Noninvasive prenatal paternity appraisal method

Abstract

The first problem addressed is to provide a novel noninvasive prenatal paternity appraisal method capable of genotyping a fetus using simple criteria. The present invention for solving the first problem is a noninvasive prenatal paternity appraisal method that comprises performing a two-sample appraisal step by performing fetal genotyping by a criterion a determination method or performing a three-sample appraisal step by performing fetal genotyping by a criterion a determination method or a criterion b determination method.

Description

Non-invasive prenatal paternity testing method

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

[0002] A technique for determining the paternity of a fetus and a putative father before birth is known, based on the analysis of circulating cell-free DNA (cfDNA) in the blood. By analyzing fetal circulating cell-free DNA (cfDNA), which is genetic material derived from the fetus and present in the maternal blood circulation, it is possible to perform a non-invasive prenatal paternity test (NIPPT) (see, for example, Patent Document 1).

[0003] Special Publication No. 2014-502845

[0004] Non-invasive prenatal paternity testing identifies the genotype of a fetus based on the analysis results of a cfDNA sample. However, because cfDNA contains a mixture of maternal and fetal DNA, the signal measured from a single polymorphic locus contains a mixture of signals attributable to maternal alleles and fetal alleles. In order to perform fetal genotyping, the signal attributable to the fetal allele must be estimated from the mixed signals. Therefore, a first objective of the present invention is to provide a novel non-invasive prenatal paternity testing method that enables fetal genotyping using simple criteria.

[0005] The present invention that solves the first problem is as follows: [1] A non-invasive prenatal paternity testing method, comprising a two-sample testing step and / or a three-sample testing step as defined below.

[0006] <Two-Sample Identification Step> A step of identifying the biological parent-child relationship between the putative father and the fetus based on the results of genotyping the putative father and the fetus at multiple polymorphic loci, where the genotyping of the fetus is performed according to the Criterion A determination method defined below.

[0007] (Criterion a determination method) When the allele detected with the strongest signal intensity derived from a specific polymorphic locus contained in data obtained by analysis of a circulating cell-free nucleic acid sample collected from a pregnant mother is designated as allele A, and the allele detected with the same or second strongest signal intensity as allele A is designated as allele B, determination is performed according to the following criterion a, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensity derived from the specific polymorphic locus.

[0008] <Criterion a> If 0%≦Fb{≦,<}L%, the genotype of the fetus at the specific polymorphic locus is determined to be homozygous AA. If M%{≦,<}Fb{≦,<}N%, the genotype of the fetus at the specific polymorphic locus is determined to be heterozygous AB. If Fa=Fb, genotyping of the specific polymorphic locus is not performed and the test is skipped. If Fb does not satisfy any of the above conditions, genotyping of the specific polymorphic locus is not performed and the test is skipped. (N is a number less than 50, and satisfies the relational expression 0{≦,<}L<M{≦,<}N. The notation {≦,<} indicates that either the inequality sign ≦ or < can be selected arbitrarily.)

[0009] <Three-sample identification step> A step of identifying the biological parent-child relationship between the putative father and the fetus based on the results of genotyping the biological mother, the putative father, and the fetus at multiple polymorphic loci, where the fetus is genotyped according to the Criterion A determination method and / or the Criterion B determination method defined below.

[0010] (Criterion b Determination Method) When the allele detected with the strongest signal intensity derived from a specific polymorphic locus determined to be homozygous in the mother, contained in the data obtained by analysis of the circulating cell-free nucleic acid sample, is designated as allele A, and the allele detected with the same or second strongest signal intensity as allele A is designated as allele B, the determination is performed according to the following criterion b, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensities derived from the specific polymorphic locus.

[0011] <Criterion b> If 0%≦Fb{≦,<}L%, the genotype of the fetus at the specific polymorphic locus is determined to be homozygous AA. If M%{≦,<}Fb is satisfied, the genotype of the fetus at the specific polymorphic locus is determined to be heterozygous AB. If Fa=Fb, genotyping of the specific polymorphic locus is not performed and the process is skipped. If Fb does not satisfy any of the above conditions, genotyping of the specific polymorphic locus is not performed and the process is skipped. (The relational expression 0{≦,<}L<M is satisfied. Note that the notation {≦,<} indicates that either the inequality sign ≦ or < can be selected arbitrarily.)

[0012] [2] The non-invasive prenatal paternity testing method according to [1], wherein the assessment of the presence or absence of a biological parent-child relationship comprises calculating a combined paternity index (CPI) by multiplying the paternity indexes (PI) calculated for each polymorphic locus, and making a determination of "yes," "no," or "cannot be determined" according to predetermined criteria depending on the CPI value, wherein the PI is calculated based on Table 1 below in the two-sample testing step, and the PI is calculated based on Table 1 or Table 2 below in the three-sample testing step. (In Tables 1 and 2, an allele with a relatively high signal intensity in the analysis of a circulating cell-free nucleic acid sample is represented as A, and an allele with a relatively low signal intensity is represented as B. In Table 1, k0 is the false negative rate, kb is the false positive rate, a is the frequency of occurrence of A, and b is the frequency of occurrence of B.)

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

[0014] <MC filter step> Genotyping of the mother is performed based on a sample collected from the mother, the sample containing nucleic acid containing the genetic information of the mother and substantially no nucleic acid containing genetic information of the fetus, and if a predetermined number or more of the following are observed: (i) polymorphic loci that are homozygous for alleles whose genotypes in the mother and the fetus or circulating cell-free nucleic acid are different from each other, and / or (ii) polymorphic loci whose genotype in the mother is heterozygous and whose genotype in circulating cell-free nucleic acid is homozygous, paternity testing based on the collected sample is not performed and the process is stopped.

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

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

[0017] (Verification Pattern 1) Based on the genotyping results of the fetus belonging to the specific family to be tested, and optionally the genotyping results of the mother belonging to the specific family to be tested, and the genotyping results of the putative father belonging to another family to be tested, parent-child testing is performed for this combination of putative father and fetus.

[0018] (Verification Pattern 2) Based on the genotyping results of the fetus belonging to the other family to be tested, and optionally the genotyping results of the biological mother of the fetus who belongs to the other family to be tested, and the genotyping results of the putative father who belongs to the specific family to be tested, parent-child testing is performed for this combination of putative father and fetus.

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

[0020] [6] The non-invasive prenatal paternity testing method according to [5], wherein after genotyping of the putative father, the fetus, and optionally the mother belonging to the specific family to be tested is completed, the results are stored in the database and updated, and the verification step includes performing the verification step by referring to the updated database, and in the verification step, the genotyping results of the specific family to be tested reflected in the database are considered to be the genotyping results of another family to be tested, and verification is performed in accordance with verification pattern 1 and / or verification pattern 2.

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

[0022] Furthermore, in the field of blood relationship testing, one person tests a sample of a potential blood relative taken from a specific family to be tested and a sample from a specific individual. In this case, sample mix-ups can occur, resulting in incorrect blood relationship testing results. One method for preventing such sample mix-ups is to perform a double check by conducting the same test twice, but this has the problem of doubling the testing costs. In view of this problem, a second object of the present invention is to provide a new technology for verifying erroneous determinations due to sample mix-ups in blood relationship testing. The present invention, which solves the second object, is as follows.

[0023] [1] A method for verifying erroneous determination due to sample mix-up in blood relationship testing between a specific person and a pseudo-blood relative, based on samples collected from a set of specific subject families, including the specific person and, optionally, the specific person's biological blood relatives, who are the subject of testing for the existence or nonexistence of a biological blood relationship, the method comprising the verification step defined below.

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

[0025] (Verification pattern 1) Based on the genotyping results of the specific person belonging to the specific family to be verified, and optionally the genotyping results of a biological blood relative of the specific person belonging to the specific family to be verified, and the genotyping results of a pseudo-blood relative belonging to another family to be verified, blood relationship verification is performed for the combination of this pseudo-blood relative and the specific person.

[0026] (Verification pattern 2) Based on the genotyping results of a specific person belonging to another family to be verified, and optionally the genotyping results of a biological blood relative of the specific person belonging to another family to be verified, and the genotyping results of the pseudo-blood relative belonging to the specific family to be verified, blood relationship verification is performed for the combination of this pseudo-blood relative and the specific person.

[0027] [2] The pseudo-blood relative is a pseudo-parent, the specific person is a child, a biological blood relative of the specific person is a biological parent of the child who is of a different sex from the pseudo-parent, the blood relationship testing is a parent-child testing, and a method for verifying an erroneous determination due to a mix-up of samples in a paternity testing between the pseudo-parent and the child based on samples collected from a set of specific subject families including the pseudo-parent, the child, and optionally the biological parent, who are the subjects of testing for the existence of a biological parent-child relationship, the verification method described in [1], characterized in that it includes a verification step defined below.

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

[0029] (Verification Pattern 1) Parent-child testing is performed for this combination of pseudo-parent and child based on the genotyping results of the child belonging to the specific family to be tested, and optionally, the genotyping results of the biological parent belonging to the specific family to be tested, and the genotyping results of a pseudo-parent belonging to another family to be tested.

[0030] (Verification Pattern 2) Parent-child testing is performed for this combination of pseudo-parent and child based on the genotyping results of the child belonging to the other family to be tested, and optionally the genotyping results of the child's biological parent who belongs to the other family to be tested, and the genotyping results of the pseudo-parent who belongs to the specific family to be tested.

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

[0032] [4] The verification method according to any one of [1] to [3], wherein the blood relationship testing is based on the results of genotyping three people: the pseudo-blood relative who is the subject of the testing to determine whether or not there is a biological parent-child relationship; the specific person; and the biological blood relative.

[0033] [5] The verification method according to any one of [1] to [4], wherein in the verification step, verification is performed according to verification pattern 1 and / or verification pattern 2 by referring to a database storing genotyping results between pseudo-relatives and a specific person who belong to one or more of the other families to be verified. [6] The verification method according to [2], wherein in the verification step, verification is performed according to verification pattern 1 and / or verification pattern 2 by referring to a database storing genotyping results between pseudo-parents, children, and optionally the biological parents of the children, who belong to one or more of the other families to be verified.

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

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

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

[0037]

[10] The verification method according to any one of [1] to [9], wherein the genotyping of the pseudo-kin and the genotyping of the specific individual are performed at different locations and / or at different times, and / or by different operators.

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

[0039] Furthermore, according to the present invention which solves the second problem, it is possible to verify with high accuracy whether or not an erroneous determination has occurred due to the wrong sample being taken.

[0040] 1 shows a flowchart of an embodiment including a two-sample identification step and a verification step. 2 shows a flowchart of an embodiment including an MC filter step, a three-sample identification step, and a verification step. 3 shows a schematic diagram of verification pattern 1 in which two-sample identification is performed by referencing a database. 4 shows a schematic diagram of verification pattern 2 in which two-sample identification is performed by referencing a database. 5 shows a schematic diagram of verification pattern 1 in which three-sample identification is performed by referencing a database. 6 shows a schematic diagram of verification pattern 2 in which three-sample identification is performed by referencing a database. 7 shows a diagram in which circulating cell-free nucleic acid samples were analyzed and the signal intensities of alleles were blotted for each polymorphic locus. The vertical axis represents the ratio of a signal indicating the presence of a specific allele to the sum of signal intensities derived from the specific polymorphic locus.

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

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

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

[0044] (Criterion a determination method) When the allele detected with the strongest signal intensity derived from a specific polymorphic locus contained in data obtained by analysis of a circulating cell-free nucleic acid sample collected from a pregnant mother is designated as allele A, and the allele detected with the same or second strongest signal intensity as allele A is designated as allele B, determination is performed according to the following criterion a, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensity derived from the specific polymorphic locus.

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

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

[0047] From the viewpoint of preventing misidentification of the signal of the maternal allele as the fetal allele, N is preferably set to 40 or less, more preferably 30 or less, and even more preferably 25 or less. This makes it possible to exclude polymorphic loci that are likely to misidentify the signal of the maternal allele as the fetal allele from the basis of paternity testing, thereby reducing the possibility of erroneous determination.

[0048] Furthermore, a condition may be added to the above-mentioned criterion a that, if the noise derived from a specific polymorphic locus exceeds a certain criterion, the specific polymorphic locus is skipped without being genotyped. That is, when the alleles detected with the third and fourth strongest signal intensities derived from a specific polymorphic locus contained in the data obtained by analyzing a circulating cell-free nucleic acid sample are designated as allele C and allele D, respectively, the following skip condition may be added to the above-mentioned criterion a, using the ratio of the signal intensity of allele C (Fc) and the ratio of the signal intensity of allele D (Fd) to the sum of the signal intensities derived from the specific polymorphic locus as indicators: If K{≦,<}Fc+Fd is satisfied, the specific polymorphic locus is skipped without being genotyped.

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

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

[0051] (Criterion b Determination Method) When the allele detected with the strongest signal intensity derived from a specific polymorphic locus determined to be homozygous in the mother, contained in the data obtained by analysis of the circulating cell-free nucleic acid sample, is designated as allele A, and the allele detected with the same or second strongest signal intensity as allele A is designated as allele B, the determination is performed according to the following criterion b, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensities derived from the specific polymorphic locus.

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

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

[0054] Furthermore, a condition may be added to the above-mentioned criterion b that, if the noise derived from a specific polymorphic locus exceeds a certain criterion, the specific polymorphic locus is skipped without being genotyped. That is, when the alleles detected with the third and fourth strongest signal intensities derived from a specific polymorphic locus contained in the data obtained by analyzing a circulating cell-free nucleic acid sample are designated as allele C and allele D, respectively, the following skip condition may be added to the above-mentioned criterion b, using the ratio of the signal intensity of allele C (Fc) and the ratio of the signal intensity of allele D (Fd) to the sum of the signal intensities derived from the specific polymorphic locus as indicators: If K{≦,<}Fc+Fd is satisfied, the specific polymorphic locus is skipped without being genotyped.

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

[0056] In the present invention, the analytical method for nucleic acid samples for genotyping is not particularly limited. Preferred examples of analytical methods that can distinguish and detect single nucleotide variations (SNVs) at polymorphic loci include base sequence analysis, mass spectrometry, digital PCR, SNV microarrays, and real-time PCR. Specific examples of base sequence analysis methods include next-generation sequencers (NGS). Preferred examples of polymorphic loci to be analyzed include single nucleotide polymorphism loci.

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

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

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

[0060]

[0061]

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

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

[0064] In the present invention, it is preferable to make a determination of "positive," "negative," or "undecidable" according to predetermined criteria depending on the CPI value, for example, as shown below. ４ ≦CPI...Result: "affirmative" 10 －６ ≦CPI<10 ４ ...Result: "Undetermined" CPI<10 －６ ...Result: "Negation"

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

[0066] After calculating the CPI, the probability of paternity (POP) may be calculated. POP can be calculated using the following formula: where P represents the prior probability of paternity.

[0067] In embodiments that include a three-sample authentication step, it is preferred to include an MC filter step as defined below (FIG. 2).

[0068] <MC filter step> Genotyping of the mother is performed based on a sample collected from the mother, the sample containing nucleic acid containing the genetic information of the mother and substantially not containing nucleic acid containing genetic information of the fetus, and if the following (i) and / or (ii) are observed in a predetermined number of times or more, the paternity test based on the collected sample is stopped without being performed: (i) a polymorphic locus where the mother and the fetus or the circulating cell-free nucleic acid are homozygous for alleles whose genotypes are different from each other, (ii) a polymorphic locus where the genotype in the mother is heterozygous and the genotype in the circulating cell-free nucleic acid is homozygous

[0069] A circulating cell-free nucleic acid sample contains nucleic acids derived from the mother as the major nucleic acid. Therefore, when analyzing a circulating cell-free nucleic acid sample and genotyping the fetus or circulating cell-free nucleic acid, it is generally unlikely that the genotypes of the mother and fetus or circulating cell-free nucleic acid will meet the conditions (i) and / or (ii) above. In such cases, sample mix-up is suspected.

[0070] The MC filter process is performed to stop the analysis without executing the three-sample analysis process if a predetermined number of such cases of suspected sample mix-up are observed (Figure 2), in order to prevent incorrect analysis results and unnecessary work.

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

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

[0073] Genotyping of circulating cell-free nucleic acids to identify the genotype of circulating cell-free nucleic acids will now be described with reference to Figure 7. A circulating cell-free nucleic acid sample contains major nucleic acids derived from the mother and minor nucleic acids derived from the fetus. Therefore, when a circulating cell-free nucleic acid sample is analyzed and the ratio of a signal indicating the presence of a specific allele to the total signal intensity derived from a specific polymorphic locus is blotted, the ratio is distributed between 0-25%, 35-65%, and 75-100%, as shown in Figure 7. Assuming no external contamination with DNA from other individuals, signals with a ratio of 5% or greater at each polymorphic locus are considered to be true signals.

[0074] Based on this, when the allele detected with the strongest signal intensity derived from a specific polymorphic locus contained in the data obtained by analysis of the circulating cell-free nucleic acid sample is designated as allele A, and the allele detected with the same or second strongest signal intensity as allele A is designated as allele B, genotyping of the circulating cell-free nucleic acid is performed according to the following criterion c, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the total signal intensity derived from the specific polymorphic locus.

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

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

[0077] In addition, if the ratio does not fall into any of the above categories, genotyping of the specific allele may be skipped without being performed.

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

[0079] In one embodiment of the present invention, the above-mentioned "genotyping of the mother based on a sample collected from the mother, which contains nucleic acid containing genetic information of the mother and is substantially free of nucleic acid containing genetic information of the fetus" and "genotyping of circulating cell-free nucleic acids based on a circulating cell-free nucleic acid sample collected from the mother" are performed, followed by an MC filter step.

[0080] The stopping condition employed in the MC filtering step may be either or both of (i) and (ii) above.

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

[0082] In one embodiment of the present invention, if a decision to stop is not made in the MC filter process, the analysis based on the collected samples continues (FIG. 2).On the other hand, if a decision to stop is made in the MC filter process, the analysis based on the collected samples is stopped without being executed (FIG. 2).

[0083] In normal legal testing, a witness is required for all sample collection. However, when the MC filter process is performed, it is possible to determine whether or not the maternal nucleic acid sample is mistaken for the circulating cell-free nucleic acid sample. This eliminates the need for a witness to be present when the maternal blood sample is collected, which is necessary for legal testing. This has the advantage that legal testing can be performed with only the presence of a witness for the maternal genomic DNA sample.

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

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

[0086]

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

[0088] 1 and 2 show an embodiment in which the verification step is performed regardless of the results of the two-sample authentication step or the three-sample authentication step. In such an embodiment, it is possible to verify whether the negative or positive results of the two-sample authentication step or the three-sample authentication step include any erroneous determinations due to sample mix-ups. If it is determined in the verification step that sample mix-ups have occurred, it can be determined that the results of the two-sample authentication step or the three-sample authentication step are unreliable.

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

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

[0091] Specific embodiments of the verification step will be described in detail in the following section on the verification method invention.

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

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

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

[0095] For example, in the case of assessing a biological sibling relationship, a specific person and their pseudo-siblings constitute the family to be assessed. In this case, biological blood relatives, such as the specific person's biological parents or siblings, may optionally be included in the family to be assessed. Furthermore, in the case of assessing the relationship between biological grandparents and grandchildren, a pseudo-grandfather or pseudo-grandmother and their grandchild constitute the family to be assessed. In this case, biological blood relatives, such as the grandchild's biological parents or a biological grandmother or grandfather of a different gender from the pseudo-blood relative, the pseudo-grandfather or pseudo-grandmother, may optionally be included in the family to be assessed.

[0096] Furthermore, when parentage testing is performed, the family to be tested includes a pseudo-parent and a child, who are the subjects of testing to determine whether or not there is a biological parent-child relationship. The family to be tested may also optionally include a biological parent of the child, who is of a different gender from the pseudo-parent. In other words, the family to be tested may include only two people, the pseudo-parent and the child, or it may include three people: the pseudo-parent, the child, and the biological parent. In other words, the verification method of the present invention is applicable to both the two-sample testing process and the three-sample testing process described above.

[0097] The "biological parent of the child, who is of a different sex from the pseudo parent" is the "biological mother of the child" if the pseudo parent is a "pseudo father," and is the "biological father of the child" if the pseudo parent is a "pseudo mother." In other words, the verification method of the present invention is applicable to both paternity testing and mother-child testing. Note that an embodiment may also be adopted in which a parent (father or mother) is included as a specific person constituting the family to be tested, and a pseudo child is included as a pseudo blood relative.

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

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

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

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

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

[0103] If genotyping of a family member and genotyping of a specific individual are performed by a single person at the same time and in the same place, there is a possibility that the sample of the entire family will be mixed up instead of just a portion of the family. If a sample mix-up occurs, it may not be possible to definitively detect the sample mix-up in the verification process described below.

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

[0105] In this way, when genotyping of the kin and the specific individual is performed at different locations, at different times, and by different operators, the likelihood of the same sample being mixed up in each genotyping is reduced. By implementing this type of genotyping and then performing verification using the verification process described below, the extremely advantageous effect of significantly improving verification accuracy can be obtained.

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

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

[0108] (Verification Pattern 1) Parentage testing is performed for this combination of pseudo parent and child based on the genotyping results of the child belonging to the specific family to be tested, and optionally, the genotyping results of the biological parent belonging to the specific family to be tested, and the genotyping results of the pseudo parent belonging to the other family to be tested. (Verification Pattern 2) Parentage testing is performed for this combination of pseudo parent and child based on the genotyping results of the child belonging to the other family to be tested, and optionally, the genotyping results of the biological parent of the child belonging to the other family to be tested, and the genotyping results of the pseudo parent belonging to the specific family to be tested.

[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 tested and a pseudo-parent belonging to another family to be tested. The parentage testing performed here may be a two-sample test or a three-sample test.

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

[0111] In the three-sample testing in verification pattern 1, parentage testing is performed based on the genotyping results of three individuals: a child belonging to a specific family to be tested, the child's biological parent who belongs to the specific family to be tested, and a putative parent who belongs to another family to be tested. The specific aspects of this testing can be directly applied to the above description of the 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 tested and a pseudo-parent belonging to a specific family to be tested. The parentage testing performed here may be a two-sample test or a three-sample test.

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

[0114] In the three-sample testing in Verification Pattern 2, parentage testing is performed based on the genotyping results of three individuals: a child belonging to another family to be tested, the child's biological parent who belongs to another family to be tested, and a putative parent who belongs to a specific family to be tested. The specific aspects of this testing can be directly applied to the above description of the three-sample testing process.

[0115] Furthermore, if the paternity test to be verified for sample mix-up is a two-sample test, the paternity test conducted in the verification process is not limited to a two-sample test, and there is no problem with conducting a three-sample test in the verification process.

[0116] Conversely, if the paternity test to be verified for sample mix-up is a three-sample test, the paternity test conducted in the verification process is not limited to a three-sample test, and there is no problem with conducting a two-sample test in the verification process.

[0117] If verification pattern 1 and / or verification pattern 2 produces a test result that affirms a biological parent-child relationship, it can be determined that there is a high possibility that a sample from a specific family to be tested has been mistaken for a sample from another family to be tested. In other words, there is a high possibility that a sample from a subject who belongs to a specific family to be tested, and who has a biological parent-child relationship, has been treated as belonging to a different family to be tested, and it can be determined that the test result of the paternity test being verified contains an erroneous determination.

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

[0119] If, through the verification process, it is determined that the results of the paternity test being verified contain an incorrect judgment due to the misidentification of a sample taken from a specific family to be tested with a sample taken from another family to be tested, a final conclusion of "indeterminable" 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 analyzed are stored will be described with reference to FIGS. 3 to 6 as appropriate.

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

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

[0123] The database pre-stores the results of genotyping for offspring CA-CX and putative fathers AFA-AFX, each belonging to a plurality of sets of families A-X to be authenticated that are different from the specific families Y and Z to be authenticated (FIG. 3). After genotyping for offspring CY and putative father AY, belonging to family Y to be authenticated, and for offspring CZ and putative father AZ, belonging to family Z to be authenticated, is completed, the database is updated before the verification step is performed, and the genotyping results for offspring CY and putative father AFY, and for offspring CZ and putative father AFZ, are stored in the database (FIG. 3). In other words, the verification step is performed by referencing the database in which the genotyping results for offspring CA-CZ and putative fathers AFA-AFZ are stored.

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

[0125] In the following, to avoid duplication, the explanation will be limited to the case where paternity testing is performed based on the genotyping results of the child CZ and the putative fathers AFA to AFZ separately. Needless to say, the following explanation also applies when paternity testing is performed based on the genotyping results of the child CY and the putative fathers AFA to AFZ separately.

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

[0127] Furthermore, if a "positive" result is obtained in the paternity test being verified, then a "positive" result should also be obtained in the verification of the putative father AFZ stored in the database through an update. On the other hand, unless there are special circumstances such as the putative father AFZ having been registered in the database as one of the putative fathers AFA to AFY because he was the subject of a paternity test in another case, a "negative" result should be obtained in the verification of the putative fathers AFA to AFY stored in the database. In other words, if a "positive" result is obtained in the paternity test being verified, then in the verification process, the putative father AFZ stored in the database through an update will function as a positive control, while the putative fathers AFA to AFY will function as negative controls unless there are special circumstances as described above, and therefore it can be confirmed that the verification process itself is being carried out normally.

[0128] Conversely, if a "negative" result is obtained in the paternity test that is the subject of verification, a "negative" result should also be obtained in the verification of the putative father AFZ stored in the database by updating. In other words, in the verification process, the putative father AFZ stored in the database by updating acts as a negative control, making it possible to confirm that the verification process itself is being carried out normally.

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

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

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

[0132] In the following, to avoid duplication, the explanation will be limited to the case where paternity testing is performed based on the genotyping results of the putative father AFZ and the children CA to CZ. Needless to say, the following explanation also applies to the case where paternity testing is performed based on the genotyping results of the putative father AFY and the children CA to CZ.

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

[0134] Furthermore, if a "positive" determination is obtained in the paternity testing that is the subject of verification, a "positive" result should also be obtained in the verification of child CZ stored in the database through an update. On the other hand, unless there are special circumstances such as child CZ having been registered in the database as one of children CA to CY because it was the subject of a paternity testing in another case, a "negative" result should be obtained in the verification of children CA to CY stored in the database. In other words, if a "positive" determination is obtained in the paternity testing that is the subject of verification, child CZ stored in the database through an update will act as a positive control in the verification process, while children CA to CY will act as negative controls unless there are special circumstances as described above, thereby confirming that the verification process itself is being carried out normally.

[0135] Conversely, if a negative result is obtained in the parent-child test that is the subject of verification, a negative result should also be obtained in the verification of the child CZ stored in the database by updating. In other words, in the verification process, the child CZ stored in the database by updating acts as a negative control, making it possible to confirm that the verification process itself is being carried out normally.

[0136] 5 shows an embodiment for executing Verification Pattern 1. In this embodiment, the verification process targets parent-child tests (paternity tests) conducted on two target families: a specific target family Y including three members: child CY, child CY's biological mother MY, and pretended father AFY; and a specific target family Z including three members: child CZ, child CZ's biological mother MZ, and pretended father AFZ.

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

[0138] The database pre-stores the genotyping results for children CA-CX, their respective biological mothers MA-MX, and their respective biological fathers AFA-AFX, each belonging to a plurality of separate families A-X, each different from the specific families Y and Z (FIG. 5). After genotyping is complete for child CY, biological father AFY, and mother MZ belonging to family Y, as well as child CZ, biological father AFZ, and mother MZ belonging to family Z, the database is updated before the verification step is performed, and the genotyping results for child CY, biological father AFY, and mother MY, as well as child CZ, biological father AFZ, and mother MZ, are stored in the database (FIG. 5). In other words, the verification step is performed by referencing the database in which the genotyping results for children CA-CZ, biological fathers AFA-AFZ, and mothers MA-MZ are stored.

[0139] In the embodiment of Figure 5, paternity testing is performed 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. That is, paternity testing (three-sample testing) is performed for each of the following combinations: 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; and child CY, mother MY, and putative father AFZ. Furthermore, in the embodiment of Figure 5, paternity testing is similarly performed independently 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.

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

[0141] If the existence of a biological parent-child relationship between child CZ and any of the putative fathers AFA-AFY is confirmed, it can be determined that the combination of samples of child CZ, mother MZ, and putative father AFZ, which had been treated as having been obtained from a specific set of family Z to be tested, was mistaken for a sample obtained from a member of another family to be tested. In other words, it can be determined that the results of the paternity test performed by analyzing the samples of child CZ, mother MZ, and putative father AFZ belonging to family Z to be tested contain an error. Note that if the above-mentioned MC filtering process is passed, the mix-up of the samples of child CZ and mother MZ does not need to be taken into consideration.

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

[0143] Conversely, if a "negative" result is obtained in the paternity test that is the subject of verification, a "negative" result should also be obtained in the verification of the putative father AFZ stored in the database by updating. In other words, in the verification process, the putative father AFZ stored in the database by updating acts as a negative control, making it possible to confirm that the verification process itself is being carried out normally.

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

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

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

[0147] If the existence of a biological parent-child relationship between putative father AFZ and any of children CA-CY is confirmed, it can be determined that the combination of samples of child CZ, mother MZ, and putative father AFZ, which had been treated as having been obtained from a specific set of family Z to be tested, was mistaken for a sample obtained from a member of another family to be tested. In other words, it can be determined that the results of the paternity test performed by analyzing the samples of child CZ, mother MZ, and putative father AFZ belonging to family Z to be tested contain an error. Note that if the above-mentioned MC filtering process is passed, the mix-up of the samples of child CZ and mother MZ does not need to be taken into consideration.

[0148] Furthermore, if a "positive" result is obtained in the paternity testing that is the subject of verification, a "positive" result should also be obtained in the verification of child CZ and mother MZ that have been stored in the database through an update. On the other hand, unless there are special circumstances such as child CZ and mother MZ having been registered in the database as one of children CA-CY and mothers MA-MY because they were the subject of a paternity testing in another case, a "negative" result should be obtained in the verification of children CA-CY and mothers MA-MY that have been stored in the database. In other words, if a "positive" result is obtained in the paternity testing that is the subject of verification, child CZ and mother MZ that have been stored in the database through an update will function as positive controls in the verification process, while children CA-CY and mothers MA-MY will function as negative controls unless there are special circumstances as described above. This confirms that the verification process itself is being carried out normally.

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

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

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

[0152] In the above-mentioned cases 1 to 3, it is possible to discover that a sample mix-up has occurred by going through the verification process. However, in the case of case 4, where there is in fact no biological parent-child relationship between child CY and putative father AFY, and between child CZ and putative father AFZ, it is not possible to discover that a sample mix-up has occurred even after going through the verification process. However, in this case, although an error in the subject's DNA profile due to the sample mix-up is included, the final determination result of denying the biological parent-child relationship remains unchanged.

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

[0154] The verification method of the present invention can be applied to both parentage testing conducted before the birth of a child and parentage testing conducted after the birth of a child. When the parentage testing to which the verification method of the present invention is applied is conducted before the birth of a child, an MC filter step may be included as described above. Furthermore, when the parentage testing to which the verification method of the present invention is applied is conducted after the birth of a child, a filter similar to the MC filter described above can also be used. Below, the two cases of paternity testing and maternal-child testing will be described separately.

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

[0156] <Postnatal MC filter> Genotyping of the child based on a sample containing nucleic acid including the child's genetic information collected from the child after birth, and genotyping of the mother based on a sample containing nucleic acid including the mother's genetic information collected from the child's biological mother, are carried out, and paternity testing based on the collected samples is stopped without being carried out if the following (i) is observed more than a predetermined number of times: (i) a polymorphic locus in which the mother and the child are homozygous for alleles whose genotypes are different from each other;

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

[0158] Furthermore, in a paternity test (maternity test) that is carried out by analyzing samples collected from three people, namely, the child after birth, the child's biological father, and the adoptive mother, it is preferable to include a post-natal FC filter step defined below.

[0159] <Postnatal FC filter> Genotyping of the child based on a sample containing nucleic acid containing the child's genetic information collected from the child after birth, and genotyping of the father based on a sample containing nucleic acid containing the father's genetic information collected from the child's biological father, are carried out, and paternity testing based on the collected samples is stopped without being carried out if the following (i) is observed more than a predetermined number of times: (i) a polymorphic locus in which the father and the child are homozygous for alleles whose genotypes are different from each other;

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

[0161] Furthermore, as mentioned above, the verification method of the present invention is not limited to parent-child testing, but can be applied to the verification of any kind of blood relationship testing. The "appearance father" and "child" in Figures 3 and 4 can be replaced with any "appearance relative" and "specific person," respectively, depending on the structure of the family being verified in the blood relationship testing to be verified. Furthermore, the "appearance father," "child," and "biological mother" in Figures 5 and 6 can be replaced with any "appearance relative," "specific person," and "biological blood relative," respectively, depending on the structure of the family being verified in the blood relationship testing to be verified.

[0162] <3> Specific Embodiment of Parentage Testing A parentage testing method employing the detection method of the present invention will be described with reference to FIGS. 1 and 2.

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

[0164] The database to be referenced in the verification process contains pre-stored genotyping results for another family to be verified. This embodiment also includes updating the database by storing the genotyping results after completing genotyping for two members of a specific family to be verified ( 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 all at once. In this case, the updated database will contain the genotyping results for children CA-CZ and adoptive fathers AFA-AFZ, as shown in FIGS. 3 to 6 .

[0165] After genotyping of two members of a specific 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. Specific explanation will be given for an embodiment in which there are two sets of appraisal target families to be verified, as shown in Figures 3 to 6, and 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 results of child CY and the genotyping results of each of the putative fathers AFA to AFZ stored in the updated database, parentage testing is performed for each combination of child CY and putative fathers AFA to AFZ.

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

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

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

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

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

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

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

[0175] In other words, the updated database will always contain a positive control that will produce a ``positive'' result or a negative control that will produce a ``negative'' result, so it will be possible to check whether any errors have occurred in the verification process itself.

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

[0177] In this embodiment, the updated database is referenced, and in the verification process, verification is performed according to verification pattern 1 and / or verification pattern 2. Specific explanation will be given according to an embodiment in which there are two sets of appraisal target families to be verified, as shown in Figures 3 to 6, and 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 results of child CY, and optionally the genotyping results of mother MY, and the genotyping results of each of the putative fathers AFA to AFZ stored in the updated database, parent-child testing is performed for each combination of child CY and putative father AFA to AFZ.

[0179] (Verification pattern 2-1) Based on the genotyping results of the putative father AFY, 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, parentage testing is performed for each combination of the putative father AFY and the children CA to CZ.

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

[0181] (Verification pattern 2-2) Based on the genotyping results of the 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, parentage testing is performed for each combination of the putative father AFZ and the children CA to CZ.

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

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

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

Claims

1. A non-invasive prenatal paternity testing method using maternal blood, comprising a two-sample testing step and / or a three-sample testing step as defined below. <Two-sample testing step> A step of testing the presence or absence of a biological parent-child relationship between the putative father and the fetus based on the results of genotyping the putative father and the fetus at a plurality of polymorphic loci. However, the genotyping of the fetus is performed according to the criterion a determination method as defined below. (Criterion a determination method) When the allele detected with the strongest signal intensity originating from a specific polymorphic locus contained in data obtained by analysis of a circulating cell-free nucleic acid sample collected from a pregnant mother is denoted as allele A, and the allele detected with the same or second strongest signal intensity as allele A is denoted as allele B, the following criterion a is used as an index to determine the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the sum of the signal intensities originating from the specific polymorphic locus. <Criteria a> - If 0%≦Fb{≦,<}L%, the genotype of the fetus at the specific polymorphic locus is determined to be homozygous AA. - If M%{≦,<}Fb{≦,<}N%, the genotype of the fetus at the specific polymorphic locus is determined to be heterozygous AB. - If Fa=Fb, skip without genotyping the specific polymorphic locus. - If Fb does not satisfy any of the above conditions, skip without genotyping the specific polymorphic locus. (N is a number 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 selected arbitrarily.) <Three-Sample Identification Process> A process of identifying the presence or absence of a biological parent-child relationship between the putative father and the fetus based on the results of genotyping the biological mother, the putative father, and the fetus at multiple polymorphic loci. However, genotyping of said fetus is performed according to the criterion a method described above or the criterion b method defined below.(Criterion b determination method) When the allele detected with the strongest signal intensity derived from a specific polymorphic locus determined to be homozygous in the mother, contained in the data obtained by analysis of the circulating cell-free nucleic acid sample, is denoted as allele A, and the allele detected with the same or the second strongest signal intensity as allele A is denoted as allele B, the determination is performed according to the following criterion b, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the sum of the signal intensities derived from the specific polymorphic locus. <Criterion b> - If 0%≦Fb{≦,<}L%, the genotype of the fetus at the specific polymorphic locus is determined to be homozygous AA. - If M%{≦,<}Fb is satisfied, the genotype of the fetus at the specific polymorphic locus is determined to be heterozygous AB. - If Fa=Fb, skip without genotyping the specific polymorphic locus. - If Fb does not satisfy any of the above conditions, skip without genotyping the specific polymorphic locus. (The relationship 0{≦,<}L<M is satisfied. Note that the notation {≦,<} indicates that either the inequality sign ≦ or < can be selected arbitrarily.) 2. The non-invasive prenatal paternity testing method of claim 1, wherein the assessment of the presence or absence of a biological parent-child relationship includes calculating a combined paternity index (CPI) by multiplying the paternity indexes (PI) calculated for each polymorphic locus, and making a determination of "positive," "negative," or "undeterminable" in accordance with predetermined criteria depending on the CPI value, wherein in the two-sample testing step, the PI is calculated based on the following Table 1, and in the three-sample testing step, the PI is calculated based on the following Table 1 or Table 2. (In Tables 1 and 2, an allele with a relatively high signal intensity in the analysis of a circulating cell-free nucleic acid sample is represented as A, and an allele with a relatively low signal intensity is represented as B. In Table 1, k0 is the false negative rate, kb is the false positive rate, a is the frequency of occurrence of A, and b is the frequency of occurrence of B.) 3. The non-invasive prenatal paternity testing method according to claim 1 or 2, comprising the three-sample identification step and also comprising an MC filtering step defined below: <MC filtering step> Genotyping the mother is performed based on a sample collected from the mother, the sample containing nucleic acid containing genetic information of the mother and substantially not containing nucleic acid containing genetic information of the fetus, and when a predetermined number or more of (i) polymorphic loci in which the mother and the fetus or the circulating cell-free nucleic acid have different genotypes, which are homozygous alleles, and / or (ii) polymorphic loci in which the genotype in the mother is heterozygous and the genotype in the circulating cell-free nucleic acid is homozygous, are observed, the paternity testing based on the collected sample is not performed and the process is stopped.

4. The non-invasive prenatal parent-child testing method according to claim 3, wherein genotyping for identifying the genotype of the circulating cell-free nucleic acid is performed by the method specified below. <Genotyping of circulating cell-free nucleic acid> When the allele detected with the strongest signal intensity from a specific polymorphic locus contained in the data obtained by analysis of the circulating cell-free nucleic acid sample is denoted as allele A, and the allele detected with the same or second strongest signal intensity as that of allele A is denoted as allele B, genotyping is performed according to the following criterion c, using as indicators the ratio of the signal intensity of allele A (Fa) and the ratio of the signal intensity of allele B (Fb) to the sum of the signal intensities from the specific polymorphic locus. <Criterion c> - If X{≦,<}Fa{≦,<}Y%, the genotype of the circulating cell-free nucleic acid at the specific polymorphic locus is determined to be heterozygous AB. If Z% {≦,<} Fa≦100 is satisfied, the genotype at the specific polymorphic locus of the circulating cell-free nucleic acid is determined to be homozygous AA. If Fa=Fb is satisfied, the genotype at the specific polymorphic locus is determined to be heterozygous AB. (The relational expression X{≦,<} Y<Z is satisfied. Note that the notation {≦,<} indicates that either the inequality sign ≦ or < can be selected arbitrarily.) 5. The non-invasive prenatal paternity testing method according to claim 1 or 2, characterized in that a verification step defined below is carried out based on a sample collected from a specific family to be tested. <Verification step> In order to verify erroneous determination due to mistaking a sample collected from another family to be tested for the specific family to be tested, verification is carried out according to verification pattern 1 and / or verification pattern 2 defined below. (Verification pattern 1) A paternity test is carried out for the combination of the pretend father and the fetus based on the result of genotyping of the fetus belonging to the specific family to be tested, and optionally, the result of genotyping of the mother belonging to the specific family to be tested, and the result of genotyping of the pretend father belonging to the other family to be tested. (Verification pattern 2) A paternity test is carried out for the combination of the pretend father and the fetus based on the result of genotyping of the fetus belonging to the other family to be tested, and optionally, the result of genotyping of the biological mother of the fetus belonging to the other family to be tested, and the result of genotyping of the pretend father belonging to the specific family to be tested.

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

7. A non-invasive prenatal parent-child testing method as described in claim 6, wherein after genotyping of the putative father, the fetus, and optionally the mother belonging to the specific family to be tested is completed, the results are stored in the database and updated, and the verification process includes performing the verification process by referring to the updated database, and in the verification process, the genotyping results of the specific family to be tested reflected in the database are regarded as the genotyping results of the other family to be tested, and verification is performed in accordance with verification pattern 1 and / or verification pattern 2.

8. The non-invasive prenatal paternity testing of claim 5, wherein the genotyping of the putative father and the genotyping of the fetus are performed at different locations and / or at different times, and / or by different operators.

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