WO2024048659A1

WO2024048659A1 - 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

Info

Publication number: WO2024048659A1
Application number: PCT/JP2023/031568
Authority: WO
Inventors: 英雄明石; 一幸沼倉; 伸太郎成田
Original assignee: 国立大学法人秋田大学
Priority date: 2022-08-30
Filing date: 2023-08-30
Publication date: 2024-03-07

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

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 detecting and/or quantifying human genomic DNA, and renal cell carcinoma or prostate cancer. This invention relates to a method for assisting in predicting the presence or absence of.

Cell-free DNA (cfDNA) is a trace amount of DNA released into peripheral blood when cells die or are destroyed, and its association with cancer has long been suggested. For example, cfDNA has already attracted attention as a tumor marker for renal cell carcinoma (for example, Non-Patent Document 1). In Non-Patent Document 1, the size distribution of cfDNA in cancer patient plasma was investigated using a microfluidic chip and next-generation sequencing, and the results suggested that fragmented cfDNA may serve as a tumor marker for renal cell carcinoma. .

However, at present, it is technically difficult to apply such knowledge to the clinical setting of cancer diagnosis. This is because, at present, the only way to analyze the size distribution of cfDNA is to comprehensively analyze genome information using next-generation sequencing, but such analysis requires extremely expensive specialized equipment. It requires experts to operate and analyze it. Although next-generation sequencing devices are sometimes installed in university facilities that specialize in genetic analysis, they are rarely installed in general hospitals or medical testing institutions. Furthermore, next-generation sequence analysis requires a great deal of cost and time, so it is not realistic to use next-generation sequence analysis for general cancer diagnosis. From the above, in order to put into practical use the diagnosis of renal cell carcinoma using fragmented cfDNA as an indicator, it is necessary to develop a method that can easily and inexpensively quantify fragmented cfDNA using more general testing equipment. has been done.

Here, the Alu sequence is a repetitive sequence specific to primates, and is known to exist in the human genome in more than 1 million copies, accounting for more than 10% of the human genome. Since the total length of the human genome is about 3 billion nucleotide pairs, a simple calculation shows that there is an average of 1 copy of the Alu sequence for every 3000 nucleotide pairs of human genomic DNA. Due to these characteristics, the possibility of measuring blood cfDNA concentration using the human Alu sequence quantitative PCR method (hereinafter also referred to as "Alu-qPCR") and applying it to cancer diagnosis has been shown for some time (for example, patent Literatures 1, 2, Non-Patent Literature 2). However, a method for quantifying minute amounts of cfDNA and examining its size distribution by Alu-qPCR has not been established.

Under these circumstances, the present inventors developed an extremely sensitive Alu-qPCR using a primer/probe set previously 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 the Alu sequence, and furthermore, by using them, the conventional technology can be improved. It has been disclosed that fragmented human genomic DNA can be specifically detected with a sensitivity of more than 1,000 times.

International Publication No. 2016/028316 pamphlet International Publication No. 2006/128192 pamphlet Patent No. 6892695

If fragmented cfDNA of a predetermined size can be detected with high sensitivity from blood samples etc., it will be possible to apply it to cancer diagnosis using them as indicators. Renal cell carcinoma is the second most common urological malignant tumor after prostate cancer and bladder cancer, and its incidence is approximately 2.5 per 100,000 people, with a male to female ratio of 2 to 3. It is said that it tends to be more common in men with a score of 1. There are no effective tumor markers for renal cell carcinoma, and the common diagnostic methods are CT, ultrasound, and MRI imaging, but it is difficult to detect because there are no characteristic early symptoms. is the current situation. Until now, there has been no accurate method for quantifying trace amounts of cfDNA, and in particular, there has been little progress in research focusing on the size distribution of low-molecular-weight cfDNA, which is likely to increase in cancer-bearing patients. Furthermore, prostate-specific antigen (PSA) and the like are used as tumor markers for prostate cancer, but there are problems such as low specificity, and a prostate biopsy is performed for definitive diagnosis. For this reason, there has been a strong demand for the development of a diagnostic method using blood tests that is less invasive and less burdensome to the patient.

Therefore, the problem of the present invention is to design primer pairs and probes for quantitatively measuring low-molecular-weight cfDNA for each size, and to quantify cfDNA by using the primer pairs and probes. The object of the present invention is to provide a method for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer.

In 2016, the present inventors developed an ultra-sensitive human genome detection method (hereinafter referred to as "ultra-sensitive Alu-qPCR") that targets the Alu sequence in the human genome (see Patent Document 3, Non-Patent Document 3). ). The ultra-high-sensitivity Alu-qPCR described above is characterized in that it can accurately quantify low-molecular-weight human genomic DNA (<100 bp).

In the course of intensive study to solve the above problems, the present inventors applied the ultra-high sensitivity Alu-qPCR described above to design primers and probes that can quantify small-molecular-weight cfDNA by size, and detected human genomic DNA. and/or it has been found that it can be quantified. Furthermore, we discovered that by using a method to detect and/or quantify 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 biomarker exists. Ta. Furthermore, they discovered that by using a method for detecting and/or quantifying human genomic DNA using the above primers and probes, it is possible to assist in predicting the presence or absence of prostate cancer using cfDNA as an indicator, and completed the present invention. did.

That is, the present invention is as follows.
[1] For human Alu detection, which is composed of the following (a) forward primer and (b) reverse primer, or the following (a') forward primer and (b) reverse primer. PCR primer pair.
(a) A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 1;
(a') A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to 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 a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 3;
[2] A PCR probe for human Alu detection consisting 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 are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 4;
[3] A PCR probe for detecting human Alu consisting of the PCR primer pair for detecting human Alu according to [1] above and (c) according to [2] above; or a PCR probe for detecting human Alu according to [1] above. A PCR primer pair 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 to the nucleotide sequence shown in SEQ ID NO: 5; A PCR probe for human Alu detection consisting of;
A PCR primer/probe set for human Alu detection, comprising:
[4] Detecting human genomic DNA in the test sample, including the step of performing PCR using the PCR primer pair and probe set for human Alu detection described in [1] above, using the DNA extracted from the test sample as a template. and/or methods of quantification.
[5] Detection and detection of human genomic DNA in the test sample, including the step of performing PCR using the PCR primer/probe set for human Alu detection described in [3] above, using the DNA extracted from the test sample as a template. / or method of quantification.
[6] A method for detecting and/or quantifying human genomic DNA in a test sample using the PCR primer/probe set for human Alu detection described in [3] above, which comprises the following (I) to (III). A method, including a process.
(I) Performing real-time PCR using the primer/probe set using the DNA extracted from the test sample as a template;
(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 standard curve;
(III) calculating the amount of human genomic DNA in the test sample from the calibration curve;
[7] A method for assisting in predicting the presence or absence of renal cell cancer 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 subject by the method for detecting and/or quantifying human genomic DNA according to any one of [4] to [6] above;
(B) A step of assisting in predicting that the subject has renal cell cancer or prostate cancer when the amount of human genomic DNA quantified in step (A) is greater than or equal to 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 subject by a method including the following steps (I') to (III');
(I') Performing real-time PCR using the DNA extracted from the test sample as a template and a primer/probe set that amplifies or detects a 63bp, 106bp, and/or 137bp human Alu sequence;
(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 standard curve;
(III) calculating the amount of human genomic DNA in the test sample from the calibration curve;
(B') A step of assisting in predicting that the subject has renal cell carcinoma or prostate cancer when the amount of human genomic DNA quantified in step (A') is greater than or equal to a predetermined threshold;
[9] The presence or absence of renal cell carcinoma or prostate cancer in the subject described in [7] or [8] above, wherein the threshold value is a value determined by an AUC value calculated from ROC curve analysis. methods to assist in predicting.

By using the primer pair and probe set of the present invention, it becomes possible to specifically detect and/or measure approximately 0.1 to 10 fg of human genomic DNA. Furthermore, by using the primer pair and probe set described above, it becomes possible to detect and/or quantify human genomic DNA with high sensitivity without using next-generation sequencing. Furthermore, by using the primer pair and probe set described above, it becomes possible to predict the presence or absence of renal cell carcinoma or prostate cancer in a minimally invasive and simple manner.

FIG. 2 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". FIG. 6 is a diagram showing the positions of the Alu model sequence (SEQ ID NO: 6), "137 bp Alu detection primer/probe set" and "181 bp Alu detection primer/probe set". FIG. 3 is a diagram showing the results of a calibration curve created based on real-time PCR using Alu144RH20 as a probe in Example 3. FIG. 3 is a diagram showing the results of a calibration curve created based on real-time PCR using Alu140RH16-LNA as a probe in Example 3. FIG. 4 is a diagram showing the results of a box plot of plasma cfDNA concentrations in healthy subjects and renal cell carcinoma patients in Example 4. In Example 4, each cfDNA concentration in plasma determined with the primer/probe set that amplifies and detects 63 bp, 106 bp, and 137 bp in FIG. 5 was determined using the primer/probe set that amplifies and detects 181 bp. It is a figure showing the results of dividing by the cfDNA concentration in plasma (63b/181bp, 106bp/181bp, 137bp/181bp). Hereinafter, division may be expressed by "/". FIG. 7 is a diagram showing the results of ROC analysis of cfDNA concentrations in plasma determined using primer-probe sets for amplifying and detecting 63 bp, 106 bp, 137 bp, and 181 bp in Example 5. FIG. 7 is a diagram showing ROC analysis results for 63b/181bp, 106bp/181bp, and 137bp/181bp in Example 5. In Example 5, the respective cfDNA concentrations in plasma determined with primer-probe sets that amplify and detect 63 bp, 106 bp, and 137 bp were compared with those determined with a primer-probe set that amplified and detected 181 bp. It is a figure showing the ROC analysis results of the results of reduction by cfDNA concentration (63bp-181bp, 106bp-181bp, 137bp-181bp). Hereinafter, "-" may be used to indicate a decrease. In Example 5, the threshold, sensitivity, specificity, and sensitivity + when the sensitivity is 100%, >95%, >90%, >85%, and >80% for 106bp, 106bp/181bp, and 106bp-181bp, respectively. It is a figure showing specificity. In Example 5, it is a diagram showing sensitivity, specificity, and sensitivity + specificity when sensitivity is set to 100%, >95%, >90%, >85%, >80% for 106 bp and 106 bp/181 bp. .

The PCR primer pair for human Alu detection of the present invention is composed of the following (a) forward primer and (b) reverse primer, or (a') forward primer and (b) reverse primer. PCR primer pair for human Alu detection:
(a) A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 1;
(a') A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to 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 a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 3;
If so, there are no particular limitations, and hereinafter also referred to as "the subject PCR primer pair for human Alu detection."

Furthermore, the PCR probe for detecting human Alu of the present invention has (c) the nucleotide sequence shown in SEQ ID NO: 4 (Alu140RH16-LNA); or the nucleotide sequence shown in SEQ ID NO: 4 with a deletion of 1 to 3 nucleotides; The probe is not particularly limited as long as it is a probe consisting of a substituted or added nucleotide sequence; hereinafter also referred to as "the present PCR probe for detecting human Alu".

Further, the PCR primer/probe set for detecting human Alu of the present invention is a PCR primer/probe set for detecting human Alu, comprising the PCR primer pair for detecting human Alu and the PCR probe for detecting human Alu, or There is no particular restriction on the PCR primer/probe set for human Alu detection, which includes the present human Alu detection PCR primer pair and the human Alu detection PCR probe described in (c') above. Also referred to as "Alu detection PCR primer/probe set."

In addition, the method of detecting and/or quantifying human genomic DNA in a test sample of the present invention includes the step of performing PCR using the above-mentioned PCR primer pair for detecting human Alu, using DNA extracted from the test sample as a template. A method for detecting and/or quantifying human genomic DNA in the test sample (hereinafter also referred to as "Method 1 for detecting and/or quantifying human genomic DNA"), or the above-mentioned PCR primers and probes for detecting human Alu. A method for detecting and/or quantifying human genomic DNA in the test sample (hereinafter also referred to as "Method 2 for detecting and/or quantifying human genomic DNA") includes a step of performing PCR using a set. can.

Furthermore, as a method for detecting and/or quantifying human genomic DNA in other test samples of the present invention,
(I) Performing real-time PCR using the present human Alu detection PCR primer/probe set using the DNA extracted from the test sample as a template;
(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 standard curve;
(III) calculating the amount of human genomic DNA in the test sample from the calibration curve;
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, including the steps of ``detecting and/or quantifying human genomic DNA'' can be mentioned. Also called method 3.

Furthermore, the method of assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject of the present invention includes (A) a biological sample extracted from a subject by the above method for detecting and/or quantifying human genomic DNA; Quantifying the amount of human genomic DNA in;
(B) assisting in predicting that the subject has renal cell cancer or prostate cancer when the amount of human genomic DNA quantified in step (A) is greater than or equal to a predetermined threshold;
A method for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in a test subject, which is characterized by comprising steps (A) and (B) (Also referred to as "Method 1 for assisting in predicting the presence or absence of cell cancer or prostate cancer"). Further, another embodiment of the method of assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject of the present invention is characterized by comprising the following steps (A') and (B'). , a method for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject (hereinafter also referred to as "Method 2 for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject") ).
(A') Quantifying the amount of human genomic DNA in a biological sample extracted from a subject by a method including the following steps (I') to (III');
(I') Performing real-time PCR using the DNA extracted from the test sample as a template and a primer/probe set that amplifies or detects a 63bp, 106bp, and/or 137bp human Alu sequence;
(II') Performing real-time PCR under the same conditions as the above step (I') using a standard sample prepared by serially diluting a known amount of human genomic DNA as a template to create a standard curve;
(III') calculating the amount of human genomic DNA in the test sample from the calibration curve;
(B') A step of assisting in predicting that the subject has renal cell carcinoma or prostate cancer when the amount of human genomic DNA quantified in step (A') is greater than or equal to a predetermined threshold;
As a primer/probe set for amplifying or detecting the 63 bp human Alu sequence, the nucleotide sequence shown in SEQ ID NO: 7 (Alu212F18) or the nucleotide sequence shown in SEQ ID NO: 7 in which 1 to 3 nucleotides are deleted, substituted, or a forward primer consisting of an added nucleotide sequence, and a nucleotide sequence shown in SEQ ID NO: 8 (Alu274R18) or a nucleotide in which 1 to 3 nucleotides have been deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 8 A primer pair consisting of a reverse primer consisting of the sequence and the nucleotide sequence shown in SEQ ID NO: 9 (Alu248RH18) or the nucleotide sequence shown in SEQ ID NO: 9 in which 1 to 3 nucleotides have been deleted, substituted, or added. Examples include a PCR primer/probe set for detecting human Alu, which includes a probe consisting of a nucleotide sequence. As a primer/probe set for amplifying or detecting the 106 bp human Alu sequence, the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20) or the nucleotide sequence shown in SEQ ID NO: 1 in which 1 to 3 nucleotides are deleted, substituted, or a forward primer consisting of an added nucleotide sequence, and a nucleotide sequence shown in SEQ ID NO: 10 (Alu206R20) or a nucleotide in which 1 to 3 nucleotides have been deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10. A PCR primer/probe set for detecting human Alu, comprising a primer pair composed of a reverse primer consisting of a reverse primer sequence, and the present PCR probe for detecting human Alu or the PCR probe for detecting human Alu described in (c') above. can be mentioned. The amount of genomic DNA in Methods 1 and 2 for assisting in predicting the presence or absence of renal cell carcinoma or prostate cancer in the subject is preferably the concentration of cfDNA in plasma (pg/μl).

◆ PCR Primer Pair for Detecting Human Alu In the present PCR primer pair for detecting human Alu, the nucleotide sequence shown in SEQ ID NO: 1 of the forward primer (a) is the forward primer disclosed in Patent Document 1 mentioned above. On the other hand, in the present PCR primer pair for human Alu detection, the nucleotide sequence (Alu57F20) shown in SEQ ID NO: 2, which is the forward primer (a'), was designed for the first time according to the present invention. The nucleotide sequence of the forward primer (a') was designed as a nucleotide sequence that could not be detected with the default settings of primer design software Primer 3. The forward primer (a') may be a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 with 1 to 3 nucleotides deleted, substituted, or added.

On the other hand, the nucleotide sequence (Alu237R19) shown in SEQ ID NO: 3 of the reverse primer (b) was designed for the first time according to the present invention. The nucleotide sequence shown in SEQ ID NO: 3 was designed as a nucleotide sequence that cannot be detected with the default settings of primer design software Primer 3. Note that the reverse primer (b) may be a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 with 1 to 3 nucleotides deleted, substituted, or added.

Examples of the present PCR primer pair for human Alu detection include the combination of (a) and (b) above, and the combination of (a') and (b) above. By using the pair of human Alu detection PCR primers in combination (a) and (b) above, it becomes possible to amplify the 101st to 237th sequences in the human Alu model sequence shown in SEQ ID NO: 6. Furthermore, by using the pair of human Alu detection PCR primers in combination (a') and (b) above, it becomes possible to amplify sequences 57 to 237 of the human Alu model sequence.

Model sequence of human Alu (SEQ ID NO: 6)
GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGG Y GGATCAC Y TGAGG Y CAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTC ACTAAAAAAATACAAAAAAATTAGCCGGGCGTGGTGGCG S G Y GCCTGTA R TCCCA CGCGCCACTGCACTCCAGCCTGGG Y GACAGAG Y GAGACTC Y GTCTCAAAAAAAAAAAAAAA

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.

Combinations of (a) and (b) above include (1) a forward primer consisting of the nucleotide sequence (Alu101F20) shown in SEQ ID NO: 1, and a reverse primer consisting of the nucleotide sequence (Alu237R19) shown in SEQ ID NO: 3; (2) a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 with 1 to 3 nucleotides deleted, substituted, or added; and a reverse primer consisting of a 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 a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 3; (4) A forward primer consisting of a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 1, and a forward primer consisting of a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 3. Examples include reverse primers consisting of nucleotide sequences in which nucleotides have been deleted, substituted, or added. In this specification, a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added is preferably a nucleotide sequence in which 1 or 2 nucleotides are deleted, substituted, or added. Preferred examples include nucleotide sequences in which one nucleotide is deleted, substituted, or added.

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

◆ PCR Probe for Detecting Human Alu In the present PCR probe for detecting human Alu, the PCR probe for detecting human Alu consisting of (c) is designed for the first time according to the present invention. The probe consisting of the nucleotide sequence (Alu140RH16-LNA) shown in SEQ ID NO: 4 was designed from the viewpoint of detecting as many human targets as possible. Of the nucleotide sequence shown in SEQ ID NO: 4, 2nd guanine, 3rd guanine, 4th cytosine, 13th guanine, 14th thymine, and 15th adenine are locked nucleic acids (LNA). (C+G+G+CTAATTTT+G+T+AT; "+" indicates LNA). The present PCR probe for detecting human Alu can hybridize with the 125th to 140th sequences in the human Alu model sequence shown in SEQ ID NO: 6. In the synthesis of the present PCR probe for human Alu detection, the LNA monomer can be obtained, for example, by the synthesis method described in WO 1998/039352 pamphlet.

The above PCR probe for detecting human Alu contains all or a partial sequence of the Alu sequence that can hybridize under stringent conditions with the PCR probe for detecting human Alu, such as the amplification product of the above PCR primer pair for detecting human Alu. can be detected and/or quantified. The above-mentioned PCR probe for detecting human Alu is preferably labeled with a labeling substance, and from the viewpoint of faster or more sensitive detection or quantification, it is preferably labeled with a fluorescent substance. Examples of the labeling substances other than fluorescent substances include biotin, a complex of biotin and avidin, and enzymes such as peroxidase. Examples of the fluorescent substances include fluorescent proteins such as luciferase, as well as fluorescein isothiocyanate (FITC), 6 -Carboxyfluorescein (6-FAM), 6-carboxy-4,7,2',7'-tetrachlorofluorescein (TET), 6-carboxy-4',5'-dichloro2',7'-dimethoxyfluorescein ( Preferred examples include fluorescent dyes such as Cy3, Cy5, 4,7,2',4',5,',7'-hexachloro-6-carboxyfluorescein (HEX). Moreover, the above-mentioned 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). It is particularly preferred that the 3' end of each of the 3' ends is labeled with a quencher substance (quencher fluorescent dye). Examples of fluorescent substances (reporter fluorescent dyes) include the aforementioned fluorescent dyes, and examples of quencher substances (quencher fluorescent dyes) include 6-carboxytetramethylrhodamine (TAMRA) and 6-carboxy-X-rhodamine. Rhodamine fluorescent dyes such as (ROX), [(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline (BHQ-1), [(4-(1-nitro-phenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]-aniline (BHQ-2), etc. Among them, 6-carboxyfluorescein (6-FAM) is used as a fluorescent substance (reporter fluorescent dye), and Iowa Black fluorescence quencher (IBFQ), [(4- Preferred examples include (2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline (BHQ-1).

The above-mentioned stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Specifically, 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 with lower identity hybridize with each other. Examples include conditions in which hybridization is not performed, or normal real-time PCR annealing conditions, specifically conditions in which hybridization is performed at 50°C or higher, preferably 52°C or higher, more preferably 54°C or higher, and even more preferably 56°C or higher. .

◆ PCR primer/probe set for detecting human Alu The PCR primer/probe set for detecting human Alu consists of the PCR primer pair for detecting human Alu, the PCR probe for detecting human Alu, or the PCR primer pair for detecting human Alu. and (c') a nucleotide sequence shown in SEQ ID NO: 5 (Alu144RH20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 5; There is no particular restriction as long as it includes a PCR probe for detecting Alu. By using the nucleotide sequence (Alu101F20) shown in SEQ ID NO: 1 as a forward primer, it is possible to amplify and detect the 137 bp Alu sequence from position 101 to position 237 in the human Alu model sequence shown in SEQ ID NO: 6. By using the nucleotide sequence (Alu57F20) shown in SEQ ID NO: 2 as a forward primer, it is possible to amplify and detect the 181 bp Alu sequence from position 57 to position 237 in the human Alu model sequence shown in SEQ ID NO: 6. Note that human genomic DNA can be quantified by amplifying and detecting the Alu sequence. The human genomic DNA to be quantified includes cfDNA.

◆Method for detecting and/or quantifying human genomic DNA As method 1 for detecting and/or quantifying human genomic DNA, PCR is performed using DNA extracted from the test sample as a template and the subject PCR primer pair for human Alu detection. There is no particular restriction as long as the process is included.

The above-mentioned "test sample" is not particularly limited as long as it can contain human genomic DNA, and may also contain DNA derived from non-human organisms in addition to human genomic DNA. Examples of the above-mentioned "test samples" include human biological samples, biological samples derived from non-human organisms, old samples, forensic samples, paleontological samples, etc. Among them, human cells transplanted into non-human animals Preferable examples include biological samples derived from xenograft model animals prepared in the same way and old samples derived from ancient human bones. Furthermore, examples of the above-mentioned "non-human animals" include non-human mammals such as mice, rats, guinea pigs, dogs, rabbits, pigs, goats, and cows; rodents such as mice, rats, and guinea pigs; More preferred examples include animals. Furthermore, the above-mentioned "human cells" may be any type of cells of human origin, such as human stem cells, human cancer cells, human somatic cells, human germ cells, etc. Particularly preferred examples include human stem cells such as stem cells, human hematopoietic stem cells, human neural stem cells, human iPS cells, human ES cells, and human somatic cell-derived embryonic stem cells. Note that in this specification, "old sample" refers to "unfresh sample" that has been used for several days to several million years under various environments, and the lower limit of the elapsed time is, for example, 2 days, 1 week, Examples include 2 weeks, 1 month, 3 months, 1 year, 3 years, etc., and the upper limit of elapsed time is 5 years, 10 years, 20 years, 40 years, 100 years, 200 years, 400 years, 600 years. , 5,000 years, 10,000 years, etc. In particular, "an old sample derived from ancient human bones" refers to a sample derived from human bones several years ago from the time when human DNA was detected and/or quantified; and/or several years to 2 million years ago, 500 to 10,000 years ago, 100 to 10,000 years ago, 200 to 10,000 years ago, 300 to 10,000 years ago, 400 to 10,000 years ago, 50-5000 years ago, 100-5000 years ago, 200-5000 years ago, 300-5000 years ago, 400-5000 years ago, 50-600 years ago, 100-600 years ago, 200-600 years ago, 300- Examples include samples derived from human bones from 600 years ago and 400 to 600 years ago.

In addition, methods for extracting human genomic DNA from the above test samples include "proteinase K/phenol extraction method," "proteinase K/phenol/chloroform extraction method," "alkali lysis method," "boiling method," and "magnetic bead method." ”, “spin column method”, and other known methods are not particularly limited. The above method preferably includes an RNA decomposition step using RNase, and more preferably a method including an RNA decomposition step using RNase A and RNase T. Specifically, as a method for extracting DNA from the above test sample, there is a method in which RNA degradation treatment is performed using RNase A and RNase T1 before the proteinase K treatment step in the "proteinase K/phenol extraction method". be able to.

The PCR in the above-mentioned "Method 1 for detecting and/or quantifying human genomic DNA" is not particularly limited as long as it is a PCR using the above-mentioned PCR primer pair for detecting human Alu, and the PCR amplification product is detected at the end of the cycle. It may be an end-point PCR method in which the amount of PCR amplified products is monitored in real time, or a real-time PCR method in which the increase in PCR amplification products is monitored in real time. As the endpoint PCR method, a digital PCR method can be mentioned. To detect amplified products in the endpoint PCR method, the reaction solution after PCR is directly subjected to conventional gel electrophoresis using agarose, etc., and the DNA fragments after electrophoresis are stained with ethidium bromide, fluorescent reagents, etc. An example of how to detect this can be given below. In addition, as a method for detecting amplified products in real-time PCR, a non-specific DNA intercalating dye is added to the PCR reaction solution in advance, and the double-stranded DNA of the amplified product is detected over time. The law can be exemplified. In the above intercalation method, 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) Non-specific DNA intercalating dyes such as , SYTOX Orange, and EvaGreen (registered trademark) can be used, and among them, it is preferable to use SYBR (registered trademark) Green I.

In addition, Method 2 for detecting and/or quantifying human genomic DNA uses the PCR primer/probe set for detecting human Alu in place of the PCR primer pair for detecting human Alu, and specifically, the method described above ( A PCR probe for detecting human Alu consisting of c) or (c') is used. In method 2 for detecting and/or quantifying human genomic DNA, it is preferable to use a fluorescently labeled PCR probe for human Alu detection consisting of (c) or (c') above. The type of PCR probe for human Alu detection consisting of (c) or (c') used here is not particularly limited, and examples thereof include hydrolysis probes, molecular beacon probes, cycling probes, Eprobe (registered trademark), and Qprobe. (registered trademark), Scorpion probe, hybridization-probe, etc. Among them, hydrolysis probe can be preferably mentioned. A hydrolysis probe is usually a linear oligonucleotide in which the 5' end of the 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 usually an oligonucleotide that can take 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. . Cycling probes are usually chimeric oligonucleotides consisting of RNA and DNA, with one end modified with a fluorescent substance (reporter fluorescent dye) and the other end modified with a quencher. . Eprobe is an artificial nucleic acid that usually has two fluorescent dyes in a thymine nucleotide, and when it is in a single-stranded state that is not bound to a target, fluorescence is suppressed, and when it is bound to a target, it emits fluorescence. Qprobe is usually an oligonucleotide with cytosine at the end, and the cytosine at the end is labeled with a fluorescent substance, and is also called a guanine quenching probe. A scorpion probe is an oligonucleotide that can take a hairpin loop structure, with one end of the nucleic acid probe being modified with a fluorescent substance (reporter fluorescent dye) and the 3' end being modified with a quencher. A hybridization-probe consists of a donor probe consisting of an oligonucleotide whose 3' end is modified with a fluorescent dye, and an acceptor probe consisting of an oligonucleotide whose 5' end is modified with a fluorescent dye. When bound to, both fluorescent dyes come into close proximity, and the fluorescent dye of the acceptor probe is excited by the fluorescence of the fluorescent dye of the donor probe, causing it to emit light.

The above PCR can be performed according to the method described in literature such as Molecular cloning, A laboratory manual, etc. It can also be carried out using commercially available PCR kits such as Kit (manufactured by TaKaRa) and TaqMan (registered trademark) Real-Time PCR Master Mixes (manufactured by Thermo Fisher Scientific) according to the instructions attached to the product. In addition, in the above PCR, in addition to the nucleic acid obtained from the test sample and the PCR primer pair for human Alu detection or the PCR primer/probe set for human Alu detection, various reagents necessary for the PCR reaction may be used. can. Such reagents include enzymes that polymerize nucleic acids (e.g., polymerases), substances that serve as materials for nucleic acids (e.g., dNTPs), buffers for nucleic acid amplification reactions (e.g., components with a buffering effect such as Tris-Cl, and Tween 20, etc.). In addition to one or more selected from the group consisting of Mg ²⁺ (surfactants, etc.) and Mg 2+ , other necessary reagents can be used additionally depending on the type of PCR.

When performing the above PCR, a commercially available nucleic acid amplification device can be used depending on the type of nucleic acid amplification reaction used, and in particular, a device capable of measuring probe signals (preferably fluorescent signals) can be used. A preferred example is a nucleic acid amplification device. Examples of real-time PCR devices capable of both amplifying nucleic acids and measuring fluorescent signals include Applied Biosystems' 7500 real-time PCR instrument, Bio-RAD's CFX96, and Roche's Lightcycler 480. Can be done.

In the above 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 if step (II) is followed by step (II). Although the order of (I) may be used, or step (I) and step (II) may be carried out simultaneously, it is preferable that step (I) and step (II) are carried out simultaneously. The above step (III) is carried out after carrying out steps (I) and (II).

In this specification, the "standard sample" is not particularly limited as long as it is a standard sample prepared by serially diluting a known amount of human genomic DNA, but the amount of human genomic DNA added to each tube for PCR reaction is For example, a standard sample prepared to have a concentration in the range of 1 fg to 10 ng, preferably 0.1 fg to 10 ng, more preferably 0.01 fg to 10 ng can be mentioned. Moreover, the above-mentioned "standard sample" may further contain, in addition to human genomic DNA, genomic DNA derived from a non-human organism that may be included in the test sample. Furthermore, the human genomic DNA contained in the above-mentioned "standard sample" may be fragmented depending on the type of the above-mentioned "test sample", for example, it may be fragmented into an appropriate size in the range of 20 kbp to 100 bp. human genomic DNA can be used.

In the above step (II), the method of "creating a standard curve" is, for example, by plotting the intensity level of the fluorescence signal obtained by real-time PCR using the above standard sample as a template against the amount of human genomic DNA. and a method in which the Ct value of the fluorescence signal obtained by real-time PCR using the above-mentioned standard sample as a template is plotted against the amount of human genomic DNA. Here, "Ct value" means the cycle number at which the amplification curve and the threshold intersect in real-time PCR, and when the amount of fluorescence derived from the generation of PCR amplification products reaches a certain amount (threshold). represents the number of cycles. 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 the above 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 above-mentioned standard curve.

◆Kit for detecting and/or quantifying human genomic DNA A kit for detecting and/or quantifying human genomic DNA may be provided with the above-mentioned PCR primer pair for detecting human Alu and/or PCR probe for detecting human Alu. In addition to the components generally used in this type of detection kit (eg, carrier, pH buffer, stabilizer, etc.), this kit may also contain attached documents such as instruction manuals. Furthermore, the kit of the present invention further includes human genomic DNA for use as a standard sample, and non-human organism-derived DNA for use as a negative control (e.g., mouse genomic DNA, rat genomic DNA, Guinea pig genomic DNA, etc.). You can leave it there.

◆Method to assist in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject In this method to assist in predicting the presence or absence of renal cell carcinoma or prostate cancer in a subject, biological samples include blood, serum, and plasma. , saliva, urine, etc. Furthermore, 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; for example, blood is collected from a subject, centrifuged at 3,000 rpm for 10 minutes, and the supernatant further centrifuged at 12,000 rpm for 15 minutes. can be mentioned.

The above threshold value can be determined in advance based on the amount of genomic DNA extracted from biological samples of healthy subjects and renal cell carcinoma patients or prostate cancer patients. The amount of genomic DNA is preferably 166 bp or less, more preferably 63 bp, 106 bp, or 137 bp, depending on the concentration of cfDNA in plasma (pg/μl) determined with a primer/probe set that amplifies or detects a 137 bp human Alu sequence. or a combination of these. In calculating the above threshold value, the concentration of cfDNA in the plasma is determined using a value converted by a predetermined transformation, such as square root transformation, exponential transformation, logarithmic transformation, angular transformation, probit transformation, reciprocal transformation, or power transformation. Alternatively, these converted values may be used in combination for the concentration of cfDNA.

The above threshold value can be calculated using a median value, an average value, an AUC (Area under curve) value based on ROC (Receiver Operating Characteristic) curve analysis, a value using the distance from the upper left corner, and a Youden index. The above threshold value is calculated by including the concentration of cfDNA in plasma (pg/μl) as well as indicators such as age, body mass index (BMI), weight, gender, medical history, drug prescription history, treatment history, etc. Good too. Furthermore, the threshold value may be determined by adding, subtracting, multiplying or dividing an arbitrary value to the cfDNA concentration in plasma determined with a primer/probe set that amplifies and detects a 63 bp, 106 bp, or 137 bp human Alu sequence. For example, the value divided by the plasma cfDNA concentration determined with a primer/probe set that amplifies and detects human Alu with a predetermined length of 167 bp or more, such as 167 to 332 bp, preferably 181 bp, or 63 bp, 106 bp, or 137 bp. The concentration of cfDNA in plasma determined with a primer/probe set for amplifying and detecting human Alu of a predetermined length, for example, 166 to 332 bp, preferably 181 bp, is determined using a primer/probe set for amplifying and detecting human Alu of a predetermined length. The threshold value may be calculated using the AUC value based on the value subtracted by the cfDNA concentration in the plasma, the value using the distance from the upper left corner, and the Youden index. The AUC value serving as the threshold value is, 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 higher.

The primer/probe set for amplifying and detecting the 181 bp human Alu includes the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20), or deletions or substitutions of 1 to 3 nucleotides in the nucleotide sequence shown in SEQ ID NO: 2, or a forward primer consisting of an added nucleotide sequence, and a nucleotide sequence shown in SEQ ID NO: 3 (Alu237R19) or a nucleotide in which 1 to 3 nucleotides have been deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 3. A PCR primer/probe set for detecting human Alu, comprising a primer pair composed of a reverse primer consisting of a reverse primer sequence, and the present PCR probe for detecting human Alu or the PCR probe for detecting human Alu described in (c') above. can be mentioned.

The stage of renal cell carcinoma or prostate cancer in the test subject may be stage 0, stage I, stage II, stage III, or stage IV according to the TNM classification of the International Union Against Cancer (UICC), but stage III is preferable. , or stage IV.

If the amount of human genomic DNA in the biological sample extracted from the subject is greater than or equal to a predetermined threshold, it is possible to assist in predicting that the subject has renal cell cancer 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 be assisted in predicting that the subject does not have renal cell cancer or prostate cancer.

Furthermore, genomic DNA extracted from biological samples of renal cell carcinoma patients or prostate cancer patients with different degrees of malignancy, especially 63bp, 106bp, and 137bp human Alu sequences in plasma determined with primer-probe sets that amplify or detect human Alu sequences. Setting a threshold based on the concentration of cfDNA (pg/μl) can also assist in predicting the malignancy of renal cell carcinoma or prostate cancer in a subject.

Hereinafter, the present invention will be explained in more detail 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 human Alu sequences of 63 bp, 106 bp, 137 bp, and 181 bp are referred to as "63 bp Alu detection primer/probe set" and "106 bp Alu detection primer/probe set," respectively.・Probe set", "137 bp Alu detection primer/probe set", "181 bp Alu detection primer/probe set".

[Conventional primer/probe set]
First, the present inventors have disclosed the human Alu model sequence (SEQ ID NO: 6) in the above-mentioned Patent Document 1. Furthermore, in Patent Document 1, the present inventors set criteria 1 to 15 for selecting primers and probes for Alu-qPCR, and the following two sets of primers and probes were selected based on the criteria. is disclosed.

<63bp Alu detection primer/probe set>
Forward primer (Alu212F18):
GGCGGAGGTTGCAGTGAG (SEQ ID NO: 7)
Reverse primer (Alu274R18):
GTCTCGCTCTGTCGCCCA (SEQ ID NO: 8)
Probe (antisense) (Alu248RH18):
TGCAGTGGCGCGATCTCG (SEQ ID NO: 9)

<106bp Alu detection primer/probe set>
Forward primer (Alu101F20):
GGTGAAAACCCCGTCTCTACT (SEQ ID NO: 1)
Reverse primer (Alu206R20):
GGTTCAAGCGATTCTCCTGC (SEQ ID NO: 10)
Probe (antisense) (Alu144RH20)
CGCCCGGCTAATTTTGTAT (SEQ ID NO: 5)

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

[Example 1] Design of new primers In the present invention, in order to detect human Alu sequences of different sizes, we have developed a “137bp Alu detection primer/probe set” and a “181bp Alu detection primer/probe set”. Two new primer-probe sets were designed. Note that the reverse primers for 137 bp and 181 bp detection have a common sequence, and the forward primers have different sequences (FIG. 2). Further, the probe sequence (Alu144RH20) is a common sequence for 106 bp, 137 bp, and 181 bp detection.

<137bp Alu detection primer/probe set>
Forward primer (Alu101F20):
GGTGAAAACCCCGTCTCTACT (SEQ ID NO: 1)
Reverse primer (Alu237R19):
GATCTCGGCTCACTGCAAC (SEQ ID NO: 3)
Probe (antisense) (Alu144RH20):
CGCCCGGCTAATTTTGTAT (SEQ ID NO: 5)

<181bp Alu detection primer/probe set>
Forward primer (Alu57F20):
CGGATCACTTGAGGTCAGGA (SEQ ID NO: 2)
Reverse primer (Alu237R19):
GATCTCGGCTCACTGCAAC (SEQ ID NO: 3)
Probe (antisense) (Alu144RH20):
CGCCCGGCTAATTTTGTAT (SEQ ID NO: 5)

[Example 2] Improvement of probe (Alu144RH20) The inventors used next-generation sequencing to analyze the amplified sequences using the forward primer (Alu101F20) and reverse primer (Alu206R20) designed so far. Table 1 shows the number of sequences that match Alu101F20, Alu206R20, and the probe (Alu144RH20) among the total number of 574,485 reads (168,980 types). The analysis results revealed that there were few sequences that matched the probe (Alu144RH20), and only 1.6% (7335/457560) of the sequences amplified by both primer sequences had completely matching sequences. This number was smaller than expected based on Blast analysis of the database.

Therefore, the inventors thought that if the number of perfectly matched sequences was increased by shortening the base length of the probe using LNA, good separation could be expected, especially at low concentrations. LNA is an artificial nucleic acid cross-linked with 2'-O,4'-C methylene, and when LNA is incorporated into an oligonucleotide, the thermal stability of the duplex is increased and the specificity of hybridization to the target sequence of the oligonucleotide is increased. Improves sex. Particularly in the case of qPCR, background fluorescence due to binding to sequences other than the target is reduced, increasing the signal/noise ratio (S/N ratio). It has also been shown that improved hybridization of qPCR probes to targets can increase the melting temperature (Tm) by up to 8°C per LNA monomer displacement in the intermediate salt state compared to DNA oligonucleotides. There is.

Table 2 shows the target types and number of targets in next-generation sequence data when the base length is shortened.

The probe (Alu144RH20; SEQ ID NO: 5) described by the present inventor in the above-mentioned Patent Document 1 is a 20 base (complementary strand) TaqMan type probe starting from the 125th position of the Alu model sequence shown in SEQ ID NO: 6. By increasing the number of bases to 16 bases, the number of targets increases approximately 3 to 4 times, so Alu140RH16-LNA was produced (SEQ ID NO: 4). Hereinafter, such a probe may be referred to as "Alu140RH16-LNA." Further, among the probes 140RH16-LNA, the 2nd to 4th "+G+G+C" and the 13th to 15th "+G+T+A" are LNAs.

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

As shown in Table 2, when the probe length is set to 14 bases, an approximately 20-fold increase in the number of targets can be expected. However, with 14 bases, the Tm value did not increase sufficiently even when six LNAs were introduced, so Alu138RH14 was not designed. Further, the reason why the probe starting with 125 was selected was that it would not intersect with the mouse.

When qPCR was performed using probe Alu144RH20 and probe Alu140RH16-LNA at an annealing temperature of 56°C, probe Alu140RH16-LNA showed a significantly faster rise in cycle number, and the effect of increasing the number of targets was observed. In addition, the saturation point of the trend is high, and it appears that detection can be performed at a faster number of cycles before the inhibitory substance (amplified nucleic acid) accumulates. In addition, the specificity is higher, the Tm value is increased by LNA, so the probe binds stably, and the shorter bases allow for faster binding, resulting in a smoother amplification curve and a lower value. It is thought that reliability has improved.

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

[Example 3] Confirmation of the sensitivity and specificity of the primer/probe set of the present invention (1) Real-time PCR conditions The 137 bp Alu detection primer/probe set designed in Example 1 above and the 181 bp Alu detection primer/probe set designed in Example 1 above. A probe set and the probe designed in Example 2 (Alu140RH16-LNA) were prepared. Furthermore, the Alu144RH20 probe was labeled at the 5' end with FAM and at the 3' end with BHQ1. The Alu140RH16-LNA probe was labeled at the 5' end with FAM and at the 3' end with IBFQ as a quenching label. Furthermore, as a reference (control), a 63 bp Alu detection primer/probe set and a 106 bp Alu detection primer/probe set described in the "Conventional Primer/Probe Set" column were similarly prepared. That is, four types of primer/probe sets (63 bp, 106 bp, 137 bp, and 181 bp Alu detection primer/probe sets) were used in this experiment. As the probe, Alu144RH20 was used in the data shown in FIG. 3 described later, and Alu140RH16-LNA was used in the data shown in FIG. 4.

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

PCR samples were prepared as follows using each primer/probe set and the above template. The template and sample were prepared using low-adsorption tubes and chips so as not to lose trace amounts of DNA.
Template 1μL
TaqMan Universal Master MixII, no UNG (Thermo Fisher Scientific)
10μL
Forward primer (10μM) 0.4μL
Reverse primer (10μM) 0.4μL
Probe (10μM) 0.5μL
Water 7.7μL
(Total) 20μL

Real-time PCR was performed on the PCR sample prepared as described above using LightCycler 480 (Roche). The PCR reaction was performed after heat denaturation at 95°C for 10 minutes, followed by 5 cycles of 30 seconds at 95°C, 4 minutes 30 seconds at 56°C, and 30 seconds at 72°C, followed by 30 seconds at 95°C. A cycle of 30 seconds at 56°C and 30 seconds at 72°C was repeated 45 times. The analysis was performed using the Fit point method using LightCycler (registered trademark) 480 Software release 1.5.0. FIG. 3 shows the results of a calibration curve created based on real-time PCR.

As shown in FIG. 3, extremely linear calibration curves with a correlation coefficient of 0.99 or higher were obtained from 1 ng to 10 fg for all of the 63 bp, 106 bp, 137 bp, and 181 bp Alu detection primer/probe sets. These results revealed that qPCR using the above primer-probe set can detect up to 10 fg of fragmented human genomic DNA. Using Alu-qPCR using these primer-probe sets, we were able to establish a method to detect human genomic DNA of four different sizes (63 bp, 106 bp, 137 bp, and 181 bp) or more with ultrahigh sensitivity. .

Furthermore, regarding the quantification of 106 bp, 137 bp, and 181 bp cfDNA, similar experiments were conducted using Alu140RH16-LNA instead of the probe Alu144RH20 used for the data in FIG. 3 above. The results are shown in Figure 4.

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

[Example 4] Measurement of plasma cfDNA concentration in healthy subjects and cancer patients Using the Alu detection primer/probe sets of 63 bp, 106 bp, 137 bp, and 181 bp used to create the calibration curve in Example 3, Alu detection for each size was performed. - cfDNA concentrations in plasma from healthy subjects and renal cell carcinoma patients were quantified by qPCR. As probes, Alu248R18 was used in the case of 63 bp, Alu144RH20 was used in the case of 106 bp, and Alu140RH16-LNA was used in the cases of 137 bp and 181 bp. Note that a primer pair that amplifies 63 bp will amplify a fragment of 63 bp or more. Similarly, a primer pair that amplifies 106 bp will amplify a fragment of 106 bp or more, a primer pair that amplifies 137 bp will amplify a fragment of 137 bp or more, and a primer pair that amplifies 181 bp will amplify a fragment of 181 bp or more. will be amplified. In other words, a primer pair that amplifies 63 bp cannot amplify a fragment of 62 bp or less. Similarly, a primer pair that amplifies 106 bp cannot amplify a fragment of 105 bp or less, a primer pair that amplifies 137 bp cannot amplify a fragment of 136 bp or less, and a primer pair that amplifies 181 bp cannot amplify a fragment of 180 bp or less. This means that it cannot be amplified.

Samples were 26 healthy individuals (all separate individuals), 20 renal cell carcinoma patients (15 individuals, 5 had their blood drawn for the second time at different times) for measurements of 63bp and 131bp, and kidney samples for measurements of 106bp and 181bp. Blood was collected from a total of 34 cell carcinoma patients (23 individuals, 7 had their blood drawn for the second time on different dates and times, and 2 had their blood drawn for the third time on different dates and times), centrifuged at 3,000 rpm for 10 minutes, and the supernatant was further collected. Plasma was obtained by centrifugation at 12,000 rpm for 15 minutes. Next, cfDNA was extracted (50 μl) from the obtained 200 μL of plasma using magLEAD12gC (manufactured by Precision System Sciences). The extracted cfDNA was stored at -80°C until measurement. Alu-qPCR (LightCycler 480: manufactured by Roche Diagnostics) was performed using 1 μl of the mixture.

Figure 5 shows the results of a box plot of the cfDNA concentration in plasma. Further, FIG. 6 shows the results of dividing the measured values of 63 bp, 106 bp, and 137 bp (plasma cfDNA concentration: pg/μl) in FIG. 5 by the measured value of 181 bp.

The results in Figures 5 and 6 show that for small size cfDNA (63bp, 106bp, 137bp), the plasma cfDNA concentration is higher in renal cell carcinoma patients than in healthy subjects, and for 181bp, the plasma cfDNA concentration is higher in renal cell carcinoma patients than in healthy subjects. It became clear that there was no difference. Furthermore, it was revealed that by dividing the plasma cfDNA concentrations of 63 bp, 106 bp, and 137 bp by the 181 bp plasma cfDNA concentration, there is a greater difference between healthy subjects and renal cell carcinoma patients.

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

The results of ROC analysis of plasma cfDNA concentration determined using primer/probe sets that amplify and detect 63bp, 106bp, and 137bp show that the AUC (Area under curve) values are all 0.7 or higher, especially for 106bp. It was a high value of 0.85 or more. In addition, the ROC analysis results for 63bp/181bp, 106bp/181bp, and 137bp/181bp all have AUC values of 0.8 or higher, and 63bp/181bp and 106bp/181bp have particularly high values of 0.9 or higher. It became clear that there was a greater difference between healthy subjects and renal cell carcinoma patients when dividing by the 181 bp cfDNA concentration than when each was used alone. Furthermore, the ROC analysis results for 63bp-181bp, 106bp-181bp, and 137bp-181bp all have AUC values of 0.78 or higher, and especially high values of 0.848 for 63bp-181bp and 0.921 for 106bp-181bp. Therefore, it became clear that it is better to reduce the cfDNA concentration to 181 bp than each of them alone.

[Example 6] Threshold value setting for cancer determination Based on the results of Example 5, if the threshold value is set to a value that allows 100% identification of patients, (i) From Figure 7, plasma determined with a primer/probe set that amplifies and detects 106 bp. The threshold value for the cfDNA concentration in the medium can be set to 5.3 pg/μl, and (ii) the threshold value for 106 bp/181 bp can be set to 2.71 according to FIG. When (i) and (ii) are used in combination, it has been shown that very reliable screening is possible with a sensitivity of 1.0 (34/34) and a specificity of 0.82 (28/34). (Table 3). There has never been a method that can distinguish between renal cell carcinoma patients and healthy individuals with such high probability, and the size-specific Alu-qPCR described above has been shown to be extremely useful as a minimally invasive method for diagnosing renal cell carcinoma. .

Furthermore, the threshold, sensitivity, specificity, and sensitivity + specificity when the sensitivity is 100%, >95%, >90%, >85%, >80% for 106bp, 106bp/181bp, and 106bp-181bp, respectively. It is shown in FIG. From FIG. 10, it was confirmed that sensitivity + specificity increases by setting the sensitivity to >95%. In addition, FIG. 11 shows the sensitivity, specificity, and sensitivity+specificity when both the 106 bp and 106 bp/181 bp thresholds are satisfied simultaneously. When the thresholds of 106bp and 106bp/181bp are met simultaneously, an increase in specificity is observed, and when the threshold of 106bp is 6.6 pg/μl and the threshold of 106bp/181bp is 2.73 (see Figure 10, >95%), the sensitivity increases. The specificity was 0.971 and the specificity was 0.923.

[Example 7] Application to prostate cancer The 106bp, 106bp/181bp, and 106bp-181bp of healthy individuals calculated in Example 5 were applied to prostate cancer patients. Plasma was obtained from three prostate cancer patients by 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 a primer/probe set for amplifying and detecting 106 bp and 181 bp, respectively. The results are shown in Table 4. Then, threshold values for 106 bp, 106 bp/181 bp, and 106 bp-181 bp were determined by the ROC analysis described in Example 5 based on the plasma cfDNA concentrations of 106 bp and 181 bp of the above-mentioned healthy subjects and the above-mentioned prostate cancer patients.

When the threshold for 106 bp was 6.89 and the threshold for 106 bp/181 bp was 2.71, sensitivity of 1 and specificity of 0.92 were obtained for 106 bp and 106 bp/181 bp. Furthermore, when the threshold value of 106 bp to 181 bp was set to 5.08, a sensitivity of 1 and a specificity of 0.81 were obtained. Therefore, the size-specific Alu-qPCR described above was shown to be very useful as a minimally invasive diagnosis of prostate cancer.

[Example 8] Application to monitoring after treatment in prostate cancer The concentration of cfDNA in plasma was investigated in monitoring after hormone therapy in prostate cancer patients. Leuplin PRO (Takeda Pharmaceutical Co., Ltd.) or Zytiga (Janssen Pharma Co., Ltd.) was used as hormone therapy for two prostate cancer patients, and plasma was obtained before administration and 6 months after administration. Next, 50 μl of cfDNA was extracted, and the cfDNA concentration (pg/μl) in plasma was examined by Alu-qPCR using primer and probe sets for amplifying and detecting 106 bp and 181 bp. Then, 106bp/181bp and 106bp-181bp were determined based on the plasma cfDNA concentrations of 106bp and 181bp. In addition, both of the two prostate cancer patients six months after administration were patients in whom the cancer treatment effect was confirmed based on the PSA value of the prostate cancer marker.

As shown in Table 5, all of 106bp, 106bp/181bp, and 106bp-181bp decreased six months after hormone therapy. Therefore, by calculating the cfDNA concentration in plasma determined using a primer/probe set that amplifies and detects 106bp and 181bp, and by calculating 106bp/181bp and 106bp-181bp based on these concentrations, it can be applied to monitoring after cancer treatment. .

Claims

A PCR primer pair for human Alu detection consisting of the following forward primer (a) and (b) reverse primer, or the following (a') forward primer and (b) reverse primer. .
(a) A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Alu101F20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 1;
(a') A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 (Alu57F20); or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to 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 a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 3;
A PCR probe for human Alu detection consisting 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 are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 4;
A PCR primer pair for detecting human Alu according to claim 1 and a PCR probe for detecting human Alu consisting of (c) according to claim 2; or a pair of PCR primers for detecting human Alu according to claim 1, ( c') A nucleotide sequence shown in SEQ ID NO: 5 (Alu144RH20), or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 5; for human Alu detection. PCR probe;
A PCR primer/probe set for human Alu detection, comprising:
A method for detecting and/or quantifying human genomic DNA in a test sample, comprising the step of performing PCR using the PCR primer pair for human Alu detection according to claim 1, using DNA extracted from the test sample as a template.
Detecting and/or quantifying human genomic DNA in the test sample, including the step of performing PCR using the PCR primer/probe set for human Alu detection according to claim 3, using the DNA extracted from the test sample as a template. Method.
A method for detecting and/or quantifying human genomic DNA in a test sample using the PCR primer/probe set for human Alu detection according to claim 3, comprising the following steps (I) to (III): Method.
(I) Performing real-time PCR using the primer/probe set using the DNA extracted from the test sample as a template;
(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 standard curve;
(III) calculating the amount of human genomic DNA in the test sample from the calibration curve;
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 subject by the method for detecting and/or quantifying human genomic DNA according to any one of claims 4 to 6;
(B) assisting in predicting that the subject has renal cell cancer or prostate cancer when the amount of human genomic DNA quantified in step (A) is greater than or equal to a predetermined threshold;
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 subject by a method including the following steps (I') to (III');
(I') Performing real-time PCR using the DNA extracted from the test sample as a template and a primer/probe set that amplifies or detects a 63bp, 106bp, and/or 137bp human Alu sequence;
(II') Performing real-time PCR under the same conditions as the above step (I') using 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') A step of assisting in predicting that the subject has renal cell carcinoma or prostate cancer when the amount of human genomic DNA quantified in step (A') is greater than or equal to a predetermined threshold;
The method for assisting in predicting the presence or absence of renal cell cancer or prostate cancer in a subject according to claim 7 or 8, wherein the threshold value is a value determined by an AUC value calculated from ROC curve analysis. .