A method for simultaneously measuring the amounts of T cell receptor gene rearrangement fragments (TREC), Igκ chain gene rearrangement fragments (KREC), and SMN1 gene by quantitative PCR, and a novel primer for the same purpose.

Abstract

To provide a method for simultaneously measuring amounts of TREC, KREC and SMN1, an inspection method using the same, and a kit thereof.SOLUTION: A method for simultaneously measuring amounts of T-cell receptor recombination excision circle (TREC), Igκ-deleting recombination excision circle (KREC) and SMN1 gene by quantitative PCR includes: (i) a process of mixing DNA and PCR reagent collected from a subject, where the PCR reagent includes a set of primers and a set of probes; and (ii) a process of performing quantitative PCR reaction to quantitatively determine the amounts of TREC, KREC and SMN1 gene, where the set of primers includes six primers including base sequences shown by sequence numbers 4, 5, 7, 10, 37 and 38, and the set of probes includes three probes including base sequences shown by sequence numbers 22, 23 and 28.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure generally relates to a method for simultaneously measuring the amounts of T cell receptor gene rearrangement fragments (TRECs), Igκ chain gene rearrangement fragments (KRECs), and the SMN1 gene by quantitative PCR.

Background Art

[0002] Newborn screening is a population-based screening for congenital metabolic disorders and the like in newborns. The purpose of newborn screening is to prevent physical and mental disabilities in advance through early detection and early treatment of genetic diseases such as hereditary diseases in which specific nutrients cannot be utilized from birth, or the levels of growth hormones and the like are in a deficient or excessive state, resulting in intellectual disabilities and physical growth disorders. In a specific screening method, first, a very small amount of blood is collected from the heel of a newborn on the 4th to 6th day after birth at an obstetric medical institution and placed on filter paper (dried blood spot; DBS), and then mailed to a screening center. Thereafter, various measurements are performed on the sample extracted from the dried blood filter paper at the screening center.

[0003] In Japan, newborn mass screening has been a national program since October 1977, with the cost of the tests covered by public funds. Currently, newborn mass screening in Japan targets a total of 20 diseases, including glucose metabolism disorders such as galactosemia, endocrine disorders such as congenital hypothyroidism and congenital adrenal hyperplasia, amino acid metabolism disorders such as phenylketonuria and maple syrup urine disease, organic acid metabolism disorders such as methylmalonic acidemia and propionic acidemia, and fatty acid metabolism disorders such as VLCAD and MCAD. On the other hand, for diseases that are not covered by newborn mass screening but meet five conditions, there is an optional paid test called expanded screening (also called optional screening). The five conditions are: (a) there is a treatment method, (b) there is a highly accurate test method to determine the possibility of having the disease, (c) it is relatively common among congenital metabolic disorders, (d) if detected before the disease develops, treatment effectiveness can be improved, symptoms can be prevented in advance, and severe disabilities can be prevented, and (e) there are few characteristic symptoms, making it difficult to detect in normal medical practice. Expanded screening includes screening for lysosomal storage diseases, severe combined immunodeficiency (SCID), and spinal muscular atrophy (SMA).

[0004] Severe combined immunodeficiency (SCID) is a type of congenital immunodeficiency disorder, classified as the most severe form of combined immunodeficiency affecting both humoral and cellular immunity. Typically, infants develop various severe infections, including opportunistic infections, within the first few months of life, and will die within one year of birth if hematopoietic stem cell transplantation, the definitive treatment, is not performed. The incidence is estimated to be approximately 1 in 50,000 births. To date, our understanding of SCID has advanced, and about 20 causative genes have been identified. SCID can be cured with hematopoietic stem cell transplantation, but the transplantation outcome is poor if infections are present, making it crucial to diagnose SCID before infection occurs (Non-patent Literature 1).

[0005] When new normal T cells are created, gene rearrangement usually occurs, producing circular DNA called T-cell receptor excision circles (TRECs). Similarly, when new normal B cells are created, circular DNA called Kappa-deleting recombination excision circles (KRECs) are produced, serving as a production marker. Therefore, TRECs and KRECs can be measured using PCR (polymerase chain reaction) from even very small amounts of blood (Non-Patent Literature 2). Consequently, SCID can be screened by evaluating the amount of TRECs and KRECs contained in a blood filter paper sample as an indicator of normal T and B cell maturation. In other words, the presence of TRECs indicates T cell maturation, and the presence of KRECs indicates B cell maturation; therefore, a decrease in TRECs and KRECs serves as an indicator for screening SCID. In recent years, neonatal screening using quantitative PCR to detect TRECs has become common, mainly in Europe and the United States, and its usefulness has been demonstrated. On the other hand, while widespread neonatal mass screening is desirable to improve the prognosis of SCID patients in Japan, currently, it is only available to those who request it.

[0006] Spinal muscular atrophy (SMA) is a motor neuron disease caused by the degeneration of anterior horn cells in the spinal cord, resulting in hypotonia and progressive muscle weakness, but without intellectual disability. Based on the age of onset and the highest level of motor function achieved, it is classified into Type I (never able to sit), Type II (never able to stand), Type III (able to walk), and Type IV (adult onset). In childhood-onset SMA, 95% are due to SMN1 gene mutations located on the long arm 5q13 of chromosome 5. Even in clinically presenting SMA, there are cases without SMN1 gene deletion, so-called non-5q SMA, and their frequency is higher in Types III and IV (Non-patent Literature 3). In Japan, severe cases of Type I occur in approximately 1 in 20,000 births, and the incidence of SMA that develops in infancy or childhood is said to be 1 to 2 in 100,000. In recent years, drugs for treating SMA have been approved in Japan, and early diagnosis and treatment have become increasingly important. In screening tests for SMA in newborns, the SMN1 gene contained in blood filter paper samples can be used as an indicator for evaluation (Non-patent Literature 4:003-201804-kijyun.pdf (nanbyou.or.jp)).

[0007] Regarding methods for measuring SCID by quantitative PCR using dried blood spot (DBS) discs, there are Patent Documents 1 (Patent 6761093, Sekisui), 2 (WO2021-049601, Sekisui), and 3 (JP 2022-053961, KM Biologics Patent Application). Furthermore, regarding methods for measuring SMA by quantitative PCR, there is Patent Document 4 (WO2021-020161, Sekisui). These measurements are methods for individually measuring one target disease using quantitative PCR, and Sekisui Medical offers contract testing services that measure SCID (TREC / KREC) and SMA individually. However, in expanded screening, being able to simultaneously measure the target disease offers advantages not only in terms of early diagnosis but also in terms of cost for the patient. For this reason, test kits that simultaneously measure SCID and SMA are available. Examples include PerkinElmer's NeoMDx kit (Non-Patent Literature 5: NeoMDx kit - PerkinElmer Japan) and ThermoFisher's TaqMan. TMSCID / SMA Plus Assay (Non-patent Document 6: TaqMan TM This is the SCID / SMA Plus Assay (thermofisher.com). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 6761093 [Patent Document 2] WO2021-049601 [Patent Document 3] Japanese Patent Publication No. 2022-053961 [Patent Document 4] WO2021-020161 [Patent Document 5] Special Publication 2005-514005 [Non-patent literature]

[0009] [Non-Patent Document 1] Hirokazu Kanekane, Kosuke Imai, and Tomohiro Morio: Severe Combined Immunodeficiency - From its Discovery to Future Prospects. Journal of the Japanese Society for Clinical Immunology. 40, 145-154 (2017) [Non-Patent Document 2] Chan K., Puck JM:Development of population-based screening for severe combined immunodeficiency.J Allergy Clin Immunol.115:391-398,2005. [Non-Patent Document 3] Reiko Arakawa, Kaori Hino, Yuri Kitamura, and Kayoko Saito: Genetic testing for neuromuscular diseases. Brain and Development. 50, 192-196 (2018) [Non-Patent Document 4] National Center for Rare Diseases Information website: 3 Spinal Muscular Atrophy 003-201804-kijyun.pdf (nanbyou.or.jp) [Non-Patent Document 5] PerkinElmer: NeoMDx Kit - PerkinElmer Japan [Non-Patent Document 6] ThermoFisher: TaqMan(TM) SCID / SMA Plus AssayTaqMan(TM) SCID / SMA Plus Assay(thermofisher.com) [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] There is a need for a method to simultaneously measure the levels of the TREC, KREC, and SMN1 genes, and the goal is to find a measurement method that is particularly superior in terms of specificity and robustness. [Means for solving the problem]

[0011] Novel primers and probes for TREC, KREC, and SMN1 were investigated, and the optimal ones were identified. Therefore, this disclosure includes: [Section 1] A method for simultaneously measuring the amounts of T cell receptor gene rearrangement fragments (TREC), Igκ chain gene rearrangement fragments (KREC), and the SMN1 gene by quantitative PCR, wherein: (i) A step of mixing DNA collected from a subject with a PCR reagent, wherein the PCR reagent includes a set of primers and a set of probes; (ii) A step of performing a quantitative PCR reaction to quantify the amounts of TREC, KREC and SMN1 genes, Includes, Here, the set of primers comprises six primers containing the nucleotide sequences shown in SEQ ID NOs: 4, 5, 7, 10, 37, and 38, and the set of probes comprises three probes containing the nucleotide sequences shown in SEQ ID NOs: 22, 23, and 28. method, [Section 2] The method according to item 1, wherein the three probes include a fluorescent substance and a quencher. [Item 3] The method according to item 1 or 2, wherein the DNA collected from the subject is derived from dried blood filter paper. [Item 4] The method according to any one of items 1-3, wherein at least one nucleic acid selected from six primers of the primer set and three probes of the probe set further comprises nucleic acid modification. [Item 5] An inspection method for evaluating the following risks when compared with a normal specimen, based on the amounts of TREC, KREC, and SMN1 genes measured by the method defined in any one of items 1-4: Evaluating that there is a risk of T cell depletion when TREC is low; Evaluating that there is a risk of B cell depletion when KREC is low; Evaluating that there is a risk of severe combined immunodeficiency (SCID) when TREC is low; or Evaluating that there is a risk of spinal muscular atrophy (SMA) when SMN1 is low. Inspection method [Item 6] The inspection method according to item 5, wherein the cut-off values are less than 200.0 copies / μL for SMA, less than 20.0 copies / μL for TREC, and less than 10.0 copies / μL for KREC. [Item 7] A kit for simultaneously measuring the amounts of TREC, KREC, and SMN1, comprising a primer set containing six primers including the base sequences shown in SEQ ID NO: 4, 5, 7, 10, 37, and 38, and a probe set containing three probes including the base sequences shown in SEQ ID NO: 22, 23, and 28. [Advantages of the Invention]

[0012] According to the present disclosure, the amounts of TREC, KREC, and SMN1 genes can be simultaneously measured. [Brief Description of the Drawings]

[0013] [Figure 1]Figure 1 shows the results of performing PCR with KREC amplification primers and then electrophoresis of the PCR products of those combinations. [Figure 2] Figure 2 shows the results of performing PCR with SMN1 amplification primers and electrophoresis of the PCR products of those combinations (a)-(i). [Figure 3] Figure 3 shows the results of performing PCR with SMN1 amplification primers and electrophoresis of the PCR products of those combinations (j)-(r). [Figure 4] Figure 4 shows the amplification results when using the TREC-PB1 probe (annealing temperature: 62°C). [Figure 5] Figure 5 shows the amplification results when using the TREC-PB2 probe (annealing temperature: 62°C). [Figure 6] Figure 6 shows the amplification results when using the KREC-PB1 probe (annealing temperature: 62°C). [Figure 7] Figure 7 shows the amplification results when using the KREC-PB2 probe (annealing temperature: 62°C). [Figure 8] Figure 8 shows the amplification results when using probe SMN1-PB1 (annealing temperature: 62°C). [Figure 9] Figure 9 shows the amplification results when using probe SMN1-PB2 (annealing temperature: 62°C). [Figure 10] Figure 10 shows the amplification results when using probe SMN1-PB3 (annealing temperature: 62°C). [Figure 11] Figure 11 shows the amplification results when using probe SMN1-PB7 (annealing temperature: 60°C). Only single assays were performed. [Figure 12] Figure 12 shows the amplification results when using probe SMN1-PB8 (annealing temperature: 63°C). [Figure 13] Figure 13 shows the amplification results when using the SMN1-PB9 probe (annealing temperature: 63°C). [Figure 14]Figure 14 shows the results of simultaneous measurement of TREC, KREC, and SMN1 using various primers and probes (Table 9). [Figure 15] Figure 15 shows the results of simultaneous measurement of TREC, KREC, and SMN1 using various primers and probes (Table 10). [Figure 16-1] Figure 16 shows the results of measuring TREC, KREC, and SMN1 using the method established in Example 5 for each patient sample and a sample from a healthy individual, using 1.5 mmφ dried blood filter paper collected from neonatal dried blood filter paper. Figure 16-1 shows the results when using a sample obtained from a healthy individual. [Figure 16-2] Figure 16-2 shows the results when using samples obtained from patients with T-cell deficiency. [Figure 16-3] Figure 16-3 shows the results when using samples obtained from patients with SCID. [Figure 16-4] Figure 16-4 shows the results when using samples obtained from patients with B-cell deficiency. [Figure 16-5] Figure 16-5 shows the results when using samples obtained from patients with SMN1 deficiency (SMN2: 2 copies). [Figure 16-6] Figure 16-6 shows the results when using samples obtained from patients with SMN1 deficiency (SMN2: 3 copies). [Figure 17] Figure 17 shows the results of measuring SMA by collecting 1.5 mmφ dried blood filter paper from neonatal dried blood filter paper of normal-grade samples (n=500) using the method established in Example 5. [Figure 18] Figure 18 shows the results of measuring TREC by collecting 1.5 mmφ dried blood filter paper from neonatal dried blood filter paper of normal-grade samples (n=500) using the method established in Example 5. [Figure 19] Figure 19 shows the results of measuring KREC by collecting 1.5 mmφ dried blood filter paper from neonatal dried blood filter paper of normal-grade samples (n=500) using the method established in Example 5. [Figure 20]Figure 20 shows a comparison between the method described herein and the use of prior art. [Figure 21] Figure 21 shows the algorithm for determining the target of inspection in TREC. [Figure 22] Figure 22 shows the algorithm for determining the subject of inspection in KREC. [Figure 23] Figure 23 shows the algorithm for determining whether SMA is a target for testing. [Modes for carrying out the invention]

[0014] In its first aspect, this disclosure relates to a method for simultaneously measuring the amounts of T cell receptor gene rearrangement fragments (TREC), Igκ chain gene rearrangement fragments (KREC), and the SMN1 gene by quantitative PCR.

[0015] In this disclosure, “simultaneous” measurement means measuring two or more targets in the same reaction system. In this disclosure, “individually” measurement means measuring two or more targets in different reaction systems. Two or more targets refer to two or more different target sequences in a PCR reaction. For example, if the amount of TREC and SMN1 genes is measured by reacting a sample with a PCR reagent in one PCR tube (i.e., the same reaction system), TREC and SMN1 genes are measured “simultaneously.” For example, “simultaneous” measurement involves using two or more different primer pairs in the same reaction system. Also, for example, if the amount of TREC and SMN1 genes is measured by reacting a sample with a PCR reagent in two different PCR tubes (i.e., different reaction systems), TREC and SMN1 genes are measured “individually.”

[0016] In this disclosure, simultaneous measurement of the amounts of TREC, KREC, and SMN1 genes may result in the detection of none or all of them. For example, if they are not detected or are below the detection limit, it indicates that the corresponding nucleic acid is present in trace amounts or is not present in the sample.

[0017] The samples used in this disclosure are not particularly limited as long as they contain nucleic acids. The nucleic acids are not particularly limited, but are DNA or RNA, and preferably DNA. In one embodiment, the sample is a sample containing nucleic acids taken from a subject, and is preferably a sample derived from a living organism. The sample derived from a living organism may be derived from a newborn, but is not particularly limited. The sample derived from a living organism may include a blood sample, for example, taken from the heel. For example, dried blood filter paper, which is filter paper containing a blood sample that has been dried, may be used. The amount of sample used can be appropriately determined by a person skilled in the art, taking into consideration the amount of nucleic acids contained in the sample. When dried blood filter paper is used as the sample, the size of the punch piece cut from the dried blood filter paper can be appropriately determined by a person skilled in the art, taking into consideration the amount of blood contained. The size of the punched pieces is, for example, approximately 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, or 4.0 mm in diameter. For example, the number of punched pieces used is 1, 2, 3, 4, 5, or 6. While increasing the amount of filter paper (e.g., number of sheets) increases the amount of DNA to be measured, it also reduces the DNA extraction efficiency and increases the amount of blood-derived PCR inhibitors introduced into the reaction system. Therefore, it is preferable to use a small amount of filter paper (e.g., number of sheets).

[0018] The PCR reagents used in this disclosure include reagents necessary for performing the PCR reaction or for detecting the amplified product. The PCR reagents include at least a set of primers and a set of probes. The PCR reagents may further include polymerase and dNTPs (deoxynucleoside triphosphate). Typically, the PCR reagents also include a buffer. The PCR reagents can be appropriately selected and used from those known to those skilled in the art.

[0019] Where a primer or probe is referred to by an SEQ ID NO: in this disclosure, the primer or probe shall contain the nucleotide sequence indicated by that SEQ ID NO:. The primers or probes of this disclosure may have 1, 2, or 3 substitutions, deletions, insertions, and / or additions to the referred sequence.

[0020] In this disclosure, the base lengths of the primers and probes used in the PCR reaction are not particularly limited, and include, for example, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, 30 bases, 31 bases, 32 bases, 33 bases, 34 bases, and 35 bases. In this disclosure, the primers and probes used in the PCR reaction may include known modifications, such as LNA (Locked nucleic acid) modification, 2'-O-methoxyethyl (2'-MOE), 2'-O-methyl (2'-OMe), and 5-methylcytidine (5-Me-dC). The LNA modification is intended to increase the binding affinity of the primer and probe to their respective targets. LNA-modified nucleic acids are indicated herein by adding a "+" to the left of the nucleic acid's name. For example, if the second nucleotide "T" of a nucleic acid represented by the base sequence ATGC is LNA-modified, it is indicated herein as A+TGC.

[0021] The nucleotide sequences of the primers used in this disclosure include, for example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 5, 7, 10, 37, and 38. Preferably, the nucleotide sequences of the primers used in this disclosure consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 5, 7, 10, 37, and 38. A primer pair in this disclosure refers to a combination of primers for amplifying a target nucleic acid, and for example, three primer pairs are used, with the primer pair for TREC being a combination of SEQ ID NOs: 4 and 5, the primer pair for KREC being a combination of SEQ ID NOs: 7 and 10, and the primer pair for SMN1 being a combination of SEQ ID NOs: 37 and 38.

[0022] The nucleotide sequences of the probes used in this disclosure include, for example, a nucleotide sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 28. Preferably, the nucleotide sequences of the probes used in this disclosure consist of a nucleotide sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 28. The set of probes in this disclosure refers to a set of probes for detecting a target nucleic acid, for example, three probes are used, the probe for TREC is SEQ ID NO: 22, the probe for KREC is SEQ ID NO: 23, and the probe for SMN1 is SEQ ID NO: 28.

[0023] The probes used in this disclosure may include a fluorescent substance and a quencher. Those skilled in the art can appropriately select the fluorescent substance and quencher. The fluorescent substance includes Cy5, 6FAM, SUN, Cy3, HEX, and Yakima Yellow, and is preferably Cy5, 6FAM, and SUN. The fluorescent substance is preferably labeled at the 5' end of the probe. The quencher includes 3IAbRQSp and 3IABkFQ. The quencher is preferably labeled at the 3' end of the probe.

[0024] In this disclosure, unless otherwise specified, "qPCR," "quantitative PCR," and "real-time PCR" are used interchangeably. Quantitative PCR generally refers to a method for transiently or over time monitoring the process of generating amplification products in PCR. In this disclosure, when detection is performed by quantitative PCR, for example, the target nucleic acid amplified after the PCR reaction (hereinafter also simply referred to as the amplification product) is detected by optical means. The detection of the amplification product may be performed, for example, after the completion of the PCR reaction, or in parallel with the PCR reaction process. If performed in parallel, the detection of the amplification product can be performed, for example, over time. Over time detection (monitoring) may be continuous or discontinuous (intermittent).

[0025] In one embodiment of this disclosure, multiplex PCR is performed. The PCR conditions are not particularly limited as long as an amplified fragment can be obtained, and can be set as appropriate. The temperature change at each step in the PCR reaction can be automatically controlled, for example, using a thermal cycler. For example, the number of amplification cycles can also be set as appropriate depending on the nucleic acid amplification enzyme used and the length of the amplified nucleic acid. For example, the number of amplification cycles can range from 10 to 100 cycles, with the upper limit of this range being 90, 80, 70, 60, 50, or 40, and the lower limit of this range being 15, 20, 25, 30, or 35. The PCR reaction can be carried out, for example, at 1 cycle / 95°C (10 minutes), 45 cycles / 94°C (30 seconds), or 1 cycle / 60°C (1 minute). The amplified product produced by the PCR reaction can be detected, for example, by detecting the fluorescence intensity generated from the amplified product. The fluorescence detection method is not particularly limited, but conventionally known methods include the intercalator method, probe method, and cycling probe method. In this disclosure, a probe method is preferably used, and more preferably the TaqMan® probe method. Detection of fluorescence intensity can be performed, for example, with a fluorometer. Generally, an apparatus comprising both a PCR reaction unit (e.g., a thermal cycler) and an optical system unit (e.g., a fluorometer) is used.

[0026] The method for quantifying target nucleic acids described herein involves generating an amplification product corresponding to the target nucleic acid in a sample, detecting the amplification product by optical means, and quantifying the amplification product. The target nucleic acid contained in the sample can be quantified by counting the number of PCR cycles required to produce a predetermined amount of the amplification product. Alternatively, the target nucleic acid can be quantified by converting the copy number of 1 μL of whole blood in dried filter paper blood from a calibration curve of Cq values ​​calculated from a standard.

[0027] In one embodiment, the sample is processed by either an elution method or an extraction method. For example, in the extraction method, a commercially available kit, NucleoSpin Tissue XS (TAKARA U0901B:74090.250), can be used, and in the elution method, a commercially available kit, the DNA Extract All Reagents Kit from Thermo Fisher SCIENTIFIC, can be used.

[0028] A second aspect of this disclosure relates to a testing method for evaluating disease risk compared to a normal sample based on the amounts of TREC, KREC, and SMN1 genes. The method of this disclosure may be used to measure the amounts of TREC, KREC, and SMN1 genes in a sample, thereby evaluating the disease risk in a subject. The method of this disclosure is preferable from the viewpoint of early diagnosis, cost, or process because it allows for the simultaneous measurement of the amounts of TREC, KREC, and SMN1 genes, and therefore allows for the evaluation of at least one amount selected from the group consisting of TREC, KREC, and SMN1 genes. The disease is not particularly limited as long as it is a disease that depends on the amounts of TREC, KREC, and SMN1 genes. For example, the disease may be T-cell deficiency, B-cell deficiency, severe combined immunodeficiency (SCID), and spinal muscular atrophy (SMA). Preferably, the disease is severe combined immunodeficiency (SCID) or spinal muscular atrophy (SMA).

[0029] In this disclosure, “assessing risk” means evaluating or determining whether a person has or is likely to develop a disease. Assessing risk may aid in the diagnosis of a disease. The evaluation or determination may be based on a cutoff value or a comparison with a normal sample value. The method of this disclosure enables simultaneous mass screening. Subjects whose risk has been assessed by the method of this disclosure may undergo further testing or examination at a specialized medical facility.

[0030] In one embodiment, a low TREC level is assessed as a risk of T-cell deficiency; a low KREC level is assessed as a risk of B-cell deficiency; a low TREC level is assessed as a risk of severe combined immunodeficiency (SCID); or a low SMN1 level is assessed as a risk of spinal muscular atrophy (SMA). Low levels may also include the absence of detection of the target PCR product.

[0031] In one embodiment, the cutoff values ​​for SMA are less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, and 4000 copies / μL. Preferably, the cutoff value is less than 200.0 copies / μL.

[0032] In one embodiment, the cutoff values ​​for TREC are less than 5, 10, 15, 20, 25, 30, 35, and 40 copies / μL. Preferably, the cutoff value is less than 20.0 copies / μL.

[0033] In one embodiment, the cutoff value for KREC is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies / μL. Preferably, the cutoff value is less than 10.0 copies / μL.

[0034] In one embodiment of the present disclosure, for example, detection specificity is improved, the robustness of the PCR reaction is improved, or the retest rate is reduced. In another embodiment of the present disclosure, the false positive rate is reduced.

[0035] A third aspect of this disclosure relates to a kit for simultaneously measuring the amounts of TREC, KREC, and SMN1. The kit of this disclosure is for simultaneously measuring the amounts of TREC, KREC, and SMN1, and includes a set of primers comprising six primers comprising the nucleotide sequences shown in SEQ ID NOs. 4, 5, 7, 10, 37, and 38, and a set of probes comprising three probes comprising the nucleotide sequences shown in SEQ ID NOs. 22, 23, and 28. The kit of this disclosure may further include instructions. The kit may include other primers or probes within the range that allows for the simultaneous measurement of the amounts of TREC, KREC, and SMN1. The kit may also include probes, other reagents and containers used in PCR, real-time PCR.

[0036] In this specification, "includes" also includes "essentially consists of" and "consists of."

[0037] In this specification, "approximately" means a range of ±10%, preferably ±5%.

[0038] The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited in any way by these examples. [Examples]

[0039] [Example 1] Testing methods, etc. (1) Specimens, reagents, equipment, etc. This disclosure uses a fluorescently labeled probe method, also known as the TaqMan probe method, to detect the target gene by quantitative PCR. For the extraction method, a commercially available kit, NucleoSpin Tissue XS (TAKARA U0901B:74090.250), was used. For the elution method, a commercially available kit, the DNA Extract All Reagents Kit from Thermo Fisher SCIENTIFIC, was used. The standard material was prepared by the Japan Gene Research Institute Co., Ltd. The sequences are summarized in the table below. [Table 1]

[0040] Other samples, reagents, etc. used in the examples are as follows: [Specimen] • Specimen: Neonatal dried blood filter paper [qPCR reagents and equipment] Improved direct method: • qPCR kit: Ampdirect Plus + BIOTAQ Shimadzu 241-08890-92 • qPCR plate: MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 mL, ABI, Cat#4346907 • qPCR plate seal: MicroAmp 96-Optical Adhesive film, ABI, Cat#4360954 or #4311971 • Preparation plate seal: BIO BIK sealing film B-TS • qPCR equipment: Quant Studio 5 Real-time PCR System, ABI, QS5-96F • Thermal cycler (for heating): Thermal cycler ASTEC

[0041] (2) Measurement procedure The measurement procedure in this embodiment is as follows: The DNA solution obtained by eluting or extracting DNA from dried blood filter paper is brought into a clean bench and dispensed in 5 μL portions into the wells of a 96 microplate for quantitative PCR reaction. Four separately prepared concentrations of standard substances are dispensed in 5 μL portions into two wells. Then, a master mix is ​​prepared by adding primers and probes, and 15 μL is dispensed into each well. After securely attaching the 8-strip flat cap, the plate is centrifuged, and quantitative PCR is performed using a Thermo Fisher Quant Studio 5 real-time PCR system. The instrument settings can be Cy5 (Reporter) / None (Quencher) for TRECs genes, FAM (Reporter) / None (Quencher) for KRECs genes, and VIC (SUN) (Reporter) / None (Quencher) for SMN1 genes. The PCR reaction was performed at 1 cycle / 95°C (10 min), 45 cycles / 94°C (30 sec), and 62°C (1 min). After the reaction is complete, the fluorescence value corresponding to the volume is measured. Next, PCR reactions were performed on DNA standards containing known amounts of TRECs gene sequences, KRECs gene sequences, and SMN1 gene sequences. The amounts (copies / μL) of the TRECs, KRECs, and SMN1 genes were then quantified using a calculation formula based on a calibration curve obtained from the fluorescence values ​​corresponding to the amounts. [Extraction method] For the extraction method, we used the commercially available NucleoSpin Tissue XS kit (TAKARA U0901B:74090.250). After investigating the number of dried blood filter papers to use (3, 2, and 1), we decided to cut out one disk from the dried blood filter paper using a 3.2 mm diameter puncher, and performed DNA extraction on this disk following the kit protocol. Specifically, one disk was placed in a 1.5 mL microtube, Buffer T1 (160 μL) was added, and vortexing for 5 seconds twice was performed. After incubation at 94°C for 10 minutes in a block incubator, it was returned to room temperature, proteinase K solution (16 μL) was added, and after vortexing, it was gently spun down. Then, it was incubated at 56°C for 1 hour. During this time, vortexing was performed approximately every 10 minutes to improve extraction efficiency. After returning to room temperature and spinning down, the solution was transferred to a new 1.5 mL microtube using a micropipette, Buffer B3 (160 μL) was added, vortexed for 5 seconds twice, incubated at 70°C for 5 minutes, then lightly vortexed, returned to room temperature, and spun down. Subsequently, 160 μL of 96-100% ethanol was added, vortexed for 5 seconds twice, and lightly spun down. Then, the solution was transferred to a NucleoSpin Tissue XS Column in a Collection Tube, centrifuged at 11,000 xg for 1 minute, the flow-through was removed, and a new Collection Tube was reattached. Next, Buffer B5 (50 μL) was added to the NucleoSpin Tissue XS Column, centrifuged at 11,000xg for 1 minute, then Buffer B5 (50 μL) was added to the NucleoSpin Tissue XS Column again, centrifuged at 11,000xg for 2 minutes, and the Collection Tube was removed. Finally, for DNA extraction, a new 1.5 mL microtube was attached, 20 μL of milliQ sterile water warmed to 70°C was added, and the DNA extract was obtained by centrifuging at 11,000xg for 1 minute. [Elution method] For the elution method, a commercially available kit, the DNA Extract All Reagents Kit from Thermo Fisher SCIENTIFIC, was used. [Results of the Punch-Out Filter Paper Quantity Study] [Table 2] Increasing the number of filter papers would increase the amount of DNA obtainable for measurement, but it was anticipated that this would also decrease the DNA extraction efficiency and increase the amount of blood-derived PCR inhibitors introduced into the reaction system. As a result, as shown in the table above, a tendency was observed for the measured value to decrease as the number of filter papers increased. Therefore, in order to eliminate differences in DNA extraction rate and the influence of PCR inhibitors for each sample, it was decided to construct a testing system using one 3.2 mm filter paper.

[0042] (3) Analysis operations The analysis was performed using the Design and Analysis Software included with the Quant Studio 5 Real-Time PCR System, ABI, QS5-96F.

[0043] [Example 2] Primer design (1) Primer for TREC amplification The forward (F) and reverse (R) primers for TREC amplification were arranged in the same sequence as in Japanese Patent Application No. 2020-160873 (KMB Patent Application). The sequences of the TREC amplification primers are shown in Table 3. [Table 3] (2) Primer for KREC amplification Three types each of the Forward (F) and Reverse (R) sequences for KREC were designed as shown in Table 4, and PCR was performed on all nine combinations ((-)F3×R4, (a)F3×R6, (b)F3×R8, (c)F5×R4, (d)F5×R6, (e)F5×R8, (f)F7×R4, (g)F7×R6, (h)F7×R8) and confirmed by electrophoresis (Figure 1). As a result, we selected (a) F3×R6, (d) F5×R6, and (g) F7×R6, which had fewer non-specific bands and stronger signals in the band in question. Further investigation involved evaluating simultaneous reactions with other targets, and we decided to use combination (d). [Table 4] (3) Primer for SMN1 amplification Three forward (F) and six reverse (R) sequences of SMN1 were designed as shown in Table 5, and 18 combinations of these sequences ((a)F1×R4, (b)F1×R5, (c)F1×R6, (d)F2×R4, (e)F2×R5, (f)F2×R6, (g)F3×R4, (h)F3×R5, (i)R3×R6, (j)F1×R13, (k)F1×R14, (l)F1×R15, (m)F2×R13, (n)F2×R14, (o)F2×R15, (p)F3×R13, (q)F3×R14, (r)F3×R15) were subjected to PCR and confirmed by electrophoresis. As a result, no non-specific bands were found, and the signal intensity was higher for the combination (j)~(r) than for the combination (a)~(i). Therefore, several combinations were selected from (j)~(r) and experiments such as simultaneous reactions with other targets were conducted, and (r)F3×R15 was finally determined (Figures 2 and 3). [Table 5]

[0044] [Example 3] Probe design Using the TREC, KREC, and SMN1 primers determined in Example 2, detection probes for TREC, KREC, and SMN1 were selected and fabricated. Probe designs were created using a web tool, referencing the three-dimensional structure and Tm value, and were first confirmed by single measurements of TREC, KREC, and SMN1. (1) TREC detection probe Two probes were created for TREC detection: TREC-PB1, which has a sequence similar to that of JP 2022-053961 (KM Biologics patent application), and TREC-PB2, which incorporates LNA (Locked Nucleic Acid: first reported by Wengel et al. in 1998; Patent Document 5: JP 2005-514005). Nucleic acids modified with fluorescent substances and quenchers were used for the TREC detection probe sequences listed in Table 6. As a result of testing, TREC-PB1 was found to be incompatible with other targets when reacted simultaneously, resulting in weak amplification and therefore not adopted (Figures 4 and 5). As a result, TREC-PB2 showed superior reactivity compared to TREC-PB1. [Table 6] (2) KREC detection probe Five probes (KREC-PB1 to 5) were prepared for KREC detection. Nucleic acids modified with fluorescent agents and quenchers were used for the KREC detection probe sequences listed in Table 7. Although sequences for KREC-PB3~5 were designed, in silico testing revealed that the sequences had LNAs on a hairpin structure, which made PCR suppression highly likely, so they were not adopted. When comparing KREC-PB1 and KREC-PB2, KREC-PB2 was found to be incompatible with other targets when reacted simultaneously, and clear amplification could not be confirmed, so KREC-PB1 was adopted (Figures 6 and 7). [Table 7] (3) SMN1 detection probe Nine probes (SMN1-PB1 to 9) were created for SMN1 detection. The sequences of the SMN1 detection probes are listed in Table 8. SMN1-PB4 to 6 were designed but were not adopted after in silico testing because a large amount of LNA would be present on the hairpin structure, raising concerns about unexpected structural abnormalities. SMN1-PB7 was not adopted after testing only in a single SMN1 assay, as it showed many waveform abnormalities and could not obtain stable amplification. After testing SMN1-PB1, 2, 3, 8, and 9 in co-reactions with other targets, SMN1-PB1 showed the best balance and stable amplification, and had the most superior reactivity (Figure 8-13). [Table 8]

[0045] [Example 4] Simultaneous measurement of TREC, KREC, and SMN1 (1) Simultaneous measurement of TREC, KREC, and SMN1 was performed using the primers and detection probes for TREC, KREC, and SMN1 determined in Examples 2 and 3. Table 9 lists the sequences of the primers and detection probes for TREC, KREC, and SMN1. As probes, TREC-PB2 was modified with Cy5 at the 5' end and 3IAbRQSp at the 3' end; KREC-PB1 was modified with 6FAM at the 5' end and 3IABkFQ at the 3' end; and SMN1-PB1 was modified with SUN at the 5' end and 3IABkFQ at the 3' end. Confirmation revealed that amplification of SMN1 was unsuccessful (Figure 14). [Table 9]

[0046] [Example 5] Simultaneous measurement of TREC, KREC, and SMN1 (2) Since amplification of SMN1 was unsuccessful in Example 4, we decided to add LNA to the SMN1 primer to improve its specificity. In this case, the LNA was introduced at a location where the sequences of SMN1 and SMN2 differed. Table 10 lists the sequences of the primers and detection probes for TREC, KREC, and SMN1. As probes, TREC-PB2 was modified with Cy5 at the 5' end and 3IAbRQSp at the 3' end, KREC-PB1 was modified with 6FAM at the 5' end and 3IABkFQ at the 3' end, and SMN1-PB1 was modified with SUN at the 5' end and 3IABkFQ at the 3' end. Confirmation confirmed that amplification of SMN1 was successful (Figure 15). [Table 10]

[0047] [Example 6] Using the method established in Example 5, 1.5 mmφ dried blood filter paper was collected from neonatal dried blood filter paper, and when each patient sample was measured, it was confirmed that they could be clearly distinguished (Figure 16).

[0048] [Example 7] Using the method established in Example 5, 1.5 mmφ dried blood filter paper was collected from neonatal dried blood filter paper of normal-judged samples (n=500), and SMA was checked. The cutoff value was <200.0 copies / μL, and the mean was 18733.3 copies / μL (Figure 17). Similarly, TREC was checked, and the cutoff value was 20.0 copies / μL, and the mean was 308.1 copies / μL (Figure 18). The cutoff value for KREC was 10.0 copies / μL, and the mean was 161.6 copies / μL (Figure 19).

[0049] [Example 8] Comparison with previous products Using the method established in Example 5, the prior art products PerkinElmer's NeoMDx kit (NeoMDx kit - PerkinElmer Japan) and Thermo Fisher's TaqMan were used. TMSCID / SMA Plus Assay (TaqMan) TM A comparison was made with SCID / SMA Plus Assay (Thermo Fisher) (Figure 20). Comparison of this method with prior art confirmed that the technology disclosed hereof exhibits high fluorescence intensity and low variability for all three target genes. In contrast, PerkinElmer showed low SMN1 fluorescence intensity, and Thermo Fisher showed high TREC baseline and variability, indicating the superiority of the method disclosed hereof.

[0050] [algorithm] Figure 21-23 shows an example of the algorithm used to determine the TREC, KREC, and SMA targets in simultaneous screening. [Industrial applicability]

[0051] According to this disclosure, the amounts of TREC, KREC, and SMN1 can be measured simultaneously, enabling efficient detection of DNA in the target.

Claims

1. A method for simultaneously measuring the amounts of T cell receptor gene rearrangement fragments (TREC), Igκ chain gene rearrangement fragments (KREC), and the SMN1 gene by quantitative PCR, the following: (i) A step of mixing DNA collected from a subject with a PCR reagent, wherein the PCR reagent includes a set of primers and a set of probes; (ii) A step of performing a quantitative PCR reaction to quantify the amounts of TREC, KREC and SMN1 genes. Includes, Here, the set of primers includes six primers containing the nucleotide sequences shown in SEQ ID NOs: 4, 5, 7, 10, 37, and 38, and the set of probes includes three probes containing the nucleotide sequences shown in SEQ ID NOs: 22, 23, and 28. method.

2. The method according to claim 1, wherein the three probes include a fluorescent substance and a quencher.

3. The method according to claim 1, wherein the DNA collected from the subject is derived from dried blood filter paper.

4. The method according to claim 1, wherein at least one nucleic acid selected from the set of six primers and the set of three probes further comprises nucleic acid modification.

5. A testing method for evaluating the following risks when compared to a normal sample, based on the amounts of TREC, KREC, and SMN1 genes measured by the method defined in claim 1: A low TREC level indicates a risk of T-cell deficiency; A low KREC level indicates a risk of B-cell deficiency; If TREC is low, assess the risk of severe combined immunodeficiency (SCID); or A low SMN1 level is used to assess the risk of spinal muscular atrophy (SMA). Testing method.

6. The testing method according to claim 5, wherein the cutoff values ​​are less than 200.0 copies / μL for SMA, less than 20.0 copies / μL for TREC, and less than 10.0 copies / μL for KREC.

7. A kit for simultaneously measuring the amounts of TREC, KREC, and SMN1, comprising a set of primers containing six primers with nucleotide sequences shown in SEQ ID NOs: 4, 5, 7, 10, 37, and 38, and a set of probes containing three probes with nucleotide sequences shown in SEQ ID NOs: 22, 23, and 28.

8. A set of primers for use in the method according to claim 1, the set of primers comprising two primers having the nucleotide sequences shown in SEQ ID NOs: 4 and 5.

9. A set of primers for use in the method according to Claim 1, the set of primers comprising two primers having the nucleotide sequences shown in SEQ ID NOs: 7 and 10.

10. A set of primers for use in the method according to Claim 1, the set of primers comprising two primers having the nucleotide sequences shown in SEQ ID NOs: 37 and 38.

11. A kit for use in the method of Claim 1, comprising a set of primers comprising two primers comprising the nucleotide sequences shown in SEQ ID NOs: 4 and 5, and a probe comprising the nucleotide sequence shown in SEQ ID NOs:

22.

12. A kit for use in the method of Claim 1, comprising a set of primers comprising two primers comprising the nucleotide sequences shown in SEQ ID NOs: 7 and 10, and a probe comprising the nucleotide sequence shown in SEQ ID NOs:

23.

13. A kit for use in the method according to claim 1, comprising a set of primers comprising two primers comprising the nucleotide sequences shown in SEQ ID NOs: 37 and 38, and a probe comprising the nucleotide sequence shown in SEQ ID NOs: 28.

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