determination method) Based on the 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 or second-strongest signal intensity as the signal intensity of allele A 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, perform according to the following reference a.

[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 identification process> A process of identifying the existence or non - existence of the biological parent - child relationship between the putative father and the fetus based on the genotyping results of the biological mother, the putative 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) Derived 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 or the second - strongest signal intensity as that of allele A is designated as allele B. When so denoted, 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, perform according to the following criterion b.

[0011] <Criterion b> · When 0% ≤ Fb {≤, <} L%, it is determined that the genotype of the fetus at the specific polymorphic locus is homozygous AA · When M% {≤, <} Fb, it is determined that the genotype of the fetus at the specific polymorphic locus is heterozygous AB · When Fa = Fb, 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 identification of the existence or non - existence of the biological parent - child relationship is The combined paternity index (CPI) is obtained by multiplying the paternity indices (PIs) determined for each polymorphic locus, and a determination of either "affirmative", "negative", or "indeterminable" is made according to a predetermined criterion based on the value of the CPI. In the two-sample identification step, the PI is determined based on Table 1 below. In the three-sample identification step, the PI is determined based on Table 1 or Table 2 below. The non-invasive prenatal paternity testing method according to [1].

Table 1

Table 2

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

[0014] <MC filter step> Genotyping of the mother is performed 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. (i) A polymorphic locus that is homozygous for an allele in which the genotype of the mother and the fetus or cell-free circulating nucleic acid are different from each other. And / or (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. When the number of observations is equal to or greater than a preset specified number, the paternity test based on the collected sample is stopped without execution.

[0015] [4] A non-invasive prenatal paternity testing method according to any one of [1] to [3], characterized by performing a verification process defined below based on a sample collected from a specific family to be tested. Prenatal paternity testing method.

[0016] <Verification process> In order to verify a misjudgment due to a mix-up with a sample collected from a family to be tested different from the specific family to be tested, verification is performed according to a verification pattern 1 and / or a verification pattern 2 defined below.

[0017] (Verification pattern 1) Based on the result of genotyping of the fetus belonging to the specific family to be tested, Optionally, the result of genotyping of the mother belonging to the specific family to be tested, and the result of genotyping of the alleged father belonging to the different family to be tested, a paternity test is performed for the combination of this alleged father and the fetus.

[0018] (Verification pattern 2) Based on the result of genotyping of the fetus belonging to the different family to be tested, Optionally, the result of genotyping of the biological mother of the fetus belonging to the different family to be tested, and the result of genotyping of the alleged father belonging to the specific family to be tested, a paternity test is performed for the combination of this alleged father and the fetus.

[0019] [5] In the verification process, Referring to a database storing the genotyping results of the putative father, the fetus, and optionally the biological mother of the fetus, belonging to one or two or more of the other identification target families, performing verification according to the verification pattern 1 and / or the verification pattern 2, the non-invasive prenatal paternity testing method according to [4].

[0020] [6] After the genotyping of the putative father, the fetus, and optionally the mother, belonging to the specific identification target family, is completed, the results are stored in and updated to the database. In the verification step, the verification step is executed by referring to the updated database. In the verification step, regarding the genotyping results of the specific identification target family reflected in the database, considering them as the genotyping results of the other identification family, performing verification according to the verification pattern 1 and / or the verification pattern 2, the non-invasive prenatal paternity testing method according to [5].

[0021] [7] The genotyping of the putative father and the genotyping of the fetus are Performed at different locations and / or different times respectively, and / or Performed by different operators respectively, the non-invasive prenatal paternity testing according to any one of [4] to [6].

[0022] Also, at the site of the blood relationship identification business, one person in charge examines the samples of the putative blood relatives and the samples of specific individuals collected from a specific set of identification target families. In this case, sample mix-ups may occur, resulting in incorrect blood relationship identification results. As a method to prevent such sample mix-ups, the same test may be performed twice for double-checking, but there is a problem that the test cost doubles. In view of such problems, a second object of the present invention is to provide a new technique for verifying misjudgment due to sample mix-ups in blood relationship identification. The present invention for solving the above second object is as follows.

[0023] [1] A method for verifying an incorrect determination due to a sample mix-up in the blood relationship identification between a specific person and a putative blood relative, which is a subject of identification of the existence of a biological blood relationship, the specific person, and optionally the biological blood relative of the specific person, based on a sample collected from a specific set of identification target families including the putative blood relative, the specific person, and optionally the biological blood relative of the specific person, the method comprising a verification process defined below.

[0024] <Verification process> To verify an incorrect determination due to a sample mix-up with a sample collected from an identification target family different from the specific identification target family, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below.

[0025] (Verification pattern 1) Based on the genotyping result of the specific person belonging to the specific identification target family, optionally, the genotyping result of the biological blood relative of the specific person belonging to the specific identification target family, and the genotyping result of the putative blood relative belonging to the other identification target family, a blood relationship identification is performed for this combination of the putative blood relative and the specific person.

[0026] (Verification pattern 2) Based on the genotyping result of the specific person belonging to the other identification target family, optionally, the genotyping result of the biological blood relative of the specific person belonging to the other identification target family, and the genotyping result of the putative blood relative belonging to the specific identification target family, a blood relationship identification is performed for this combination of the putative blood relative and the specific person.

[0027] [2] The putative blood relative is a putative parent, the specific person is a child, and the biological blood relative of the specific person is the biological parent of the child, who is of a different gender from the putative parent. The blood relationship identification is a parentage test, A method for verifying an incorrect determination due to a sample mix-up in the parentage test between a putative parent and a child, based on samples collected from a specific set of identification target families including the putative parent, the child, and optionally the biological parent, which are the subjects of the identification of the existence or non-existence of a biological parent-child relationship. The method is characterized by including a verification process defined below, and is the verification method described in [1].

[0028] <Verification process> In order to verify an incorrect determination due to a sample mix-up with samples collected from an identification target family different from the specific identification target family, verification is performed according to verification pattern 1 and / or verification pattern 2 defined below.

[0029] (Verification pattern 1) Based on the genotyping result of the child belonging to the specific identification target family, Optionally, the genotyping result of the biological parent belonging to the specific identification target family, And the genotyping result of the putative parent belonging to the different identification target family, A parentage test is performed for this combination of putative parent and child.

[0030] (Verification pattern 2) Based on the genotyping result of the child belonging to the different identification target family, Optionally, the genotyping result of the biological parent of the child belonging to the different identification target family, And the genotyping result of the putative parent belonging to the specific identification target family, A parentage test is performed for this combination of putative parent and child.

[0031] [3] The verification method according to [1] or [2], wherein the blood relationship identification includes an identification performed based on the genotyping results of two individuals, namely the putative blood relative and the specific person, which are the subjects of the identification of the existence or non-existence of a biological blood relationship.

[0032] [4] The verification method according to any one of [1] to [3], wherein the blood relationship identification includes an identification performed based on the genotyping results of three persons, namely, the putative blood relative who is the subject of identification of the existence or non-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 in the verification step, verification according to the verification pattern 1 and / or the verification pattern 2 is performed by referring to a database storing the genotyping results of a putative blood relative and a specific person belonging to one or more of the other identification target families. [6] In the verification step, The verification method according to [2], wherein in the verification step, verification according to the verification pattern 1 and / or the verification pattern 2 is performed by referring to a database storing the genotyping results of a putative parent, a child, and optionally the biological parent of the child belonging to one or more of the other identification target families.

[0034] [7] The verification method according to [5], including storing and updating the results in the database after the genotyping of the putative blood relative and the specific person belonging to the specific identification target family is completed.

[0035] [8] The verification method according to [6], including storing and updating the results in the database after the genotyping of the putative parent, the child, and optionally the biological parent belonging to the specific identification target family is completed.

[0036] [9] The verification step includes executing the verification step by referring to the updated database, and regarding the genotyping results of the specific identification target family reflected in the database as the genotyping results of the other identification family, and performing verification according to the verification pattern 1 and / or the verification pattern 2. The verification method according to [7] or [8].

[0037]

[10] Typing of the putative blood relative and typing of the specific person are performed at different locations and / or different times, and / or performed by different operators, respectively, and the verification method according to any one of [1] to [9].

Advantages of the Invention

[0038] According to the present invention that solves the first problem, based on the results of genotyping of two persons, namely the putative father and the fetus, non-invasive prenatal paternity testing can be simply carried out with clear criteria of criterion a or criterion b.

[0039] In addition, according to the present invention that solves the second problem, misjudgment due to sample mix-up can be verified with high accuracy.

Brief Description of the Drawings

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

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

[0042] The present invention includes a two-sample identification step and / or a three-sample identification step defined below.

[0043] <Two-sample identification step> The two-sample identification step is a step of identifying the existence or non-existence of a biological parent-child relationship between the alleged father and the fetus based on the results of genotyping of the alleged father and the fetus at a plurality of polymorphic loci. In the two-sample identification step, the genotyping of the fetus is performed according to the reference a determination method defined below.

[0044] (Reference a determination method) Based on the data obtained by analyzing the circulating cell-free nucleic acid sample collected from the 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 or second-strongest signal intensity as the signal intensity of allele A 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 indices, the following reference a is used.

[0045] <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.

[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 derived from a specific polymorphic locus included in the data obtained by analyzing the cell-free nucleic acid sample are denoted as allele C and allele D, respectively, using 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 as indicators, the following skip conditions may be added to the above-mentioned standard a. ·When K satisfies K{≦,<}Fc + Fd, skip without performing genotyping of the specific polymorphic locus.

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

[0050] <3 - sample Identification Step> The 3 - sample identification step is a step of identifying the existence or non - existence of the biological parent - child relationship between the putative father and the fetus based on the genotyping results of the biological mother, the putative father, and the fetus at a plurality of polymorphic loci. In the 3 - sample identification step, the genotyping of the fetus is performed according to the above - mentioned reference a determination method and / or the reference b determination method defined below.

[0051] (Reference 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 or the second - strongest signal intensity as the signal intensity of allele A 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, perform according to the following reference b.

[0052] <Reference b> ·When 0%≦Fb{≦,<}L% is satisfied, it is determined that the genotype at the specific polymorphic locus of the fetus is homozygous AA. ·When M%{≦,<}Fb is satisfied, it is determined that the genotype at the specific polymorphic locus of the fetus 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.

[0053] (satisfies the following.) 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 one of the inequality signs ≦ and < 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. There is no limit to the upper limit value of M.

[0054] In addition, 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, using 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 as indices, the following skip condition may be added to the above-mentioned standard b. ·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, for example, any value of 10 or less, preferably 5 or less.

[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 include nucleotide sequence analysis, mass spectrometry, digital PCR, SNV microarray, real-time PCR, and the like. 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] When performing the genotyping of the putative father and the mother's genotyping in the case of the three-sample identification process, it can be easily done by conventional methods. For example, it can be easily done by analyzing samples usually used in genetic tests, such as samples of buccal mucosal cells collected from the oral cavity.

[0058] When performing the two-sample identification process, based on the fetal genotyping result determined by the above method and the genotyping result of the putative father, the existence or non-existence of the biological parent-child relationship between the fetus and the putative father is identified (Figure 1). In addition, when performing the three-sample identification process, based on the fetal genotyping result determined by the above method, the genotyping result of the putative father, and the genotyping result of the biological mother of the fetus, the existence or non-existence of the biological parent-child relationship between the fetus and the putative father is identified (Figure 1, Figure 2).

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

[0060]

Table 1

[0061]

Table 2

[0062] In Tables 1 and 2, for the analysis of circulating cell-free nucleic acid samples, the allele with relatively high signal intensity is denoted as A, and the allele with 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 occurrence frequency of A, and b is the occurrence frequency of B. Note that k0 and kb can be appropriately set according to the accuracy of the detection system. For example, they can be set to 0.1 or less, preferably 0.01 or less, and more preferably 0.001 or less.

[0063] After obtaining the PI for each single nucleotide polymorphism locus, the Combined Paternity Index (CPI) may be obtained. CPI is obtained as the product of all PIs.

[0064] Preferably, in the present invention, for example, as shown below, according to a predetermined criterion corresponding to the value of CPI, a determination of either "affirmative", "negative", or "indeterminable" is made. 10 4 ≦CPI ··· Result: "Affirmative" 10 -6 ≦CPI < 10 4 ··· Result: "Indeterminable" CPI < 10 -6 ··· Result: "Negative"

[0065] The criteria shown above are only examples, and the specific reference values of CPI for determination can be appropriately set.

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

Equation

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

[0068] <MC filter process> Perform 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 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 homozygous for alleles with different genotypes between the mother and the fetus or cell-free circulating nucleic acid (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 unlikely that the genotypes of the mother and the fetus or cell-free circulating nucleic acid meet the conditions described in the above (i) and / or (ii). In such a case, sample miscollection is suspected.

[0070] The MC filter process is performed to prevent incorrect identification result determination and wasted work by stopping the identification without performing the 3-sample identification process when such cases of suspected sample miscollection are observed to be equal to or more than a predetermined specified number (Fig. 2).

[0071] The "preset specified number" can be arbitrarily set. 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] Although the specific number of the specified number is not particularly limited, it can be set to 5 or less, more preferably 3 or less, and even more preferably 1.

[0073] Regarding the genotyping of cell-free circulating nucleic acids for identifying the genotype of cell-free circulating nucleic acids, it will be described with reference to FIG. 7. The cell-free circulating nucleic acid sample contains major nucleic acids derived from the mother and minor nucleic acids derived from the fetus. Therefore, when analyzing the cell-free circulating nucleic acid sample and plotting the ratio of the signal indicating the presence of a specific allele to the total signal intensity derived from a specific polymorphic locus, it is distributed as shown in FIG. 7 at 0 to 25%, 35 to 65%, and 75 to 100%. Assuming no contamination by DNA from others externally, the case where the above ratio at each polymorphic locus is 5% or more is treated as a true signal.

[0074] Based on this, among the data obtained by analyzing the cell-free circulating nucleic acid sample, the allele detected with the strongest signal intensity derived from a specific polymorphic locus is designated as allele A. The allele detected with the same or second-strongest signal intensity as the signal intensity of allele A 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 indicators, genotyping of cell-free circulating nucleic acids is performed according to the following criterion c.

[0075] <Criterion c> · When X{≦,<}Fa{≦,<}Y% is satisfied, it is determined that the genotype at the specific polymorphic locus of the cell-free circulating nucleic acid is AB heterozygous. · When Z%{≦,<}Fa≦100 is satisfied, it is determined that the genotype at the specific polymorphic locus of the cell-free circulating nucleic acid is AA homozygous. · When Fa = Fb is satisfied, it is determined that the genotype of the specific polymorphic locus is AB heterozygous.

[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 "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 implemented.

[0082] In one embodiment of the present invention, when a stop determination is not 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 the case of ordinary legal authentication, a witness is required when collecting all samples. However, when performing the MC filter process, since it is possible to determine whether there is a mix-up between the mother's nucleic acid sample and the cell-free circulating nucleic acid sample, the presence of a witness for collecting the maternal blood required for legal authentication becomes unnecessary, and there is an advantage that legal authentication can be performed with only the witness of the mother's Genomic DNA sample.

[0084] The present invention may also be in a form including either one or both of a two-sample authentication process and a three-sample authentication process. In the case of a form including both the two-sample authentication process and the three-sample authentication process, it may be in a form where the authentication result of either one is taken as the final authentication result, or it may include a process of comparing the authentication results of these two authentication processes. And it may also be an embodiment in which the final authentication result is obtained according to a predetermined criterion based on the pattern of agreement or disagreement of the two authentication results.

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

[0086]

Table 3

[0087] The non-invasive prenatal paternity testing method of the present invention may further include a verification process for verifying misjudgment due to sample mix-up (FIG. 1, FIG. 2).

[0088] FIG. 1 and FIG. 2 show an embodiment in which the verification process is executed regardless of the results of the two-sample authentication process or the three-sample process. In the case of such an embodiment, it is possible to verify whether the results of negating or affirming the biological parent-child relationship by the two-sample authentication process or the three-sample process include misjudgment caused by sample mix-up. If it is determined that there has been a sample mix-up in the verification process, the results of the two-sample identification process or the three-sample process can be considered unreliable.

[0089] Figures 1 and 2 are merely one embodiment, and an embodiment in which the verification process is executed according to the results of the two-sample identification process or the three-sample process may also be possible.

[0090] When two or more workers perform inspections independently, or when one worker performs multiple inspections at different locations and times, the possibility of making the same sample mix-up in all inspections is extremely low. Under such a double-check system, an embodiment in which the verification process is carried out when a "negative" judgment result is obtained in each inspection may also be possible. Conversely, under the above double-check system, when a "positive" judgment result is obtained in each inspection, the judgment result may be regarded as correct and the verification process may not be carried out. Of course, the verification process may be further carried out to form a triple-check system.

[0091] The specific embodiments of the verification process will be described in detail in the description item of the invention of the following verification method.

[0092] <2> Verification Method The present invention is a method for verifying misjudgments in blood relationship identification due to sample mix-ups, based on samples taken from a set of specific families to be identified.

[0093] The verification method of the present invention can be applied to identification methods for identifying biological parent-child relationships, biological sibling relationships, biological grandparent-grandchild relationships, and any other biological blood relationships.

[0094] As used herein, the family to be identified refers to a group including a specific person who is at least the subject of identification of the existence of a biological blood relationship and his or her putative blood relatives. The family to be identified may optionally include one or more biological blood relatives of the specific person. That is, the family to be identified may be in a form that includes only two persons, the putative blood relative and the specific person, or a form that includes three persons, the putative blood relative, the specific person, and the biological blood relative of the specific person. In other words, the verification method of the present invention is applicable to both the above-described two-sample identification step and three-sample identification step.

[0095] For example, in the case of identifying a biological sibling relationship, the specific person and the putative sibling of the specific person constitute the family to be identified. In this case, biological blood relatives such as the biological parent or sibling of the specific person may be arbitrarily included in the family to be identified. Also, in the case of identifying a biological grandparent-grandchild relationship, the putative grandfather or grandmother and the grandchild constitute the family to be identified. In this case, biological blood relatives such as the biological parent of the grandchild, or a biological grandmother or grandfather of a different gender from the putative grandfather or grandmother who is the putative blood relative, may be arbitrarily included in the family to be identified.

[0096] Also, when performing a parent-child identification, the family to be identified includes the putative parent and the child who are the subjects of identifying the existence of a biological parent-child relationship. And the family to be identified may optionally include the biological parent of the child, who is of a different gender from the putative parent. That is, the family to be identified may be in a form that includes only two persons, the putative parent and the child, or a form that includes three persons, the putative parent, the child, and the biological parent. In other words, the verification method of the present invention is applicable to both the above-described two-sample identification step and three-sample identification step.

[0097] "The biological parent of the child, who is of a different gender from the putative parent" is the "biological mother of the child" when the putative parent is the "putative father", and is the "biological father of the child" when the putative parent is the "putative mother". That is, the verification method of the present invention is applicable to both father-child identification and mother-child identification.

[0098] Hereinafter, the embodiments when the verification method of the present invention is applied to paternity testing including "child", "alleged father", and optionally "biological mother" as the family to be identified will be described focusing thereon. Of course, it should be noted that the present invention is not limited to the paternity testing described below and can be applied to the verification of any blood relationship identification.

[0099] The verification method of the present invention can also be applied to paternity testing conducted between a child after birth and an alleged parent. Naturally, it can also be applied to paternity testing conducted between a child (fetus) before birth and an alleged parent (alleged father), such as the non-invasive prenatal paternity testing method of the present invention described above.

[0100] When applying the verification method of the present invention to father-son testing, the specific mode of the father-son testing is not limited. For example, PI calculation based on Table 1 or Table 2 above may be performed. In the case where the child is after birth, "fetus" in Table 1 or Table 2 above can be read as "child after birth" and directly applied mutatis mutandis. Further, based on the calculated PI, CPI may be calculated, or POP may be obtained based on CPI.

[0101] When applying the verification method of the present invention to mother-child testing, the specific mode of the mother-child testing is not limited. For example, a Maternity Index (MI) can be calculated according to the PI calculation in father-son testing. Further, based on the calculated MI, a Combined Maternity Index (CMI) may be calculated according to the CPI calculation method. Also, based on the calculated CMI, a Probability of Maternity (POM) may be obtained according to the POP calculation method.

[0102] Of course, the verification method of the present invention is applicable to paternity testing that does not use the PI or MI calculation formulas shown in Table 1 or Table 2. For example, the verification method of the present invention can be applied to a method that uses a likelihood ratio representing the probability of a parent-child relationship other than the method using the calculation formulas in Table 1 or Table 2.

[0103] When performing genotyping of putative blood relatives and genotyping of a specific individual by the same operator at the same location and the same time, there is a possibility of misidentifying all samples of the family to be identified instead of a part of the family to be identified. If such a misidentification of samples occurs for each family to be identified, it may not be possible to definitely detect the misidentification of samples in the verification process described later.

[0104] To solve this problem, in a preferred embodiment of the verification method of the present invention, genotyping of putative blood relatives and genotyping of a specific individual are performed at different locations and / or at different times, and / or by different operators. In the case of paternity testing, genotyping of the putative parent and genotyping of the child are performed at different locations and / or at different times, and / or by different operators.

[0105] When genotyping of putative blood relatives and a specific individual is performed at different locations, different times, and by different operators in this way, the effect that the possibility of the same sample misidentification occurring in each genotyping is reduced can be obtained. After adopting such an embodiment of genotyping, by performing verification by the verification process described later, an extremely advantageous effect that the verification accuracy can be significantly improved can be obtained.

[0106] The verification method of the present invention is characterized by including a verification process defined as follows.

[0107] <Verification Process> To verify misjudgment due to misidentification with samples collected from a family to be identified different from the specific family to be identified, verification is performed according to verification pattern 1 and / or verification pattern 2 defined as follows.

[0108] (Verification Pattern 1) The result of genotyping of the child belonging to the specific family to be identified and Optionally, based on the genotyping results of the biological parent belonging to the specific family to be identified and the genotyping results of the putative parent belonging to the other family to be identified, parentage testing is performed for this combination of putative parent and child. (Verification Pattern 2) Based on the genotyping results of the child belonging to the other family to be identified, optionally, the genotyping results of the biological parent of the child belonging to the other family to be identified, and the genotyping results of the putative parent belonging to the specific family to be identified, parentage testing is performed for this combination of putative parent and child.

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

[0110] The two-sample test in Verification Pattern 1 performs parentage testing based on the genotyping results of a child belonging to a specific family to be identified and two putative parents belonging to another family to be identified. The specific mode can directly apply the description of the above two-sample testing process.

[0111] The three-sample test in Verification Pattern 1 performs parentage testing based on the genotyping results of a child belonging to a specific family to be identified, the biological parent of the child belonging to the specific family to be identified, and two putative parents belonging to another family to be identified. The specific mode can directly apply the description of the above three-sample testing process.

[0112] Verification Pattern 2 is a pattern in which parentage testing is performed for a combination of a child belonging to another family to be identified and a putative parent belonging to a specific family to be identified. The paternity test conducted here may be a two-sample test or a three-sample test.

[0113] For the two-sample test in verification pattern 2, a paternity test is performed based on the genotyping results of a child belonging to another family to be identified and two putative parents belonging to a specific family to be identified. The specific manner can directly apply the description of the above two-sample test process.

[0114] For the three-sample test in verification pattern 2, a paternity test is performed based on the genotyping results of a child belonging to another family to be identified, the biological parent of the child belonging to another family to be identified, and three putative parents belonging to a specific family to be identified. The specific manner can directly apply the description of the above three-sample test process.

[0115] In addition, when the paternity test for verifying the presence or absence of sample mix-up is a two-sample test, the paternity test performed in the verification process is not necessarily limited to a two-sample test. There is no problem at all in performing a three-sample test in the verification process.

[0116] Conversely, when the paternity test for verifying the presence or absence of sample mix-up is a three-sample test, the paternity test performed in the verification process is not necessarily limited to a three-sample test. There is no problem at all in performing a two-sample test in the verification process.

[0117] In verification pattern 1 and / or verification pattern 2, if an identification result that affirms a biological parent-child relationship appears, it can be judged that there is a high possibility that the samples of a specific family to be identified and the samples of another family to be identified have been mixed up. That is, it is highly possible that the sample of the subject belonging to the specific family to be identified with a biological parent-child relationship has been treated as belonging to a different family to be identified, and it can be judged that the identification result of the paternity test to be verified includes 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 paternity test performed on a set of target families for identification may also be a target for verification in the verification process. Further, as shown in FIG. 3, the paternity tests performed on two or more sets of target families for identification can also be used as targets for verification in the verification process.

[0123] The database stores in advance the results of genotyping of children CA to CX and putative fathers AFA to AFX, which belong to a plurality of different sets of other target families for identification A to X, different from the specific target families for identification Y and Z (FIG. 3). In addition, after the genotyping of child CY belonging to the target family for identification Y and putative father AY, and child CZ belonging to the target family for identification Z and putative father AZ is completed, the database is updated before the verification process is carried out, and the results of genotyping of child CY and putative father AFY, and child CZ and putative father AFZ are stored in the database (FIG. 3). That is, the verification process is carried out by referring to the database storing the results of genotyping of children CA to CZ and putative fathers AFA to AFZ.

[0124] In the embodiment of FIG. 3, a paternity test is performed based on the genotyping result of child CY and each of the genotyping results of putative fathers AFA to AFZ stored in the database. That is, a paternity test is performed for each combination of child CY and putative father AFA, child CY and putative father AFB, ··· child CY and putative father AFX, child CY and putative father AFY, and child CY and putative father AFZ. In addition, in the embodiment of FIG. 3, independently of this, a paternity test is similarly performed based on the genotyping result of child CZ and each of the genotyping results of putative fathers AFA to AFZ stored in the database.

[0125] Hereinafter, in order to avoid duplication of description, the case of performing a paternity test based on each of the genotyping results of child CZ and putative fathers AFA to AFZ will be described in detail. Needless to say, the following description is also applicable to the case of performing a paternity test based on each of the genotyping results of child CY and putative fathers AFA to AFZ.

[0126] If it is confirmed that there is a biological parent-child relationship between the child CZ and any of the putative fathers AFA to AFY, it can be determined that there was a mistake in the combination of the samples of the child CZ and the putative father AFZ, which were treated as being obtained from a specific set of identified family Z, with the samples obtained from a person belonging to another identified family. That is, it can be determined that the identification result of the paternity test performed by analyzing the samples of the child CZ and the putative father AFZ belonging to the identified family Z contains an error.

[0127] Also, in the paternity test to be verified, if a "positive" determination result has been obtained, a "positive" result should also be obtained in the verification targeting the putative father AFZ stored in the database by the update. On the other hand, unless there is a special circumstance such that the putative father AFZ was registered in the database as one of the putative fathers AFA to AFY for reasons such as being the subject of a paternity test in another case, a "negative" result should be obtained in the verification targeting the putative fathers AFA to AFY stored in the database. That is, in the paternity test to be verified, if a "positive" determination result has been obtained, in the verification process, the putative father AFZ stored in the database by the update acts as a positive control, while the putative fathers AFA to AFY act as negative controls unless there are the above-mentioned special circumstances. Therefore, it can be confirmed that the verification process itself is being executed normally.

[0128] Conversely, in the paternity test to be verified, if a "negative" determination result has been obtained, a "negative" result should also be obtained in the verification targeting the putative father AFZ stored in the database by the update. That is, in the verification process, since the putative father AFZ stored in the database by the update acts as a negative control, it can be confirmed that the verification process itself is being executed normally.

[0129] Note that, prior to verification, FIG. 3 shows an embodiment of updating the database based on the genotyping results of child CY belonging to the family Y to be identified, putative father AFY, and child CZ belonging to the family Z to be identified, putative father AFZ. However, it goes without saying that valid verification can also be performed using a database in which only the genotyping results of children CA to CX and putative fathers AFA to AFX are stored in advance without updating the database.

[0130] Next, FIG. 4 shows an embodiment of executing verification pattern 2.

[0131] The database configuration of the embodiment in FIG. 4 is the same as that in FIG. 3. In the embodiment of FIG. 4, paternity testing is performed based on the genotyping result of putative father AFY and each of the genotyping results of children CA to CZ stored in the database. That is, paternity testing is performed for each combination of putative father AFY and child CA, putative father AFY and child CB, ··· putative father AFY and child CX, putative father AFY and child CY, putative father AFY and child CZ. Also, in the embodiment of FIG. 4, independently of this, paternity testing is similarly performed based on the genotyping result of putative father AFZ and each of the genotyping results of children CA to CZ stored in the database.

[0132] Hereinafter, in order to avoid duplication of explanation, the description will be focused on the case of performing paternity testing based on each of the genotyping results of putative father AFZ and children CA to CZ. Needless to say, the following explanation is also valid for the case of performing paternity testing based on each of the genotyping results of putative father AFY and children CA to CZ.

[0133] If the existence of a biological parent-child relationship is affirmed between putative father AFZ and any of children CA to CY, it can be determined that there has been a mix-up with a sample obtained from a person belonging to another family to be identified in the combination of samples of child CZ and putative father AFZ that had been treated as being obtained from a specific set of family Z to be identified. That is, it can be determined that the paternity test results obtained by analyzing the samples of the child CZ belonging to the family Z to be identified and the putative father AFZ contain errors.

[0134] In addition, in the paternity test to be verified, when a "positive" determination result is obtained, a "positive" result should also be obtained in the verification targeting the child CZ stored in the database by the update. On the other hand, unless there is a special circumstance where the child CZ was registered in the database as one of the children CA to CY for reasons such as being the subject of a paternity test in another case, a "negative" result should be obtained in the verification targeting the children CA to CY stored in the database. That is, in the paternity test to be verified, when a "positive" determination result is obtained, in the verification process, the child CZ stored in the database by the update acts as a positive control, while the children CA to CY act as negative controls unless there are the above-mentioned special circumstances. Therefore, it can be confirmed that the verification process itself is being executed normally.

[0135] Conversely, in the paternity test to be verified, when a "negative" determination result is obtained, a "negative" result should also be obtained in the verification targeting the child CZ stored in the database by the update. That is, in the verification process, since the child CZ stored in the database by the update acts as a negative control, it can be confirmed that the verification process itself is being executed normally.

[0136] Next, an embodiment of executing the verification pattern 1 is shown in FIG. 5. In this embodiment, the paternity tests (father-son tests) performed on two sets of families to be identified, namely, the specific family Y including the child CY, the biological mother MY of the child CY, and the putative father AFY, and the specific family Z including the child CZ, the biological mother MZ of the child CZ, and the putative father AFZ, are the verification targets in the verification process.

[0137] Naturally, the paternity test performed on a set of target families for identification may also be a verification target in the verification process. Also, as shown in FIG. 5, the paternity tests performed on two or more sets of target families for identification can also be used as verification targets in the verification process.

[0138] In the database, the genotyping results of children CA to CX, their biological mothers MA to MX, and putative fathers AFA to AFX, which belong to multiple sets of different target families for identification A to X that are different from the specific target families for identification Y and Z, are stored in advance (FIG. 5). Also, after the genotyping of child CY and putative father AFY, mother MZ belonging to the target family for identification Y, and child CZ and putative father AFZ, mother MZ belonging to the target family for identification Z is completed, the database is updated before the verification process is carried out, and the genotyping results of child CY, putative father AFY, mother MY, and child CZ, putative father AFZ, mother MZ are stored in the database (FIG. 5). That is, the verification process is carried out by referring to the database in which the genotyping results of children CA to CZ, putative fathers AFA to AFZ, and mothers MA to MZ are stored.

[0139] In the embodiment of FIG. 5, based on the genotyping results of child CY and mother MY and the genotyping results of putative fathers AFA to AFZ stored in the database, a paternity test is carried out. That is, paternity tests (three-sample tests) are carried out for each combination of child CY, mother MY, and putative father AFA; child CY, mother MY, and putative father AFB; ··· child CY, mother MY, and putative father AFX; child CY, mother MY, and putative father AFY; child CY, mother MY, and putative father AFZ. Also, in the embodiment of FIG. 5, independently of this, based on the genotyping results of child CZ and mother MZ and the genotyping results of putative fathers AFA to AFZ stored in the database, a paternity test is similarly carried out.

[0140] The following will focus on the case of performing paternity testing based on the genotyping results of child CZ, mother MZ, and putative fathers AFA to AFZ respectively, in order to avoid duplicate explanations. Needless to say, the following explanations are also applicable to the case of performing paternity testing based on the genotyping results of child CY, mother MY, and putative fathers AFA to AFZ respectively.

[0141] If the existence of a biological parent-child relationship is affirmed between child CZ and any of the putative fathers AFA to AFY, it can be determined that there was a mix-up with samples obtained from someone belonging to another family to be identified, in the combination of samples of child CZ, mother MZ, and putative father AFZ that were treated as being obtained from a specific family Z to be identified. That is, it can be determined that the paternity testing results obtained by analyzing the samples of child CZ, mother MZ, and putative father AFZ belonging to family Z to be identified contain errors. In addition, when passing through the above-mentioned MC filter process, the mix-up of samples of child CZ and mother MZ does not need to be considered.

[0142] Also, in the paternity testing to be verified, if a "positive" judgment result is obtained, a "positive" result should also be obtained in the verification targeting the putative father AFZ stored in the database by the update. On the other hand, unless there is a special circumstance where a person (including ancestors and descendants) having a biological parent-child relationship with child CZ was registered in the database as one of the putative fathers AFA to AFY for reasons such as being the subject of paternity testing in another case, a "negative" result should be obtained in the verification targeting the putative fathers AFA to AFY stored in the database. That is, in the paternity testing to be verified, if a "positive" judgment result is obtained, in the verification process, the putative father AFZ stored in the database by the update acts as a positive control, while the putative fathers AFA to AFY act as negative controls unless there are the above-mentioned special circumstances. Therefore, it can be confirmed that the verification process itself is being executed normally.

[0143] Conversely, in the paternity test to be verified, when a "negative" judgment result is obtained, the verification targeting the putative father AFZ stored in the database by the update should also yield a "negative" result. That is, in the verification process, since the putative father AFZ stored in the database by the update acts as a negative control, it is possible to confirm that the verification process itself is being executed properly.

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

[0145] In the embodiment of FIG. 6, paternity tests are performed based on the genotyping results of the putative father AFY and the genotyping results of the children CA to CZ and mothers MA to MZ stored in the database respectively. That is, paternity tests (3-sample tests) are performed for each combination of the putative father AFY, child CA and mother MA; putative father AFY, child CB and mother MB;... putative father AFY, child CX and mother MX; putative father AFY, child CY and mother MY; putative father AFY, child CZ and mother MZ. Also, in the embodiment of FIG. 6, independently of this, paternity tests are similarly performed based on the genotyping results of the putative father AFZ and the genotyping results of the children CA to CZ and mothers MA to MZ stored in the database respectively.

[0146] Hereinafter, to avoid duplication of explanation, the description will be focused on the case of performing paternity tests based on the genotyping results of the putative father AFZ and the children CA to CZ and mothers MA to MZ respectively. Needless to say, the following explanation is also valid for the case of performing paternity tests based on the genotyping results of the putative father AFY and the children CA to CZ and mothers MA to MZ respectively.

[0147] If the existence of a biological parent-child relationship is affirmed between the putative father AFZ and any one of the children CA to CY, it can be determined that there was a mix-up with samples obtained from persons belonging to another family to be identified, in the combination of samples of the child CZ, mother MZ, and putative father AFZ, which were treated as being obtained from a specific set of families Z to be identified. That is, it can be determined that the identification results of the parent-child identification performed by analyzing the samples of the child CZ, mother MZ, and putative father AFZ belonging to the family Z to be identified contain errors. Note that when the above-described MC filter process has been passed, the mix-up of samples of the child CZ and mother MZ does not need to be considered.

[0148] Also, in the parent-child identification to be verified, if a "positive" determination result has been obtained, a "positive" result should also be obtained in the verification targeting the child CZ and mother MZ stored in the database by the update. On the other hand, unless there are special circumstances such that the child CZ and mother MZ were registered in the database as any one of the children CA to CY and mothers MA to MY, which were the subjects of parent-child identification in other cases, a "negative" result should be obtained in the verification targeting the children CA to CY and mothers MA to MY stored in the database. That is, in the parent-child identification to be verified, if a "positive" determination result has been obtained, in the verification process, the child CZ and mother MZ stored in the database by the update act as positive controls, while the children CA to CY and mother MA to MY act as negative controls unless there are the above-described special circumstances, so it can be confirmed that the verification process itself is being executed normally.

[0149] Conversely, in the parent-child identification to be verified, if a "negative" determination result has been obtained, a "negative" result should also be obtained in the verification targeting the child CZ and mother MZ stored in the database by the update. That is, in the verification process, since the child CZ and the mother MZ stored in the database by the update serve as negative controls, it is possible to confirm that the verification process itself is being executed normally.

[0150] Next, the effectiveness of verification common to the embodiments shown in FIGS. 3 to 6 will be described. In the embodiments shown in FIGS. 3 to 6, paternity testing for the identification target families Y and Z is the subject of verification. Here, it is assumed that a sample mix-up has occurred between the identification target families Y and Z. At this time, the following four cases can be considered as the results of paternity testing for each of the identification target families X and Y.

[0151] Case 1: In any of the paternity tests for the identification target families Y and Z, a result affirming the biological paternity of the child and the putative father was obtained. Case 2: A result affirming the biological paternity was obtained in the paternity test for the identification target family Y, while a result negating the biological paternity was obtained in the paternity test for the identification target family Z. Case 3: A result negating the biological paternity was obtained in the paternity test for the identification target family Y, while a result affirming the biological paternity was obtained in the paternity test for the identification target family Z. Case 4: In any of the paternity tests for the identification target families Y and Z, a result negating the biological paternity was obtained.

[0152] In the cases of Cases 1 to 3 described above, it is possible to discover that there was a sample mix-up by going through the verification process. However, in the case of Case 4, and indeed, when there is no biological parent-child relationship between child CY and putative father AFY, and between child CZ and putative father AFZ, even after the verification process, it is impossible to discover that there was a sample mix-up. However, in this case, although the subject's DNA profile error caused by the sample mix-up is included, the final determination result of negating the biological parent-child relationship remains unchanged.

[0153] The embodiments shown in FIGS. 3 to 6 are embodiments regarding paternity testing. However, the present invention is not limited to paternity testing and is naturally applicable to maternity testing as well. When applying to maternity testing, in the embodiments shown in FIGS. 3 to 6, the "putative father" can be replaced with "putative mother" for application. Also, in the embodiments shown in FIGS. 5 and 6, the "biological mother" can be replaced with "biological father" for application.

[0154] The verification method of the present invention can be applied to both paternity testing performed before a child is born and paternity testing performed after a child is born. When the paternity testing to which the verification method of the present invention is applied is performed before a child is born, it may include the MC filter process as described above. Also, even when the paternity testing to which the verification method of the present invention is applied is performed after a child is born, a filter similar to the above-mentioned MC filter can be adopted. Hereinafter, two cases of paternity testing and maternity testing will be described respectively.

[0155] In paternity testing (paternity testing) performed by analyzing samples collected from a child after birth, the biological mother of the child, and a putative father, it is preferable to include the post-natal MC filter process defined below.

[0156] <postnatal MC filter> Genotyping of the child based on a sample containing a nucleic acid containing the genetic information of the child collected from the child after birth, and Genotyping of the mother based on a sample containing nucleic acid comprising the genetic information of the mother collected from the biological mother of the child, and is performed, and when the following (i) is observed in a number equal to or greater than a preset specified number, the paternity test based on the collected sample is stopped without being executed. (i) A polymorphic locus that is homozygous for alleles with different genotypes in the mother and the child

[0157] By providing a postnatal MC filter, it is not necessary for a witness to be present when collecting a sample from the biological mother even in a legal identification.

[0158] Also, in a paternity test (mother-child test) performed by analyzing samples collected from three people: a child after birth, the biological father of the child, and the putative mother, postnatal It is preferable to include an FC filter step as defined below.

[0159] <postnatal FC filter> Genotyping of the child based on a sample containing nucleic acid comprising the genetic information of the child collected from the child after birth, and Genotyping of the father based on a sample containing nucleic acid comprising the genetic information of the father collected from the biological father of the child, and is performed, and when the following (i) is observed in a number equal to or greater than a preset specified number, the paternity test based on the collected sample is stopped without being executed. (i) A polymorphic locus that is homozygous for alleles with different genotypes in the father and the child

[0160] By providing a postnatal MC filter, it is not necessary for a witness to be present when collecting a sample from the biological mother even in a legal identification.

[0161] Also, as described above, the verification method of the present invention is not limited to paternity tests, but can be applied to the verification of any kinship test. 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 process is executed, the updated database is referenced, and verification is performed according to verification pattern 1 and / or verification pattern 2. As specifically described according to the embodiment in which there are two sets of identification target families to be verified, as shown in FIGS. 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) Based on the genotyping result of child CY and the genotyping results of each of the putative fathers AFA to AFZ stored in the updated database, paternity testing is performed for each combination of child CY and putative fathers AFA to AFZ.

[0168] (Verification pattern 2-1) Based on the genotyping result of putative father AFY and the genotyping results of each of children CA to CZ stored in the updated database, paternity testing is performed for each combination of putative father AFY and children CA to CZ.

[0169] (Verification pattern 1-2) Based on the genotyping result of child CZ and the genotyping results of each of the putative fathers AFA to AFZ stored in the updated database, paternity testing is performed for each combination of child CZ and putative fathers AFA to AFZ.

[0170] (Verification pattern 2-2) Based on the genotyping result of putative father AFZ and the genotyping results of each of children CA to CZ stored in the updated database, paternity testing is performed for each combination of putative father AFZ and children CA to CZ.

[0171] That is, in the present embodiment, the genotyping result of the specific identification target family reflected in the updated database is regarded as the genotyping result of another identification family, and verification is performed according to verification pattern 1 and / or verification pattern 2.

[0172] Hereinafter, in order to avoid duplication of description, description will be given according to the embodiment of executing verification pattern 1-2 and / or 2-2. Note that the following description is naturally applicable to the embodiment of executing verification pattern 1-1 and / or 2-1.

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

[0174] Also, if the biological parent-child relationship is negated by the two-sample identification process, in the verification process, paternity testing based on the genotyping results of child CZ and putative father AFZ stored in the updated database (verification pattern 1-2), and paternity testing based on the genotyping results of putative father AFZ and child CZ stored in the updated database (verification pattern 2-2) should yield a "negative" result.

[0175] That is, the updated database will necessarily be in a state equipped with a positive control that yields an "affirmative" result or a negative control that yields a "negative" result, so it is possible to confirm whether an error has occurred in the verification process itself.

[0176] The embodiment shown in FIG. 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 biological mother of the child. Further, this embodiment includes an MC filter process (FIG. 2). After the genotyping of three individuals belonging to a specific family to be identified is completed, the results are stored in a database and updated (FIG. 2). In addition, when there are two or more families to be identified as the target of the verification process, the database may be updated in a batch by registering them at once. In this case, as shown in FIGS. 3 to 6, the genotyping results of children CA to CZ and putative fathers AFA to AFZ are stored in the updated database.

[0177] In this embodiment, the updated database is referred to, and verification is performed according to verification pattern 1 and / or verification pattern 2 in the verification process. Specifically, according to the embodiment in which there are two families to be identified as the target of verification, as shown in FIGS. 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) Based on the genotyping result of child CY, optionally, the genotyping result of mother MY, and the genotyping results of putative fathers AFA to AFZ stored in the updated database, paternity testing is performed for each combination of child CY and putative fathers AFA to AFZ.

[0179] (Verification pattern 2-1) Based on the genotyping result of putative father AFY, optionally, the genotyping results of mothers MA to MZ stored in the updated database, and the genotyping results of children CA to CZ stored in the updated database, Based on this, paternity testing is performed for each combination of the putative father AFY and children CA to CZ.

[0180] (Verification Pattern 1-2) Based on the genotyping result of child CZ and optionally, the genotyping result of mother MZ and the genotyping results of each of the putative fathers AFA to AFZ stored in the updated database, paternity testing is performed for each combination of child CZ and putative fathers AFA to AFZ.

[0181] (Verification Pattern 2-2) Based on the genotyping result of putative father AFZ and optionally, the genotyping results of each of the mothers MA to MZ stored in the updated database and the genotyping results of each of the children CA to CZ stored in the updated database, paternity testing is performed for each combination of putative father AFZ and children CA to CZ.

[0182] Similar to the embodiment of FIG. 1, also in the embodiment of FIG. 2, the genotyping result of the specific family to be identified reflected in the updated database is regarded as the genotyping result of another family to be identified, and verification is performed according to Verification Pattern 1 and / or Verification Pattern 2.

[0183] Similar to the embodiment of FIG. 1, in the embodiment of FIG. 2, the updated database is in a state where there is a positive control that will surely give a "positive" result or a negative control that will give a "negative" result if there is a corresponding family to be identified with a positive result, so it is possible to confirm whether an error has occurred in the verification process itself.

Industrial Applicability

[0184] The present invention can be applied to paternity 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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