PCR PRIMER PAIR FOR HUMAN ALu DETECTION, PCR PROBE FOR HUMAN ALu DETECTION, PCR PRIMER AND PROBE SET FOR HUMAN ALu DETECTION, METHOD FOR DETECTING AND / OR QUANTIFYING HUMAN GENOMIC DNA, AND METHOD FOR ASSISTING PREDICTION OF PRESENCE OR ABSENCE OF RENAL CELL CARCINOMA OR PROSTATE CANCER

Abstract

The present invention addresses the problem of providing a method for designing a primer pair and a probe for quantitatively measuring low-molecular cfDNA by size, quantifying cfDNA by using the primer pair and the probe, and assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer.　Prepared is a PCR primer pair for human Alu detection composed of the following (a) or (a') forward primer, and (b) reverse primer. (a) A forward primer comprising a nucleotide sequence represented by SEQ ID NO: 1 or 2; (a') a forward primer comprising a nucleotide sequence (Alu57F20) represented by SEQ ID NO: 2, or a nucleotide sequence represented by SEQ ID NO: 2 in which 1-3 nucleotides are deleted, substituted, or added; and (b) a reverse primer comprising a nucleotide sequence represented by SEQ ID NO: 3.

Description

PCR primer pair for detecting human Alu, PCR probe for detecting human Alu, PCR primer-probe set for detecting human Alu, method for detecting and / or quantifying human genomic DNA, and method for assisting in prediction of the presence or absence of renal cell carcinoma or prostate cancer

[0001] The present invention relates to a PCR primer pair for detecting human Alu, a PCR probe for detecting human Alu, a PCR primer-probe set for detecting human Alu, a method for detecting and / or quantifying human genomic DNA, and a method for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer.

[0002] Cell-free DNA (cfDNA) is a trace amount of DNA released into peripheral blood upon cell death or destruction, and its association with cancer has long been suggested. For example, cfDNA has already attracted attention as a tumor marker for renal cell carcinoma (e.g., Non-Patent Document 1). Non-Patent Document 1 investigated the size distribution of cfDNA in the plasma of cancer patients using a microfluidic chip and next-generation sequencing, suggesting that fragmented cfDNA may be a tumor marker for renal cell carcinoma.

[0003] However, at present, it is technically difficult to apply such findings to clinical cancer diagnosis. This is because, currently, the only way to analyze the size distribution of cfDNA is to perform comprehensive analysis of genomic information using next-generation sequencing, etc., but this analysis requires very expensive specialized equipment and experts to operate and analyze it. Next-generation sequencing devices are sometimes installed in university facilities specializing in genetic analysis, but are rarely available in general hospitals or medical testing institutions. Furthermore, next-generation sequencing analysis is costly and time-consuming, making it unrealistic to use it for general cancer diagnosis. For these reasons, in order to commercialize renal cell carcinoma diagnosis using fragmented cfDNA, it is necessary to develop a method that can quantify fragmented cfDNA easily and inexpensively using more conventional testing equipment.

[0004] Here, Alu sequences are repetitive sequences specific to primates, and are known to exist in more than 1 million copies in the human genome, accounting for more than 10% of the human genome. Since the total length of the human genome is approximately 3 billion nucleotide pairs, a simple calculation would suggest that an average of one copy of Alu sequences exists for every 3,000 nucleotide pairs of human genomic DNA. Based on these characteristics, the possibility of measuring blood cfDNA concentrations using quantitative PCR methods for human Alu sequences (hereinafter also referred to as "Alu-qPCR") and applying them to cancer diagnosis has previously been demonstrated (e.g., Patent Documents 1 and 2, Non-Patent Document 2). However, a method for quantifying minute amounts of cfDNA using Alu-qPCR to examine its size distribution had not yet been established.

[0005] In this context, the present inventors have previously developed a highly sensitive Alu-qPCR method using a primer-probe set designed based on original criteria (Patent Document 3 and Non-Patent Document 3). Specifically, Patent Document 3 and Non-Patent Document 3 disclose primer-probe sets that specifically amplify 63-bp and 106-bp fragments in Alu sequences, and further disclose that their use enables specific detection of fragmented human genomic DNA with a sensitivity more than 1,000 times that of conventional techniques.

[0006] International Publication No. 2016 / 028316 Pamphlet International Publication No. 2006 / 128192 Pamphlet Japanese Patent No. 6892695

[0007] Yamamoto et al., Cancer Science. 2019; 110: 617-628.Bedin et al., International Journal of Cancer 2016; 140: 1888-1898Funakoshi et al., Scientific Reports. 2017; 7, 13202

[0008] If fragmented cfDNA of a predetermined size could be detected with high sensitivity from blood samples, it could be used as an indicator for cancer diagnosis. Renal cell carcinoma (RCC) is the third most common urological malignant tumor after prostate cancer and bladder cancer, with an incidence rate of approximately 2.5 cases per 100,000 people and a male-to-female ratio of 2-3:1, suggesting a male predominance. Renal cell carcinoma does not have an effective tumor marker, and diagnostic methods typically involve imaging tests such as CT scans, ultrasound (echo), and MRI scans. However, detection is currently difficult due to the lack of characteristic early symptoms. Until now, there has been no accurate method for quantifying trace amounts of cfDNA, and little research has focused on the size distribution of low-molecular-weight cfDNA, which may be increased in cancer patients. Furthermore, while prostate-specific antigen (PSA) and other tumor markers are used for prostate cancer, they have problems such as low specificity, and prostate biopsies are required for definitive diagnosis. Therefore, there is a strong demand for the development of a diagnostic method using a blood test that is less invasive and less burdensome for patients.

[0009] Therefore, an object of the present invention is to design a primer pair and a probe for quantitatively measuring low molecular weight cfDNA by size, to quantify cfDNA by using the primer pair and the probe, and to provide a method for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer based on the quantification of cfDNA.

[0010] In 2016, the present inventors developed an ultrasensitive human genome detection method (hereinafter referred to as "ultrasensitive Alu-qPCR") that targets Alu sequences in the human genome (see Patent Document 3 and Non-Patent Document 3). The ultrasensitive Alu-qPCR is characterized by its ability to accurately quantify low-molecular-weight human genomic DNA (<100 bp).

[0011] In the course of intensive research to solve the above-mentioned problems, the present inventors have designed primers and probes that can quantify small molecular weight cfDNA by size by applying the above-mentioned ultrasensitive Alu-qPCR, and have found that human genomic DNA can be detected and / or quantified. Furthermore, they have found that by using a method for detecting and / or quantifying human genomic DNA, it is possible to use cfDNA as an indicator to assist in predicting the presence or absence of renal cell carcinoma, for which no effective biomarkers exist. Furthermore, they have found that by using a method for detecting and / or quantifying human genomic DNA using the above-mentioned primers and probes, it is possible to use cfDNA as an indicator to assist in predicting the presence or absence of prostate cancer, and have completed the present invention.

[0012] That is, the present invention is as follows: [1] A PCR primer pair for detecting human Alu, which is composed of the following forward primer (a) and reverse primer (b), or composed of the following forward primer (a') and reverse primer (b): (a) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20), or the nucleotide sequence shown in SEQ ID NO: 1 with one to three nucleotides deleted, substituted, or added; (a') a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20), or the nucleotide sequence shown in SEQ ID NO: 2 with one to three nucleotides deleted, substituted, or added; (b) a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19), or the nucleotide sequence shown in SEQ ID NO: 3 with one to three nucleotides deleted, substituted, or added; [2] a PCR probe for detecting human Alu, which is composed of the following (c): (c) the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA); or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 4; [3] a PCR probe for detecting human Alu, comprising the PCR primer pair for detecting human Alu described in [1] above and (c) described in [2] above; or a PCR probe for detecting human Alu, comprising the PCR primer pair for detecting human Alu described in [1] above and (c') the nucleotide sequence shown in SEQ ID NO: 5 (Alu144RH20), or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 5. [4] A method for detecting and / or quantifying human genomic DNA in a test sample, comprising the step of performing PCR using DNA extracted from the test sample as a template and the PCR primer pair and probe set for detecting human Alu described in [1] above.[5] A method for detecting and / or quantifying human genomic DNA in a test sample, comprising a step of performing PCR using the PCR primer-probe set for detecting human Alu described in [3] above, using DNA extracted from the test sample as a template. [6] A method for detecting and / or quantifying human genomic DNA in a test sample using the PCR primer-probe set for detecting human Alu described in [3] above, comprising the following steps (I) to (III): (I) performing real-time PCR using the primer-probe set, using DNA extracted from the test sample as a template; (II) performing real-time PCR under the same conditions as in step (I), using standard samples prepared by serially diluting known amounts of human genomic DNA as templates, to create a calibration curve; (III) calculating the amount of human genomic DNA in the test sample from the calibration curve; [7] A method for assisting in prediction of the presence or absence of renal cell carcinoma or prostate cancer in a test subject, comprising the following steps (A) and (B): (A) quantifying the amount of human genomic DNA in a biological sample extracted from a subject by the method for detecting and / or quantifying human genomic DNA described in any one of [4] to [6] above; (B) assisting in predicting whether the subject has renal cell carcinoma or prostate cancer when the amount of human genomic DNA quantified in step (A) is equal to or greater than a predetermined threshold; [8] a method for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject, characterized by comprising the following steps (A') and (B'):(A') quantifying the amount of human genomic DNA in a biological sample extracted from a test subject by a method comprising the following steps (I') to (III'): (I') performing real-time PCR using a primer-probe set that amplifies or detects 63 bp, 106 bp, and / or 137 bp human Alu sequences, with DNA extracted from the test sample as a template; (II) performing real-time PCR under the same conditions as in step (I) above, with a standard sample prepared by serially diluting a known amount of human genomic DNA as a template, to create a calibration curve; (III) calculating the amount of human genomic DNA in the test sample from the calibration curve; (B') assisting in predicting that the test subject has renal cell carcinoma or prostate cancer, when the amount of human genomic DNA quantified in step (A') is equal to or greater than a predetermined threshold; [9] A method for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject according to [7] or [8] above, characterized in that the threshold value is a value determined by the AUC value calculated from ROC curve analysis.

[0013] The primer pair and probe set of the present invention enables specific detection and / or measurement of approximately 0.1 to 10 fg of human genomic DNA. Furthermore, the use of the primer pair and probe set enables highly sensitive detection and / or quantification of human genomic DNA without the use of next-generation sequencing. Furthermore, the use of the primer pair and probe set enables minimally invasive and simple prediction of the presence or absence of renal cell carcinoma or prostate cancer.

[0014] 1 is a diagram showing the positional relationship between the Alu model sequence (SEQ ID NO: 6) and the "63 bp Alu detection primer-probe set" and the "106 bp Alu detection primer-probe set." It is a diagram showing the positions of the Alu model sequence (SEQ ID NO: 6) and the "137 bp Alu detection primer-probe set" and the "181 bp Alu detection primer-probe set." It is a diagram showing the results of a calibration curve prepared based on real-time PCR using Alu144RH20 as the probe in Example 3. It is a diagram showing the results of a calibration curve prepared based on real-time PCR using Alu140RH16-LNA as the probe in Example 3. It is a diagram showing the results of box plots of cfDNA concentrations in plasma from healthy subjects and renal cell carcinoma patients in Example 4.

[0033] In Example 4, this figure shows the results of dividing the respective cfDNA concentrations in plasma determined using primer-probe sets that amplify and detect 63 bp, 106 bp, and 137 bp in FIG. 5 by the cfDNA concentration in plasma determined using a primer-probe set that amplifies and detects 181 bp (63b / 181bp, 106bp / 181bp, 137bp / 181bp). Hereinafter, division may be represented by " / ". In Example 5, this figure shows the results of ROC analysis of the respective cfDNA concentrations in plasma determined using primer-probe sets that amplify and detect 63 bp, 106 bp, 137 bp, and 181 bp. In Example 5, this figure shows the results of ROC analysis of 63b / 181bp, 106bp / 181bp, and 137bp / 181bp. In Example 5, the respective cfDNA concentrations in plasma determined with a primer-probe set amplifying and detecting 63 bp, 106 bp, and 137 bp were subtracted by the cfDNA concentration in plasma determined with a primer-probe set amplifying and detecting 181 bp (63 bp - 181 bp, 106 bp - 181 bp, 137 bp - 181 bp). Hereinafter, subtraction may be represented by "-". In Example 5, the threshold, sensitivity, specificity, and sensitivity + specificity are shown when the sensitivity is set to 100%, > 95%, > 90%, > 85%, and > 80% for 106 bp, 106 bp / 181 bp, and 106 bp - 181 bp, respectively.FIG. 10 shows the sensitivity, specificity, and sensitivity+specificity for 106 bp and 106 bp / 181 bp in Example 5 when the sensitivity is set to 100%, >95%, >90%, >85%, and >80%.

[0015] The PCR primer pair for detecting human Alu of the present invention is not particularly limited, as long as it is composed of the following forward primer (a) and reverse primer (b), or composed of the forward primer (a') and reverse primer (b): (a) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20); or the nucleotide sequence shown in SEQ ID NO: 1 with one to three nucleotides deleted, substituted, or added; (a') a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20); or the nucleotide sequence shown in SEQ ID NO: 2 with one to three nucleotides deleted, substituted, or added; or (b) a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19); or the nucleotide sequence shown in SEQ ID NO: 3 with one to three nucleotides deleted, substituted, or added; and hereinafter, these primers are also referred to as the "present PCR primer pair for detecting human Alu."

[0016] Furthermore, the PCR probe for detecting human Alu of the present invention is not particularly limited as long as it is a probe consisting of (c) the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA); or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 4, and hereinafter may also be referred to as the "PCR probe for detecting human Alu of the present invention."

[0017] Furthermore, the PCR primer-probe set for detecting human Alu of the present invention is not particularly limited as long as it is a PCR primer-probe set for detecting human Alu comprising the present PCR primer pair for detecting human Alu and the present PCR probe for detecting human Alu, or a PCR primer-probe set for detecting human Alu comprising the present PCR primer pair for detecting human Alu and the PCR probe for detecting human Alu described in (c') above, and hereinafter may also be referred to as the "present PCR primer-probe set for detecting human Alu."

[0018] Other examples of the method of the present invention for detecting and / or quantifying human genomic DNA in a test sample include a method for detecting and / or quantifying human genomic DNA in the test sample, which comprises a step of performing PCR using the present PCR primer pair for detecting human Alu using DNA extracted from the test sample as a template (hereinafter also referred to as "Method 1 for detecting and / or quantifying human genomic DNA"), and a method for detecting and / or quantifying human genomic DNA in the test sample, which comprises a step of performing PCR using the present PCR primer-probe set for detecting human Alu (hereinafter also referred to as "Method 2 for detecting and / or quantifying human genomic DNA").

[0019] Furthermore, another method of the present invention for detecting and / or quantifying human genomic DNA in a test sample can include a method for detecting and / or quantifying human genomic DNA in a test sample using the present PCR primer-probe set for detecting human Alu, comprising the steps of: (I) performing real-time PCR using DNA extracted from the test sample as a template and the present PCR primer-probe set for detecting human Alu; (II) performing real-time PCR under the same conditions as in step (I) above, using a standard sample prepared by serially diluting a known amount of human genomic DNA as a template, to create a calibration curve; and (III) calculating the amount of human genomic DNA in the test sample from the calibration curve; and hereinafter this method is also referred to as "Method 3 for detecting and / or quantifying human genomic DNA."

[0020] Furthermore, the method of the present invention for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject includes the following steps (A) and (B): (A) quantifying the amount of human genomic DNA in a biological sample extracted from the subject by the above-mentioned method for detecting and / or quantifying human genomic DNA; and (B) assisting in the prediction that the subject has renal cell carcinoma or prostate cancer if the amount of human genomic DNA quantified in step (A) is equal to or greater than a predetermined threshold (hereinafter also referred to as "Method 1 for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in the subject"). Another embodiment of the method of the present invention for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject includes a method of assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject, characterized by comprising the following steps (A') and (B') (hereinafter also referred to as "Method 2 for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in the subject").(A') quantifying the amount of human genomic DNA in a biological sample extracted from a test subject by a method comprising the following steps (I') to (III'): (I') performing real-time PCR using a primer-probe set that amplifies or detects 63 bp, 106 bp, and / or 137 bp human Alu sequences, with DNA extracted from the test sample as a template; (II') performing real-time PCR under the same conditions as in step (I') above, with a standard sample prepared by serially diluting a known amount of human genomic DNA as a template, to create a calibration curve; (III') calculating the amount of human genomic DNA in the test sample from the calibration curve; (B') assisting in predicting that the test subject has renal cell carcinoma or prostate cancer, when the amount of human genomic DNA quantified in step (A') is equal to or greater than a predetermined threshold; An example of a primer-probe set for amplifying or detecting a 63-bp human Alu sequence is a PCR primer-probe set for detecting human Alu, which includes a primer pair composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO:7 (Alu212F18) or the nucleotide sequence shown in SEQ ID NO:7 with one to three nucleotides deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO:8 (Alu274R18) or the nucleotide sequence shown in SEQ ID NO:8 with one to three nucleotides deleted, substituted, or added, and a probe consisting of the nucleotide sequence shown in SEQ ID NO:9 (Alu248RH18) or the nucleotide sequence shown in SEQ ID NO:9 with one to three nucleotides deleted, substituted, or added.An example of a primer-probe set for amplifying or detecting a 106-bp human Alu sequence is a PCR primer-probe set for detecting human Alu, comprising a primer pair consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20) or the nucleotide sequence shown in SEQ ID NO: 1 with one to three nucleotides deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 10 (Alu206R20) or the nucleotide sequence shown in SEQ ID NO: 10 with one to three nucleotides deleted, substituted, or added, and the present human Alu detection PCR probe or the human Alu detection PCR probe described in (c') above. In the present methods 1 and 2 for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject, the amount of genomic DNA is preferably expressed as the concentration (pg / μl) of cfDNA in plasma.

[0021] ◆ PCR primer pair for detecting human Alu In the present PCR primer pair for detecting human Alu, the nucleotide sequence of the forward primer (a) shown in SEQ ID NO: 1 is the forward primer disclosed in Patent Document 1. On the other hand, in the present PCR primer pair for detecting human Alu, the nucleotide sequence (Alu57F20) shown in SEQ ID NO: 2, which is the forward primer (a'), was designed for the first time by the present invention. The nucleotide sequence of this forward primer (a') was designed as a nucleotide sequence that cannot be detected using the default settings of Primer 3, a primer design software. Note that the forward primer (a') may also be a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 in which 1 to 3 nucleotides have been deleted, substituted, or added.

[0022] On the other hand, the nucleotide sequence (Alu237R19) of the reverse primer (b) shown in SEQ ID NO: 3 was designed for the first time by the present invention. This nucleotide sequence shown in SEQ ID NO: 3 was designed as a nucleotide sequence that cannot be detected using the default settings of primer 3, which is a primer design software. Note that the reverse primer (b) may also be a reverse primer consisting of a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 3.

[0023] Examples of the present PCR primer pair for detecting human Alu include the combination of (a) and (b) above, and the combination of (a') and (b) above. By using the PCR primer pair for detecting human Alu having the combination of (a) and (b) above, it is possible to amplify the sequence from bases 101 to 237 in the model sequence of human Alu shown in SEQ ID NO: 6. Furthermore, by using the PCR primer pair for detecting human Alu having the combination of (a') and (b) above, it is possible to amplify the sequence from bases 57 to 237 in the model sequence of human Alu.

[0024] Model sequence of human Alu (SEQ ID NO: 6) GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGYGGATCACYTGAGGYCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAATTAGCCGGGCGTGGTGG CGSGYGCCTGTARTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAG TGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGYGACAGAGYGAGACTCYGTCTCAAAAAAAAAAAA

[0025] In the model sequence of human Alu shown in SEQ ID NO: 6, Y represents C or T, S represents C or G, and R represents A or G.

[0026] Examples of the combinations of (a) and (b) above include: (1) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20) and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19); (2) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 in which 1 to 3 nucleotides have been deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3; (3) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 in which 1 to 3 nucleotides have been deleted, substituted, or added; and (4) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 in which 1 to 3 nucleotides have been deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 in which 1 to 3 nucleotides have been deleted, substituted, or added. In this specification, a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added preferably refers to a nucleotide sequence in which 1 or 2 nucleotides have been deleted, substituted, or added, and more preferably refers to a nucleotide sequence in which 1 nucleotide has been deleted, substituted, or added.

[0027] Examples of the combinations of (a') and (b) above include: (1) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20) and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19); (2) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 in which 1 to 3 nucleotides have been deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3; (3) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 in which 1 to 3 nucleotides have been deleted, substituted, or added; and (4) a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 in which 1 to 3 nucleotides have been deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 in which 1 to 3 nucleotides have been deleted, substituted, or added.

[0028] Human Alu Detection PCR Probe: Among the present human Alu detection PCR probes, the human Alu detection PCR probe (c) was designed for the first time by the present invention. The probe consisting of the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA) was designed with the aim of detecting as many human targets as possible. In the nucleotide sequence shown in SEQ ID NO: 4, the second guanine, the third guanine, the fourth cytosine, the thirteenth guanine, the fourteenth thymine, and the fifteenth adenine are locked nucleic acid (LNA) (C+G+G+CTAATTTTT+G+T+AT; "+" indicates LNA). This human Alu detection PCR probe hybridizes to the sequence from 125 to 140 in the human Alu model sequence shown in SEQ ID NO: 6. In synthesizing this human Alu detection PCR probe, the LNA monomer can be obtained, for example, by the synthesis method described in WO 1998 / 039352.

[0029] The present PCR probe for detecting human Alu can detect and / or quantify all or a partial sequence of an Alu sequence that can hybridize under stringent conditions to the present PCR probe for detecting human Alu, such as an amplification product using the present PCR primer pair for detecting human Alu. The present PCR probe for detecting human Alu is preferably labeled with a labeling substance, and from the viewpoint of more rapid or more sensitive detection or quantification, is preferably labeled with a fluorescent substance. Examples of the labeling substance other than fluorescent substances include biotin, a complex of biotin and avidin, and enzymes such as peroxidase. Preferred examples of the fluorescent substance include fluorescent proteins such as luciferase, as well as fluorescent dyes such as fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), 6-carboxy-4,7,2',7'-tetrachlorofluorescein (TET), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), Cy3, Cy5, 4,7,2',4',5,',7'-hexachloro-6-carboxyfluorescein (HEX). Furthermore, the present PCR probe for detecting human Alu is more preferably double-labeled with a fluorescent substance (reporter fluorescent dye) and a quencher substance (quencher fluorescent dye), and it is particularly preferred that the 5'-end is labeled with a fluorescent substance (reporter fluorescent dye) and the 3'-end is labeled with a quencher substance (quencher fluorescent dye).Examples of fluorescent substances (reporter fluorescent dyes) include the above-mentioned fluorescent dyes, and examples of quencher substances (quencher fluorescent dyes) include rhodamine fluorescent dyes such as 6-carboxytetramethylrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX), and black hole quenchers such as [(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline (BHQ-1) and [(4-(1-nitro-phenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]-aniline (BHQ-2). Among these, 6-carboxyfluorescein (6-FAM) is preferred as a fluorescent substance (reporter fluorescent dye), and quencher substances (quencher fluorescent dyes) are preferred. Suitable examples include Black Fluorescence Quencher (IBFQ) and [(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline (BHQ-1).

[0030] The stringent conditions refer to conditions under which so-called specific hybrids are formed but non-specific hybrids are not formed. Specifically, these conditions include conditions under which DNAs having an identity of 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% hybridize with each other, and DNAs having lower identities do not hybridize with each other, or conditions for annealing in conventional real-time PCR, specifically, hybridization conditions at 50°C or higher, preferably 52°C or higher, more preferably 54°C or higher, and even more preferably 56°C or higher.

[0031] Human Alu Detection PCR Primer-Probe Set The present human Alu detection PCR primer-probe set is not particularly limited as long as it comprises the present human Alu detection PCR primer pair and the present human Alu detection PCR probe, or the present human Alu detection PCR primer pair and (c') a human Alu detection PCR probe consisting of the nucleotide sequence (Alu144RH20) shown in SEQ ID NO: 5, or a nucleotide sequence in which one to three nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 5. Using the nucleotide sequence (Alu101F20) shown in SEQ ID NO: 1 as a forward primer allows for the amplification and detection of a 137-bp Alu sequence from positions 101 to 237 in the human Alu model sequence shown in SEQ ID NO: 6. Using the nucleotide sequence (Alu57F20) shown in SEQ ID NO: 2 as a forward primer allows for the amplification and detection of a 181-bp Alu sequence from positions 57 to 237 in the human Alu model sequence shown in SEQ ID NO: 6. Human genomic DNA can be quantified by amplifying and detecting the Alu sequence. The human genomic DNA to be quantified includes cfDNA.

[0032] Method for detecting and / or quantifying human genomic DNA Method 1 for detecting and / or quantifying human genomic DNA is not particularly limited as long as it includes a step of performing PCR using DNA extracted from a test sample as a template and the present human Alu detection PCR primer pair.

[0033] The "test sample" is not particularly limited as long as it contains human genomic DNA, and may further contain DNA derived from a non-human organism in addition to human genomic DNA. Examples of the "test sample" include human biological samples, biological samples derived from non-human organisms, ancient samples, forensic samples, and paleontological samples. Preferred examples include biological samples derived from xenotransplant model animals prepared by transplanting human cells into non-human animals, and ancient samples derived from ancient human bones. Examples of the "non-human animal" include non-human mammals such as mice, rats, guinea pigs, dogs, rabbits, pigs, goats, and cows, with rodents such as mice, rats, and guinea pigs being more preferred. Furthermore, the "human cells" may be any type of human-derived cell, such as human stem cells, human cancer cells, human somatic cells, and human germ cells. Particularly preferred examples include human stem cells, such as human mesenchymal stem cells, human hematopoietic stem cells, human neural stem cells, human iPS cells, human ES cells, and human somatic cell-derived embryonic stem cells. In this specification, the term "old sample" refers to a "non-fresh sample" that has been in a variety of environments for a period of several days to several million years or more, and examples of lower limits of the time that has passed include 2 days, 1 week, 2 weeks, 1 month, 3 months, 1 year, and 3 years, while examples of upper limits of the time that has passed include 5 years, 10 years, 20 years, 40 years, 100 years, 200 years, 400 years, 600 years, 5,000 years, and 10,000 years. In particular, "old samples derived from ancient human bones" refers to samples derived from human bones that are several years or more old from the time when human DNA was detected and / or quantified, and examples of such old samples include samples derived from human bones that are several years to 2 million years old, 500,000 to 10,000 years old, 1 million to 10,000 years old, 200 to 10,000 years old, 300 to 10,000 years old, 400 to 10,000 years old, 50 to 5,000 years old, 100 to 5,000 years old, 200 to 5,000 years old, 300 to 5,000 years old, 400 to 5,000 years old, 50 to 600 years old, 100 to 600 years old, 200 to 600 years old, 300 to 600 years old, and 400 to 600 years old from the time when human DNA was detected and / or quantified.

[0034] Furthermore, the method for extracting human genomic DNA from the test sample is not particularly limited as long as it is a known method such as the "proteinase K / phenol extraction method," "proteinase K / phenol / chloroform extraction method," "alkaline lysis method," "boiling method," "magnetic bead method," or "spin column method." Among the above methods, a method including an RNA degradation step using RNase is preferred, and a method including an RNA degradation step using RNase A and RNase T is even more preferred. Specifically, an example of a method for extracting DNA from the test sample is the "proteinase K / phenol extraction method," in which RNA degradation treatment is performed using RNase A and RNase T1 prior to the proteinase K treatment step.

[0035] The PCR in the "Method 1 for detecting and / or quantifying human genomic DNA" is not particularly limited as long as it uses the PCR primer pair for detecting human Alu. It may be an end-point PCR method in which the PCR amplification product is detected at the end of a cycle, or a real-time PCR method in which the increase in the PCR amplification product is monitored in real time. An example of an end-point PCR method is digital PCR. An example of a method for detecting an amplification product in end-point PCR is to subject the reaction solution after PCR to conventional gel electrophoresis using agarose or the like, and detect the DNA fragments after electrophoresis using ethidium bromide staining, a fluorescent reagent, or the like. An example of a method for detecting an amplification product in real-time PCR is the intercalation method, in which a nonspecific DNA intercalating dye is added to the PCR reaction solution in advance, and the double-stranded DNA of the amplification product is detected over time. In the intercalation method, nonspecific DNA intercalating dyes such as SYBR (registered trademark) Green I, SYBR (registered trademark) Green II, SYBR (registered trademark) Gold, BEBO, YO-PRO-1, LCGreen (registered trademark), SYTO-9, SYTO-13, SYTO-16, SYTO-60, SYTO-62, SYTO-64, SYTO-82, POPO-3, TOTO-3, BOBO-3, TO-PRO-3, YO-PRO-1, PicoGreen (registered trademark), SYTOX Orange, and EvaGreen (registered trademark) can be used. Among these, it is preferable to use SYBR (registered trademark) Green I.

[0036] Furthermore, the present method 2 for detecting and / or quantifying human genomic DNA uses the present PCR primer-probe set for detecting human Alu instead of the present PCR primer pair for detecting human Alu, specifically, a PCR probe for detecting human Alu consisting of (c) or (c') above. In the present method 2 for detecting and / or quantifying human genomic DNA, it is preferable to use a fluorescently labeled PCR probe for detecting human Alu consisting of (c) or (c') above. The type of PCR probe for detecting human Alu consisting of (c) or (c') above used here is not particularly limited, and examples include hydrolysis probes, molecular beacon probes, cycling probes, Eprobe (registered trademark), Qprobe (registered trademark), Scorpion probes, hybridization probes, etc., with hydrolysis probes being particularly preferred. Hydrolysis probes are typically linear oligonucleotides in which the 5' end of a nucleic acid probe is modified with a fluorescent substance (reporter fluorescent dye) and the 3' end is modified with a quencher. A molecular beacon probe is typically an oligonucleotide that can form a stem-loop structure, with the 5' end of the nucleic acid probe modified with a fluorescent substance (reporter fluorescent dye) and the 3' end modified with a quencher. A cycling probe is typically a chimeric oligonucleotide composed of RNA and DNA, with one end modified with a fluorescent substance (reporter fluorescent dye) and the other end modified with a quencher. An Eprobe is typically an artificial nucleic acid that contains two fluorescent dyes in a thymine nucleotide. Its fluorescence is suppressed in a single-stranded state when not bound to a target, but it emits fluorescence upon binding to a target. A Qprobe is typically an oligonucleotide with a cytosine terminus, with the terminal cytosine labeled with a fluorescent substance and also known as a guanine quencher probe. A scorpion probe is typically an oligonucleotide that can form a hairpin loop structure, with one end of the nucleic acid probe modified with a fluorescent substance (reporter fluorescent dye) and the 3' end modified with a quencher.The hybridization probe consists of a donor probe, which is an oligonucleotide modified with a fluorescent dye at the 3' end, and an acceptor probe, which is an oligonucleotide modified with a fluorescent dye at the 5' end. When these probes bind to a target, the two fluorescent dyes come into close proximity, and the fluorescent dye in the acceptor probe is excited by the fluorescence of the fluorescent dye in the donor probe, causing it to emit light.

[0037] The PCR can be performed according to the method described in documents such as "Molecular Cloning, A Laboratory Manual," or can be performed using commercially available PCR kits such as KOD-Plus-Neo (Toyobo Co., Ltd.), TaKaRa PCR Amplification Kit (TaKaRa), or TaqMan® Real-Time PCR Master Mixes (Thermo Fisher Scientific) according to the instructions provided with the kit. The PCR can be performed using nucleic acids obtained from a test sample, the present PCR primer pair for detecting human Alu, or the present PCR primer-probe set for detecting human Alu, as well as various reagents required for the PCR reaction. These reagents include enzymes that polymerize nucleic acids (e.g., polymerases), nucleic acid raw materials (e.g., dNTPs), buffers for nucleic acid amplification reactions (e.g., components with buffering properties such as Tris-Cl, surfactants such as Tween 20, etc.), and Mg ２＋ In addition, depending on the type of PCR, necessary reagents can also be used additionally.

[0038] When performing the PCR, a commercially available nucleic acid amplifier can be used depending on the type of nucleic acid amplification reaction used, and among these, a nucleic acid amplifier equipped with a device capable of measuring a probe signal (preferably a fluorescent signal) is preferred. Examples of real-time PCR instruments capable of both nucleic acid amplification and fluorescent signal measurement include the 7500 real-time PCR instrument manufactured by Applied Biosystems, the CFX96 manufactured by Bio-RAD, and the Light Cycler 480 manufactured by Roche.

[0039] In the present method 3 for detecting and / or quantifying human genomic DNA, steps (I) and (II) may be performed in the order of step (I) followed by step (II), or step (II) followed by step (I), or steps (I) and (II) may be performed simultaneously, but steps (I) and (II) are preferably performed simultaneously. Step (III) is performed after steps (I) and (II).

[0040] As used herein, the term "standard sample" is not particularly limited as long as it is prepared by serially diluting a known amount of human genomic DNA, but examples include standard samples prepared so that the amount of human genomic DNA added to each PCR reaction tube is in the range of, for example, 1 fg to 10 ng, preferably 0.1 fg to 10 ng, and more preferably 0.01 fg to 10 ng. Furthermore, the "standard sample" may further contain, in addition to human genomic DNA, genomic DNA derived from a non-human organism that may be contained in the test sample. Furthermore, the human genomic DNA contained in the "standard sample" may be fragmented depending on the type of the "test sample." For example, human genomic DNA fragmented to an appropriate size in the range of 20 kbp to 100 bp can be used.

[0041] Examples of methods for "creating a calibration curve" in step (II) include plotting the intensity level of the fluorescent signal obtained by real-time PCR using the standard sample as a template against the amount of human genomic DNA, and plotting the Ct value of the fluorescent signal obtained by real-time PCR using the standard sample as a template against the amount of human genomic DNA. Here, "Ct value" refers to the cycle number at which the amplification curve intersects with a threshold in real-time PCR, and represents the cycle number at which the amount of fluorescence derived from the generation of PCR amplification products reaches a certain predetermined amount (threshold). The Ct value decreases as the amount of target DNA initially contained in the sample increases, and conversely, increases as the amount of target DNA contained in the sample decreases. In step (III), the amount of human genomic DNA contained in the test sample can be calculated by comparing the intensity level or Ct value of the fluorescent signal obtained by real-time PCR of the test sample with the calibration curve.

[0042] A kit for detecting and / or quantifying human genomic DNA may be prepared by including the above-described PCR primer pair for detecting human Alu and / or the PCR probe for detecting human Alu. This kit may include components typically used in this type of detection kit (e.g., carrier, pH buffer, stabilizer, etc.) as well as an accompanying document such as an instruction manual. Furthermore, the kit of the present invention may further include human genomic DNA for use as a standard sample and DNA derived from a non-human organism (e.g., mouse genomic DNA, rat genomic DNA, guinea pig genomic DNA, etc.) for use as a negative control.

[0043] Method for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject In the method for assisting in the prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject, examples of biological samples include blood, serum, plasma, saliva, and urine. Examples of human genomic DNA include cfDNA and exosome DNA. It is desirable to determine the concentration of cfDNA in plasma. Known methods can be used to obtain plasma, but examples include a method in which blood is drawn from a subject, centrifuged at 3,000 rpm for 10 minutes, and the supernatant is further centrifuged at 12,000 rpm for 15 minutes.

[0044] The threshold can be determined in advance based on the amount of genomic DNA extracted from biological samples of healthy individuals and patients with renal cell carcinoma or prostate cancer. The amount of genomic DNA can be determined based on the plasma cfDNA concentration (pg / μl) determined using a primer-probe set that amplifies or detects a human Alu sequence of preferably 166 bp or less, more preferably 63 bp, 106 bp, or 137 bp, or a combination thereof. In calculating the threshold, the plasma cfDNA concentration may be transformed using a predetermined method, such as square root transformation, exponential transformation, logarithmic transformation, angular transformation, probit transformation, reciprocal transformation, or power transformation, or the cfDNA concentration may be combined with these transformed values.

[0045] The threshold can be calculated using the median, mean, AUC (area under curve) value based on ROC (Receiver Operating Characteristic) curve analysis, a value using the distance from the upper left corner, or the Youden index. The threshold may be calculated based on the plasma cfDNA concentration (pg / μl) as well as other indicators such as age, body mass index (BMI), weight, sex, medical history, drug prescription history, and treatment history. Furthermore, the threshold may be calculated by adding, subtracting, multiplying, or dividing any value with respect to the plasma cfDNA concentration determined using a primer-probe set that amplifies and detects, for example, a 63 bp, 106 bp, or 137 bp human Alu sequence. For example, a value obtained by dividing the value by the cfDNA concentration in plasma determined with a primer-probe set amplifying and detecting human Alu of a predetermined length of 167 bp or more, for example, 167 to 332 bp, preferably 181 bp, or a value obtained by subtracting the cfDNA concentration in plasma determined with a primer-probe set amplifying and detecting human Alu of a predetermined length, for example, 166 to 332 bp, preferably 181 bp, based on the AUC value, the distance from the upper left corner, or the Youden index may be used to calculate the threshold. Examples of threshold AUC values ​​include, for example, 0.7 or more, preferably 0.75 or more, more preferably 0.8 or more, even more preferably 0.84 or more, even more preferably 0.9 or more, and most preferably 0.92 or more.

[0046] An example of a primer-probe set for amplifying and detecting the 181-bp human Alu is a PCR primer-probe set for detecting human Alu, which includes a primer pair composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20) or the nucleotide sequence shown in SEQ ID NO: 2 with one to three nucleotides deleted, substituted, or added, and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19) or the nucleotide sequence shown in SEQ ID NO: 3 with one to three nucleotides deleted, substituted, or added, and the present human Alu detection PCR probe or the human Alu detection PCR probe described in (c') above.

[0047] The stage of renal cell carcinoma or prostate cancer in the subject may be any of stage 0, I, II, III, or IV according to the TNM classification of the Union for International Cancer Control (UICC), but preferably stage III or IV.

[0048] When the amount of human genomic DNA in a biological sample extracted from a subject is equal to or greater than a predetermined threshold, it can assist in predicting whether the subject has renal cell carcinoma or prostate cancer. Conversely, when the amount of human genomic DNA in a biological sample extracted from a subject is less than a predetermined threshold, it can assist in predicting whether the subject does not have renal cell carcinoma or prostate cancer.

[0049] Furthermore, by determining a threshold based on the concentration (pg / μl) of cfDNA in plasma determined using genomic DNA extracted in advance from biological samples of renal cell carcinoma or prostate cancer patients with different malignancies, particularly a primer-probe set that amplifies or detects human Alu sequences of 63 bp, 106 bp, and 137 bp, it is also possible to assist in predicting the malignancy of renal cell carcinoma or prostate cancer in a test subject.

[0050] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. In the following examples, primer-probe sets for amplifying and detecting 63 bp, 106 bp, 137 bp, and 181 bp human Alu sequences will also be referred to as the "63 bp Alu detection primer-probe set," the "106 bp Alu detection primer-probe set," the "137 bp Alu detection primer-probe set," and the "181 bp Alu detection primer-probe set," respectively.

[0051] [Conventional Primer-Probe Sets] First, the present inventors disclosed the above-mentioned human Alu model sequence (SEQ ID NO: 6) in Patent Document 1. Furthermore, in Patent Document 1, the present inventors established criteria 1 to 15 for selecting primers and probes for Alu-qPCR, and disclosed the following two primer-probe sets selected based on these criteria.

[0052] <63 bp primer-probe set for detecting Alu> Forward primer (Alu212F18): GGCGGAGGTTGCAGTGAG (SEQ ID NO: 7) Reverse primer (Alu274R18): GTCTCGCTCTGTCGCCCA (SEQ ID NO: 8) Probe (antisense) (Alu248RH18): TGCAGTGGCGCGATCTCG (SEQ ID NO: 9)

[0053] <106 bp primer-probe set for detecting Alu> Forward primer (Alu101F20): GGTGAAAACCCCGTCTCTACT (SEQ ID NO: 1) Reverse primer (Alu206R20): GGTTCAAGCGATTCTCCTGC (SEQ ID NO: 10) Probe (antisense) (Alu144RH20): CGCCCGGCTAATTTTTGTAT (SEQ ID NO: 5)

[0054] FIG. 1 shows the relationship between the Alu model sequence (SEQ ID NO: 6) and each primer and probe.

[0055] [Example 1] Design of New Primers In the present invention, two new primer-probe sets were designed to detect human Alu sequences of different sizes: a "primer-probe set for detecting 137 bp Alu" and a "primer-probe set for detecting 181 bp Alu." The reverse primers for detecting 137 bp and 181 bp share a common sequence, while the forward primers have different sequences (Figure 2). The probe sequence (Alu144RH20) is also a common sequence for detecting 106 bp, 137 bp, and 181 bp.

[0056] <137 bp primer-probe set for detecting Alu> Forward primer (Alu101F20): GGTGAAAACCCCGTCTCTACT (SEQ ID NO: 1) Reverse primer (Alu237R19): GATCTCGGCTCACTGCAAC (SEQ ID NO: 3) Probe (antisense) (Alu144RH20): CGCCCGGCTAATTTTTGTAT (SEQ ID NO: 5)

[0057] <181 bp primer-probe set for detecting Alu> Forward primer (Alu57F20): CGGATCACTTGAGGTCAGGA (SEQ ID NO: 2) Reverse primer (Alu237R19): GATCTCGGCTCACTGCAAC (SEQ ID NO: 3) Probe (antisense) (Alu144RH20): CGCCCCGGCTAATTTTTGTAT (SEQ ID NO: 5)

[0058] [Example 2] Improvement of the probe (Alu144RH20) The inventors used next-generation sequencing to analyze the amplified sequences using the previously designed forward primer (Alu101F20) and reverse primer (Alu206R20). Table 1 shows the number of sequences that match Alu101F20, Alu206R20, and the probe (Alu144RH20) out of a total of 574,485 reads (168,980 types). The analysis results revealed that there were few sequences that matched the probe (Alu144RH20), and that only 1.6% (7,335 / 457,560) of the amplified sequences using both primer sequences had a perfect match. This was a smaller number than expected based on the Blast analysis of the database.

[0059]

[0060] Therefore, the inventors proposed that shortening the probe length using LNAs would increase the number of perfectly matched sequences, thereby enabling better separation, especially at low concentrations. LNAs are artificial nucleic acids bridged with 2'-O,4'-C methylene. Incorporation of LNAs into oligonucleotides increases the thermal stability of the duplex and improves the specificity of hybridization of the oligonucleotide to its target sequence. In particular, in the case of qPCR, background fluorescence due to binding to non-target sequences is reduced, improving the signal-to-noise ratio (S / N ratio). Furthermore, improved hybridization of qPCR probes to their targets has been reported to potentially increase the melting temperature (Tm) by up to 8°C per LNA monomer substitution in the neutral salt state compared to DNA oligonucleotides.

[0061] Table 2 shows the target types and numbers of targets in next-generation sequencing data when the base length is shortened.

[0062]

[0063] The probe (Alu144RH20; SEQ ID NO: 5) described by the present inventors in the above-mentioned Patent Document 1 is a 20-base (complementary strand) TaqMan probe starting from the 125th base of the Alu model sequence shown in SEQ ID NO: 6. By increasing the number of bases to 16, the number of targets increases by approximately 3 to 4 times, and therefore Alu140RH16-LNA was prepared (SEQ ID NO: 4). Hereinafter, such a probe may be referred to as "Alu140RH16-LNA." Furthermore, in probe 140RH16-LNA, the "+G+G+C" residues at positions 2 to 4 and the "+G+T+A" residues at positions 13 to 15 are LNA.

[0064] Probe (Alu140RH16-LNA): C+G+G+CTAATTTTT+G+T+AT (SEQ ID NO: 4)

[0065] As shown in Table 2, a probe length of 14 bases is expected to increase the number of targets by approximately 20-fold. However, since the Tm value did not increase sufficiently even with the introduction of six LNAs at 14 bases, Alu138RH14 was not designed. The probe starting at 125 was selected because it would not cross-react with mice.

[0066] When qPCR was performed using probe Alu144RH20 and probe Alu140RH16-LNA at an annealing temperature of 56°C, the cycle number rise was significantly faster with probe Alu140RH16-LNA, demonstrating the effect of increasing the number of targets. Furthermore, the saturation point of the trend was higher, suggesting that detection at earlier cycle numbers was possible before the accumulation of inhibitors (amplified nucleic acids). Furthermore, the increased specificity and the increased Tm value with LNA resulted in stable probe binding, and the shorter base length allowed for more rapid binding, which is believed to have resulted in a smoother amplification curve and improved reliability of the values.

[0067] Furthermore, when qPCR was performed at an annealing temperature of 58°C, the probe Alu144RH20 did not exhibit a smooth amplification curve, suggesting that the Tm value of the probe was insufficient. However, when probe Alu140RH16-LNA was used, a stable and clean amplification curve was obtained even at 58°C. Furthermore, the difference in cycle number between 10 fg and NTC (no template control) was larger when probe Alu140RH16-LNA was used. This is thought to be due to increased sensitivity due to increased specificity in the low concentration range.

[0068] Example 3 Confirmation of Sensitivity and Specificity of Primer-Probe Sets of the Present Invention (1) Real-Time PCR Conditions The 137-bp Alu detection primer-probe set and 181-bp Alu detection primer-probe set designed in Example 1 above, as well as the probe (Alu140RH16-LNA) designed in Example 2, were prepared. Furthermore, the Alu144RH20 probe was labeled with FAM at the 5' end and BHQ1 at the 3' end. The Alu140RH16-LNA probe was labeled with FAM at the 5' end and IBFQ as a quenching label at the 3' end. Additionally, as reference (control) controls, the 63-bp Alu detection primer-probe set and the 106-bp Alu detection primer-probe set described in the "Conventional Primer-Probe Set" section were similarly prepared. That is, four types of primer-probe sets (primer-probe sets for detecting Alu of 63 bp, 106 bp, 137 bp, and 181 bp) were used in this experiment. As the probe, Alu144RH20 was used for the data shown in Figure 3, and Alu140RH16-LNA was used for the data shown in Figure 4.

[0069] A standard sample containing only fragmented human genomic DNA serially diluted 10-fold from 10 fg / μL to 1 ng / μL was used as a PCR template. Specifically, the human genome was fragmented using a Covaris DNA Shearing System M220 (Covaris). Human genome quantification was performed using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). A 10-fold dilution series was prepared from 10 ng of human genome using Easy Dilution (Takara Bio).

[0070] PCR samples were prepared using each primer-probe set and the above template as follows. The templates and samples were prepared using low-adsorption tubes and tips to prevent the loss of even traces of DNA. Template: 1 μL; TaqMan Universal Master Mix II, no. UNG (Thermo Fisher Scientific); 10 μL; Forward primer (10 μM); 0.4 μL; Reverse primer (10 μM); 0.4 μL; Probe (10 μM); 0.5 μL; Water: 7.7 μL (Total): 20 μL

[0071] Real-time PCR was performed on the PCR samples prepared as described above using a LightCycler 480 (Roche). The PCR reaction consisted of 10 minutes of heat denaturation at 95°C, followed by five cycles of 95°C for 30 seconds, 56°C for 4 minutes 30 seconds, and 72°C for 30 seconds, followed by 45 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. Analysis was performed using the LightCycler® 480 Software release 1.5.0 using the fit point method. The calibration curve generated based on real-time PCR is shown in Figure 3.

[0072] As shown in Figure 3, all of the primer-probe sets for detecting Alu (63 bp, 106 bp, 137 bp, and 181 bp) yielded highly linear calibration curves with correlation coefficients of 0.99 or greater from 1 ng to 10 fg. These results demonstrate that qPCR using the above primer-probe sets is capable of detecting fragmented human genomic DNA up to 10 fg. Alu-qPCR using these primer-probe sets established a method for ultrasensitive detection of human genomic DNA of four different sizes (63 bp, 106 bp, 137 bp, and 181 bp) or greater.

[0073] Furthermore, a similar experiment was carried out to quantify 106 bp, 137 bp, and 181 bp cfDNA using Alu140RH16-LNA instead of the probe Alu144RH20 used for the data in Figure 3. The results are shown in Figure 4.

[0074] From FIG. 4, it was confirmed that human genomic DNA (106 bp, 137 bp, and 181 bp) could be detected with ultra-high sensitivity even when Alu140RH16-LNA was used.

[0075] Example 4: Measurement of cfDNA concentrations in plasma from healthy individuals and cancer patients. Using the 63 bp, 106 bp, 137 bp, and 181 bp Alu detection primer-probe sets used to create the calibration curve in Example 3, cfDNA concentrations in plasma from healthy individuals and renal cell carcinoma patients were quantified by size-specific Alu-qPCR. The probes used were Alu248R18 for 63 bp, Alu144RH20 for 106 bp, and Alu140RH16-LNA for 137 bp and 181 bp. Note that a primer pair amplifying 63 bp will amplify fragments of 63 bp or longer. Similarly, a primer pair that amplifies 106 bp will amplify fragments of 106 bp or more, a primer pair that amplifies 137 bp will amplify fragments of 137 bp or more, and a primer pair that amplifies 181 bp will amplify fragments of 181 bp or more. In other words, a primer pair that amplifies 63 bp will not be able to amplify fragments of 62 bp or less. Similarly, a primer pair that amplifies 106 bp will not be able to amplify fragments of 105 bp or less, a primer pair that amplifies 137 bp will not be able to amplify fragments of 136 bp or less, and a primer pair that amplifies 181 bp will not be able to amplify fragments of 180 bp or less.

[0076] Blood samples were collected from 26 healthy individuals (all from different individuals), 20 renal cell carcinoma patients (15 individuals, 5 of whom had a second blood draw on a different date and time) for the 63 bp and 131 bp measurements, and 34 renal cell carcinoma patients (23 individuals, 7 of whom had a second blood draw on a different date and time, and 2 of whom had a third blood draw on a different date and time) for the 106 bp and 181 bp measurements. The samples were centrifuged at 3,000 rpm for 10 minutes, and the supernatant was further centrifuged at 12,000 rpm for 15 minutes to obtain plasma. Next, cfDNA (50 μl) was extracted from the resulting 200 μL of plasma using magLEAD12gC (Precision System Science). The extracted cfDNA was stored at -80 °C until measurement. 1 μl of the sample was used to perform Alu-qPCR (LightCycler 480: Roche Diagnostics).

[0077] The results of box plotting the cfDNA concentrations in plasma are shown in Figure 5. Furthermore, the results of dividing the measured values ​​of 63 bp, 106 bp, and 137 bp in Figure 5 (cfDNA concentrations in plasma: pg / µl) by the measured value of 181 bp are shown in Figure 6.

[0078] The results in Figures 5 and 6 revealed that plasma cfDNA concentrations of small sized cfDNA (63 bp, 106 bp, 137 bp) were higher in renal cell carcinoma patients than in healthy subjects, and that there was no difference between healthy subjects and renal cell carcinoma patients in the 181 bp cfDNA concentration. Furthermore, dividing the plasma cfDNA concentrations of 63 bp, 106 bp, and 137 bp by the plasma cfDNA concentration of 181 bp revealed a greater difference between healthy subjects and renal cell carcinoma patients.

[0079] Example 5 ROC (Receiver Operating Characteristic) Analysis Based on the results of the cfDNA concentration in plasma in Example 4, ROC analysis was performed to visualize the effectiveness of distinguishing between healthy subjects and cancer patients and to set a threshold. The ROC analysis results for each cfDNA concentration in plasma determined using primer-probe sets that amplify and detect 63 bp, 106 bp, 137 bp, and 181 bp are shown in Figure 7, the ROC analysis results for 63 bp / 181 bp, 106 bp / 181 bp, and 137 bp / 181 bp are shown in Figure 8, and the ROC analysis results for 63 bp-181 bp, 106 bp-181 bp, and 137 bp-181 bp are shown in Figure 9. The cfDNA concentration in each graph is as described at the bottom of each graph.

[0080] The ROC analysis results for plasma cfDNA concentrations determined using primer-probe sets amplifying and detecting 63bp, 106bp, and 137bp showed AUC (Area under curve) values ​​of 0.7 or higher, with 106bp being particularly high at 0.85 or higher. Furthermore, the ROC analysis results for 63bp / 181bp, 106bp / 181bp, and 137bp / 181bp all showed AUC values ​​of 0.8 or higher, with 63bp / 181bp and 106bp / 181bp being particularly high at 0.9 or higher. It was revealed that there is a difference between healthy subjects and renal cell carcinoma patients when dividing by the 181bp cfDNA concentration rather than by each alone. Furthermore, the ROC analysis results for 63bp-181bp, 106bp-181bp, and 137bp-181bp all showed AUC values ​​of 0.78 or higher, with 63bp-181bp and 106bp-181bp being particularly high at 0.848 and 0.921, respectively, demonstrating that it is better to reduce the 181bp cfDNA concentration than to use either of these alone.

[0081] Example 6: Setting the threshold for cancer detection. Based on the results of Example 5, when the threshold was set to a value that could 100% identify patients, (i) the threshold for the cfDNA concentration in plasma determined using a primer-probe set amplifying and detecting 106 bp (see Figure 7) was 5.3 pg / μL, and (ii) the threshold for 106 bp / 181 bp (see Figure 8) was 2.71. When (i) and (ii) were used in combination, highly reliable screening was demonstrated, with a sensitivity of 1.0 (34 / 34) and a specificity of 0.82 (28 / 34) (Table 3). A method capable of distinguishing renal cell carcinoma patients from healthy controls with such a high probability was previously unavailable, demonstrating the usefulness of the size-specific Alu-qPCR as a minimally invasive method for diagnosing renal cell carcinoma.

[0082]

[0083] Furthermore, Figure 10 shows the threshold, sensitivity, specificity, and sensitivity + specificity when the sensitivity was set to 100%, >95%, >90%, >85%, and >80% for 106 bp, 106 bp / 181 bp, and 106 bp-181 bp, respectively. Figure 10 confirms that setting the sensitivity to >95% increases the sensitivity + specificity. Additionally, Figure 11 shows the sensitivity, specificity, and sensitivity + specificity when both the 106 bp and 106 bp / 181 bp thresholds are met simultaneously. When both the 106 bp and 106 bp / 181 bp thresholds were met simultaneously, an increase in specificity was observed. When the 106 bp threshold was 6.6 pg / μl and the 106 bp / 181 bp threshold was 2.73 (see Figure 10, >95%), the sensitivity was 0.971 and the specificity was 0.923.

[0084] Example 7 Application to Prostate Cancer The 106 bp, 106 bp / 181 bp, and 106 bp-181 bp values ​​calculated for healthy subjects in Example 5 were applied to prostate cancer patients. Plasma was obtained from three prostate cancer patients using the method described in Example 4. Next, 50 μL of cfDNA was extracted, and the cfDNA concentration (pg / μL) in the plasma was examined by Alu-qPCR using primer / probe sets that amplify and detect the 106 bp and 181 bp values, respectively. The results are shown in Table 4. Then, based on the cfDNA concentrations of 106 bp and 181 bp in the plasma of the healthy subjects and the prostate cancer patients, the threshold values ​​for 106 bp, 106 bp / 181 bp, and 106 bp-181 bp were determined by the ROC analysis described in Example 5.

[0085]

[0086] When the threshold for 106 bp was set to 6.89 and the threshold for 106 bp / 181 bp was set to 2.71, the sensitivity and specificity for 106 bp and 106 bp / 181 bp were 1 and 0.92, respectively. Furthermore, when the threshold for 106 bp-181 bp was set to 5.08, the sensitivity and specificity were 1 and 0.81, respectively. Therefore, the size-specific Alu-qPCR was shown to be extremely useful for minimally invasive prostate cancer diagnosis.

[0087] Example 8: Application to Post-Treatment Monitoring of Prostate Cancer Plasma cfDNA concentrations were examined in prostate cancer patients during post-hormonal therapy monitoring. Two prostate cancer patients received Leuprorelin PRO (Takeda Pharmaceutical Co., Ltd.) or Zytiga (Janssen Pharmaceuticals) as hormone therapy, and plasma was obtained before and six months after administration. Next, 50 μl of cfDNA was extracted, and the plasma cfDNA concentration (pg / μl) was examined by Alu-qPCR using primer-probe sets that amplify and detect the 106 bp and 181 bp fragments, respectively. Then, 106 bp / 181 bp and 106 bp-181 bp were determined based on the plasma cfDNA concentrations of 106 bp and 181 bp. Note that both of the two prostate cancer patients six months after administration were patients for whom the effectiveness of cancer treatment was confirmed based on the PSA value of the prostate cancer marker.

[0088]

[0089] As shown in Table 5, all of 106bp, 106bp / 181bp, and 106bp-181bp decreased 6 months after hormone therapy. Therefore, the plasma cfDNA concentration determined using a primer / probe set that amplifies and detects 106bp and 181bp, and the calculation of 106bp / 181bp and 106bp-181bp based on these concentrations can be used for monitoring after cancer treatment.

Claims

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8. (A'') quantifying the amount of human genomic DNA in a biological sample extracted from a test subject by a method comprising the following steps (I'') to (III''); (I'') performing real-time PCR using a primer-probe set for amplifying or detecting a 63 bp, 106 bp, and / or 137 bp human Alu sequence, and a real-time PCR using a primer-probe set for amplifying or detecting a 181 bp human Alu sequence, using human genomic DNA in a biological sample extracted from the subject as a template; (II') performing real-time PCR under the same conditions as in the above step (I'') using standard samples prepared by serially diluting known amounts of human genomic DNA as templates to prepare a calibration curve; (III'') calculating, from the calibration curve, the amount of human genomic DNA in the biological sample extracted from the test subject based on real-time PCR using a primer-probe set that amplifies or detects a 63 bp, 106 bp, and / or 137 bp human Alu sequence, and the amount of human genomic DNA in the biological sample extracted from the test subject based on real-time PCR using a primer-probe set that amplifies or detects a 181 bp human Alu sequence; (B'') assisting in prediction that the subject has renal cell carcinoma or prostate cancer, when the amount of human genomic DNA in the biological sample extracted from the subject based on real-time PCR using a primer-probe set that amplifies or detects the 63 bp, 106 bp, and / or 137 bp human Alu sequence quantified in step (A'') is divided by or subtracted from the amount of human genomic DNA in the biological sample extracted from the subject based on real-time PCR using a primer-probe set that amplifies or detects the 181 bp human Alu sequence quantified in step (A'') and is equal to or greater than a predetermined threshold value; The method for assisting in prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject, comprising the steps (A'') and (B'').

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10. In (I'') of step (A''), A primer-probe set for amplifying or detecting a 63-bp human Alu sequence comprises a PCR primer pair for detecting human Alu, the primer pair being composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO:7 (Alu212F18); or the nucleotide sequence shown in SEQ ID NO:7 with 1 to 3 nucleotides deleted, substituted or added; and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO:8 (Alu274R18); or the nucleotide sequence shown in SEQ ID NO:8 with 1 to 3 nucleotides deleted, substituted or added; a PCR primer-probe set for detecting human Alu, the PCR probe comprising a nucleotide sequence (Alu248RH18) shown in SEQ ID NO:9; or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO:9, A primer-probe set for amplifying or detecting a 106 bp human Alu sequence comprises a PCR primer pair for detecting human Alu, the primer pair being composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20); or the nucleotide sequence shown in SEQ ID NO: 1 with 1 to 3 nucleotides deleted, substituted or added; and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 10 (Alu206R20); or the nucleotide sequence shown in SEQ ID NO: 10 with 1 to 3 nucleotides deleted, substituted or added; a PCR primer-probe set for detecting human Alu, the PCR probe comprising a nucleotide sequence (Alu144RH20) shown in SEQ ID NO:5; or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO:5, A primer-probe set for amplifying or detecting a 137 bp human Alu sequence comprises a PCR primer pair for detecting human Alu, the primer pair being composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO:1 (Alu101F20); or the nucleotide sequence shown in SEQ ID NO:1 with 1 to 3 nucleotides deleted, substituted or added; and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO:3 (Alu237R19); or the nucleotide sequence shown in SEQ ID NO:3 with 1 to 3 nucleotides deleted, substituted or added; A PCR probe for detecting human Alu, comprising the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA); or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 4; or a PCR primer-probe set for detecting human Alu, comprising: a nucleotide sequence (Alu144RH20) shown in SEQ ID NO:5; or a PCR probe for detecting human Alu consisting of a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO:5, A primer-probe set for amplifying or detecting a 181-bp human Alu sequence comprises a PCR primer pair for detecting human Alu, the primer pair being composed of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO:2 (Alu57F20); or the nucleotide sequence shown in SEQ ID NO:2 with 1 to 3 nucleotides deleted, substituted or added; and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO:3 (Alu237R19); or the nucleotide sequence shown in SEQ ID NO:3 with 1 to 3 nucleotides deleted, substituted or added; A PCR probe for detecting human Alu, comprising the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA); or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 4; or A PCR probe for detecting human Alu, which comprises the nucleotide sequence shown in SEQ ID NO:5 (Alu144RH20); or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO:

5. The method according to claim 8, which is a PCR primer-probe set for detecting human Alu, comprising:

11. The method according to claim 8 or 10, wherein the threshold value is a value determined by an AUC value calculated from an ROC curve analysis.

12. The method according to claim 8, 10 or 11, wherein the subject is a patient undergoing hormone therapy for prostate cancer, and the method aids in predicting the presence or absence of prostate cancer as a monitoring method after cancer treatment.

13. A kit comprising a PCR primer pair for detecting human Alu, the primer pair being composed of the following forward primer (a') and reverse primer (b) for amplifying or detecting a 181 bp human Alu sequence, for use in calculating an amount of human genomic DNA in the method for assisting in prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject according to claim 8. (a') a forward primer consisting of the nucleotide sequence (Alu57F20) shown in SEQ ID NO: 2; or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2; (b) a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19); or the nucleotide sequence shown in SEQ ID NO: 3 in which 1 to 3 nucleotides have been deleted, substituted, or added;

14. A kit for use in calculating an amount of human genomic DNA in the method for assisting in prediction of the presence or absence of renal cell carcinoma or prostate cancer in a subject according to claim 8, comprising a PCR primer pair for detecting human Alu, the PCR primer pair being composed of the following forward primer (a') and reverse primer (b) for amplifying or detecting a 181 bp human Alu sequence, and a PCR probe for detecting human Alu, the PCR probe being composed of the following (c). (a') a forward primer consisting of the nucleotide sequence (Alu57F20) shown in SEQ ID NO: 2; or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2; (b) a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19); or the nucleotide sequence shown in SEQ ID NO: 3 in which 1 to 3 nucleotides have been deleted, substituted, or added; (c) a PCR probe for detecting human Alu consisting of the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA); or a nucleotide sequence in which 1 to 3 nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 4;