Prostate cancer early detection kit from urine

Abstract

This invention relates to a urine-based early prostate cancer detection kit. The kit detects methylated fragments of DNA sequences located in the CpG island region of the human GSTP1 gene promoter region, specifically located at (hg38)chr11:67583458-67584482 strand=+. This invention develops a urine-based early prostate cancer detection kit that significantly improves detection sensitivity and diagnostic accuracy through the combined detection of specific fragment methylation of CpG islands in the GSTP1 gene promoter region. This eliminates the need for plasma samples and biopsies, reducing unnecessary invasive examinations and enabling early, non-invasive diagnosis of prostate cancer.

Description

Technical Field

[0001] The invention relates to the field of cancer detection, and in particular to a detection kit for early diagnosis of prostate cancer from urine. Background Art

[0002] Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate gland. It is one of the most common malignant tumors in men and seriously endangers the patient's health and quality of life. Prostate cancer has a long incubation period and progresses slowly. However, since there are no obvious symptoms in the early stages, it is easy for patients to ignore it. According to statistics, about 70% of prostate cancer patients are already in the late stage when diagnosed, which is largely attributed to the difficulty of early detection. One of the important reasons for the high mortality rate of prostate cancer patients is the low detection accuracy of early prostate cancer patients. See Wu Hanchao, Zhang Xiongwei, Wang Qiang. Research progress in prostate cancer diagnosis methods. Chinese and Foreign Medical Research. 2021, 19(07):188-190.

[0003] Prostate-specific antigen (PSA) is currently commonly used to screen for prostate cancer from plasma and is the gold standard for clinical prostate cancer screening. Although PSA testing has high sensitivity, it has low specificity. Non-malignant diseases such as prostatitis and benign prostatic hyperplasia can also lead to elevated PSA levels, resulting in false positive results. See William J. Catalona, ​​Paula C. Southwick, Kevin M. Slawin, et al., Comparison of percent free PSA, PSA density, and age-specific PSA cutoffs for prostate cancer detection and staging. Urology. 2000, 56(2):255-260. Therefore, when screening shows elevated PSA, a prostate puncture biopsy is often required to confirm whether it is prostate cancer. Puncture biopsy is an invasive detection method that causes great harm to the patient's body, and puncture biopsy has limitations such as false negatives and overdiagnosis. Studies have shown that the false negative rate of the initial puncture biopsy can be as high as 30%, which means that many patients may need to undergo multiple punctures to be diagnosed, see Chen Rui, Xie Liping, Zhou Liqun, et al. Survey report on the current status of prostate puncture biopsy in member hospitals of the Chinese Prostate Cancer Alliance, Chinese Journal of Urology. 2015, 36: 342- 345. In view of this, it is necessary to propose a detection method that can reduce the risk of physical damage to patients and has higher sensitivity and specificity to achieve the accuracy of early screening for prostate cancer.

[0004] Molecular markers, especially gene methylation markers, have shown great potential in the early diagnosis of prostate cancer in recent years due to their high specificity and stability. For example, GSTP1 gene, HOX family genes, RASSF family genes, etc. have been found to be useful for the early diagnosis of prostate cancer, see Louise Katrine Larsen, Guro Elisabeth Lind, Per Guldberg, et al., DNA-Methylation-Based Detection of Urological Cancer in Urine: Overview of Biomarkers and Considerations on Biomarker Design, Source of DNA, and Detection Technologies. International Journal of Molecular Sciences. 2019, 20(11): 2657, and Ravindran Ragavi, Panneerselvam Muthukumaran, Srividhya Nandagopal, et al., Epigenetics regulation of prostate cancer: Biomarker and therapeutic potential. Urologic Oncology: Seminars and Original Investigations. 2023, 41(8): 340-353. Prostate cells do not easily enter the urethra, and methylated genes can be better detected in urine after 20 seconds of rectal massage. Compared with plasma samples and puncture biopsies, urine has the advantages of being non-invasive and reproducible. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a detection kit for early diagnosis of prostate cancer from urine, the kit is used to detect methylated fragments of CpG island DNA sequence segments located in the promoter region of human GSTP1 gene, the specific genomic location of the methylated fragments is (hg38) chr11: 67583458 -67584482 strand=+, and the methylated fragments include at least one of the following four groups: the first group, chr11:67583460-67583479 strand=+, chr11:67583533-67583551 strand=+, chr11:67583503-67583523 strand=+; the second group, chr11:67583619-67583636 strand=+, chr11:67583681-67583697 strand=+, strand=+, chr11:67583660-67583677 strand=+, the third group, chr11:67583737-67583759 strand=+, chr11:67583794-67583810 strand=+, chr11:67583763-67583781 strand=+; and, the fourth group, cchr11:67584231-67584249 strand=+, chr11:67584315-67584338 strand=+, chr11:67584266-67584288 strand=+.

[0006] In one embodiment, the kit is at least one of a bisulfite conversion fluorescence quantitative PCR kit, a bisulfite conversion sequencing kit, and a methylation chip sequencing kit.

[0007] In one embodiment, the kit is a kit for detecting the following three fragments: the first group, chr11:67583460-67583479 strand=+, chr11:67583533-67583551 strand=+, chr11:67583503-67583523 strand=+; the second group, chr11:67583619-67583636 strand=+, chr11:67583681-67583697 strand=+, chr11:67583660-67583677 strand=+, and the fourth group, chr11:67584231-67584249 strand=+, chr11:67584315-6758338 strand=+,chr11:67584266-67584288 strand=+.

[0008] In one embodiment, the kit is a fluorescent quantitative PCR kit, which includes the following primers and probes:

[0009] .

[0010] The present invention develops a detection kit for early diagnosis of prostate cancer from urine, which is based on the combined detection of methylation of specific fragments of CpG island in the promoter region of GSTP1 gene, especially the first group, chr11:67583460-67583479 strand=+, chr11:67583533-67583551 strand=+, chr11:67583503-67583523 strand=+; the second group, chr11:67583619-67583636 strand=+, chr11:67583681-67583697 strand=+, chr11:67583660-67583677 strand=+, and, the fourth group, cchr11:67584231-67584249 strand=+ The combined detection of three groups of strand=+, chr11:67584315-67584338 strand=+, chr11:67584266-67584288 strand=+ has the best comprehensive performance in sensitivity and specificity, with a sensitivity of 91.67% and a specificity of 92.54%. This greatly improves the sensitivity and diagnostic accuracy of the detection, thereby eliminating the need for plasma samples and puncture activities, reducing unnecessary invasive examinations, and achieving early non-invasive diagnosis of prostate cancer. In addition, since the combined detection of the present invention has high sensitivity and high specificity results, the present invention can use urine detection, which has the advantages of being non-invasive and repeatable, thereby improving the convenience of diagnosis and reducing the cost of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0012] Figure 1 This is a schematic diagram of the CpG island DNA sequence segment in the promoter region of the GSTP1 gene;

[0013] Figure 2 This is a schematic diagram of the sequence segment of the genomic DNA sequence of the GSTP1 gene containing 96 CpGs after sulfite treatment. DETAILED DESCRIPTION

[0014] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with the following embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0015] The present invention is based on the CpG island DNA sequence segment in the promoter region of the human GSTP1 gene, with a specific genomic location of (hg38) chr11: 67583458 -67584482 strand=+, and the CG sequence contained therein is as follows, containing a total of 96 CGs. Figure 1 >After sulfite treatment, the sequence of this segment becomes Figure 2 , Figure 2 The merging base Y represents the base C / T. After sulfite conversion, the methylated CG sequence in the above genomic reference sequence is expected to remain as a CG sequence, while the unmethylated CG sequence is converted to a TG sequence, and all single C bases are converted to T bases. This creates conditions for distinguishing methylated and unmethylated sequences through qPCR primer probes.

[0016] In the sequence containing 96 CpGs, the inventors selected 4 sequences to design primers and probes for methylation fluorescence quantitative (qMS-PCR) for methylation detection. The boxes represent the locations of the upstream and downstream primers; the gray area sequences represent the locations of the probes, such as Figure 2 As shown; the primer probe sequences are shown in Table 1. The upstream and downstream primers each include at least 2 CG sequences. The probe includes at least 2 CG sequences. The CG numbers on the above primer probes can fully distinguish methylated DNA from unmethylated DNA.

[0017] Table 1 Four sets of qMS-PCR primers and probes were designed by selecting four sequences from the sequences containing 96 CpGs

[0018]

[0019] From the primer probes designed for the four sequences, a primer probe designed for one sequence was randomly selected for qPCR reaction. The reaction system components were from Promega, and the specific reaction system is shown in Table 2:

[0020] Table 2 A sequence qPCR reaction system of the present invention

[0021]

[0022] Fill up to 30 μL with water.

[0023] The primers and probes designed for the two sequences were mixed into one tube for duplex qPCR reaction. The reaction system components were from Promega, and the specific reaction system is shown in Table 3:

[0024] Table 3 Two-segment sequence qPCR reaction system of the present invention

[0025]

[0026] The primers and probes designed for the three sequences were mixed into one tube for triple qPCR reaction. The reaction system components were from Promega, and the specific reaction system is shown in Table 4:

[0027] Table 4 Three-segment sequence qPCR reaction system of the present invention

[0028]

[0029] Fill up to 30 μL with water.

[0030] Primers and probes designed for four sequences were mixed into one tube for quadruple qPCR reaction. The reaction system components were from Promega, and the specific reaction system is shown in Table 5:

[0031] Table 5 Four-segment sequence qPCR reaction system of the present invention

[0032]

[0033] Fill up to 30 μL with water.

[0034] The reaction conditions of all systems are the same, and the specific reaction conditions are shown in Table 6:

[0035] Table 6 PCR reaction conditions of the present invention

[0036]

[0037] Fluorescence was collected at 65°C. If the Ct value of the target gene fragment by qMS-PCR was <41, the methylation detection of the fragment was considered positive.

[0038] The qMS-PCR designed for the above four fragments was tested using 67 cases of benign prostate lesions and 48 cases of urine from prostate cancer patients. The clinical information of the cases and the specific methylation results are shown in Tables 7 to 9. The judgment criteria are: when the methylation detected by a single fragment is positive, the specimen is judged to be methylation-positive. The sensitivity, specificity, and overall consistency rate obtained by comparison with the pathological results are shown in Table 10 below.

[0039] Table 7

[0040]

[0041] Table 8

[0042]

[0043] Table 9

[0044]

[0045] Table 10. Results of fluorescence quantification of methylation of single fragments in urine

[0046]

[0047] From the above results, it can be seen that the sensitivity of a single fragment is low. In order to improve the sensitivity of the detection, the inventor tried to jointly detect the above four fragments. Considering that among the above four fragments, the total matching rate of PCR3 is low, the applicant selected PCR1+ PCR2, PCR1+ PCR2+ PCR4 and PCR1+ PCR2+ PCR3+ PCR4 combinations as the optimal detection system. Therefore, the primer probes designed for these four fragments were randomly mixed into one tube for multiple qPCR reactions, and each combination was used for 67 cases of benign prostate tissue lesions and 48 cases of prostate cancer patients' urine for testing. The clinical information of the cases and the specific methylation results are shown in Tables 7-9. The specific test results are as follows.

[0048] A multiplex combined qMS-PCR was designed for PCR1+PCR2. When the Ct value of the qMS-PCR in the double combined PCR was <41 (i.e., the methylation detected by the double system was positive), the methylation sample was judged to be positive for methylation. The results are shown in Table 11 below.

[0049] Table 11. Detection results of dual qMS-PCR combined with fluorescence quantitative system in urine

[0050]

[0051] A multiplex combined qMS-PCR was performed for the combination of PCR1+ PCR2+ PCR4. When the Ct value of the qMS-PCR in the triple combined PCR was <41 (i.e., the methylation detected by the triple system was positive), the methylation of the methylated sample was judged to be positive. The results are shown in Table 12 below.

[0052] Table 12. Detection results of triple qMS-PCR combined with fluorescence quantitative system in urine

[0053]

[0054] A multiplex combined qMS-PCR was performed for PCR1+ PCR2+ PCR3+ PCR4. When the Ct value of the qMS-PCR in the quadruple combined PCR was <41 (i.e., the methylation detected by the quadruple system was positive), the methylation of the methylated sample was judged to be positive. The results are shown in Table 13 below.

[0055] Table 13. Detection results of quadruple qMS-PCR combined with fluorescence quantitative system in urine

[0056]

[0057] It can be seen that the triple combined fluorescence quantitative PCR has a better overall consistency rate than the double and quadruple combined fluorescence quantitative PCR. Next, the triple combined PCR combination was further tested. When the Ct value of the qMS-PCR in the triple combined PCR was <41 (i.e., the methylation detected by the triple system was positive), the methylation sample was judged to be positive for methylation. The results are shown in Table 14 below.

[0058] Table 14. Results of fluorescence quantitative methylation detection in urine for random combinations of three fragments

[0059]

[0060] In the triple combined fluorescence quantitative PCR test results, the results showed that the PCR1+ PCR2+ PCR4 combination had the best detection performance, while the sensitivity, specificity and consistency rate of other combinations were worse than this combination.

[0061] In summary, analysis of the test results showed that combined detection of single fragments would significantly improve sensitivity. It can be concluded that in triple combined fluorescence quantitative PCR, the combination of PCR1+PCR2+PCR4 is the optimal detection system, and its comprehensive performance of sensitivity and specificity is optimal, with a sensitivity of 91.67% and a specificity of 92.54%.

[0062] A triple combined qMS-PCR method was then identified to detect urine samples for early non-invasive diagnosis of prostate cancer, thereby improving diagnostic accuracy and reducing unnecessary invasive examinations.

[0063] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0064] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. Use of a methylation molecular marker quantitative detection reagent in the preparation of a urine early diagnosis of prostate cancer detection kit, characterized in that: The kit is used to detect the methylated fragment of the CpG island DNA sequence segment located in the promoter region of the human GSTP1 gene, and the methylated molecular markers are composed of the following three groups: the first group, chr11:67583460-67583479 strand=+, chr11:67583533-67583551 strand=+, chr11:67583503-67583523 strand=+; the second group, chr11:67583619-67583636 strand=+, chr11:67583681-67583697 strand=+, chr11:67583660-67583677 strand=+, and, the fourth group, cchr11:67584231-67584249 strand=+, chr11:67584315-67584338 strand=+, chr11:67584266-67584288 strand=+; the genome version is hg38; the kit includes the following primers and probes: 。 2. The use according to claim 1, characterized in that: The kit is a bisulfite conversion fluorescence quantitative PCR kit.

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