CN116064790A - Markers, kits and methods of use for prostate cancer screening - Google Patents

Abstract

The invention relates to a marker for screening prostatic cancer, a kit and a using method thereof, wherein the marker is urine exosome markers PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR; the kit for screening the prostate cancer comprises a reaction solution of five markers and a beta-actin reaction solution; the sequence of the front primer in the beta-actin reaction liquid is shown as SEQ ID NO. 1, and the sequence of the rear primer is shown as SEQ ID NO. 2. The kit adopts reverse transcription polymerase chain reaction to detect five markers of PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR, and 5 markers are combined for use, so that the sensitivity and the specificity of diagnosis are greatly improved. The detection of RT-qPcr PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR of urine exosomes of 50 cases of prostate cancer shows that the ROC characteristic curve shows that the sensitivity of the kit for diagnosing the prostate cancer is 91.43%, and the specificity reaches 89.66%; this result is superior to other products in the market.

Description

Translated fromChinese

用于前列腺癌筛查的标记物、试剂盒及其使用方法Markers, kits and methods of use for prostate cancer screening

技术领域technical field

本发明属于生物技术领域，具体涉及一种用于前列腺癌筛查的标记物、试剂盒及其使用方法。The invention belongs to the field of biotechnology, and in particular relates to a marker, a kit and a use method thereof for prostate cancer screening.

背景技术Background technique

前列腺癌是目前男性面临的最常见癌症之一，是我国第六大恶性癌。在美国男性发病率第一位，死亡率第二位。前列腺癌在我国排男性恶性肿瘤的第三位。由于前列腺癌在早期没有任何症状，患者往往容易错过最佳的治疗时机，导致诊疗难度加大。常在体检时行直肠指检或检测血清PSA升高被发现。前列腺特异性抗原（prostate specific antigen 或者 PSA）是一种由前列腺上皮细胞分泌产生的一种丝氨酸蛋白酶，是一种糖蛋白。PSA及其衍生物是目前早期筛查前列腺癌最常用的生物标志物，目前已经发展出了PHI和4KScore等结合多指标联合检测结果的综合评判标准。但因PSA特异性低带来的假阳性给患者带来了大量的经济和生理负担，人们仍然在寻找更好的前列腺癌生物标志物以更好的实现前列腺癌的筛查。尚没有一种结果可靠的早筛产品，目前体检用的PSA难以满足早期筛查的要求，急需要一些崭新的技术问世。Prostate cancer is one of the most common cancers faced by men, and it is the sixth most malignant cancer in my country. In the United States, the incidence of males is the first and the death rate is the second. Prostate cancer ranks third among male malignancies in my country. Because prostate cancer does not have any symptoms in the early stage, patients often miss the best opportunity for treatment, making diagnosis and treatment more difficult. It is often found during digital rectal examination or elevated serum PSA during physical examination. Prostate specific antigen (PSA) is a serine protease secreted by prostate epithelial cells and a glycoprotein. PSA and its derivatives are currently the most commonly used biomarkers for early screening of prostate cancer. At present, comprehensive evaluation criteria such as PHI and 4KScore combined with multi-index joint detection results have been developed. However, the false positives caused by the low specificity of PSA have brought a lot of economic and physiological burden to patients, and people are still looking for better prostate cancer biomarkers to better realize the screening of prostate cancer. There is no early screening product with reliable results, and the PSA used in physical examination can hardly meet the requirements of early screening, so some new technologies are urgently needed.

非入侵性的前列腺癌的诊断方法一直是临床研究中的热点。尿液为前列腺最直接接触的液体，因此，我们认为尿液里应有大量的肿瘤外泌体。The diagnosis method of non-invasive prostate cancer has been a hot spot in clinical research. Urine is the fluid that the prostate is most directly in contact with. Therefore, we believe that there should be a large number of tumor exosomes in urine.

鉴于此，申请此专利。In view of this, apply for this patent.

发明内容Contents of the invention

为了解决现有技术存在的问题，本发明提供了一种用于前列腺癌筛查的标记物、试剂盒及其使用方法，解决穿刺活检带来的诸多问题，开发出高效准确的早诊早筛试剂盒，满足临床需求。减轻病人的痛苦，降低前列腺癌的死亡率。In order to solve the problems existing in the prior art, the present invention provides a marker for prostate cancer screening, a kit and its use method, solves many problems caused by biopsy, and develops an efficient and accurate early diagnosis and early screening method. Kits to meet clinical needs. Reduce the suffering of patients and reduce the mortality rate of prostate cancer.

本发明的目的是提供一种用于前列腺癌筛查的试剂盒。The purpose of the present invention is to provide a test kit for prostate cancer screening.

本发明的另一目的是提供上述用于前列腺癌筛查的试剂盒的使用方法。Another object of the present invention is to provide a method of using the above kit for prostate cancer screening.

根据本发明的具体实施方式的用于前列腺癌筛查的尿液外泌体标记物，所述尿液外泌体标记物为PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR。According to the specific embodiment of the present invention, the urinary exosome markers for prostate cancer screening are PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR.

尿液外泌体标记物PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR的用途，所述尿液外泌体标记物用于前列腺癌筛查。Use of urinary exosome markers PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR for prostate cancer screening.

根据本发明的具体实施方式的用于前列腺癌筛查的试剂盒，所述试剂盒包含五种标记物的反应液和β-actin反应液；β-actin反应液中的前引物序列如SEQ ID NO:1所示，后引物序列如SEQ ID NO:2所示；通过逆转录聚合酶链反应检测尿外泌体中的PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR表达水平。According to the test kit for prostate cancer screening according to the specific embodiment of the present invention, the test kit comprises the reaction solution of five markers and the β-actin reaction solution; the front primer sequence in the β-actin reaction solution is as SEQ ID Shown in NO: 1, the back primer sequence is shown in SEQ ID NO: 2; the expression levels of PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR in urinary exosomes were detected by reverse transcription polymerase chain reaction.

检测PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR的反应液所使用的试剂相同，只是引物不同。The reagents used in the reaction solutions for detecting PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR are the same, but the primers are different.

具体的，检测五种标记物的反应液均包括：PCR buffer，MgCl2，dNTP，taq聚合酶，前引物，后引物，探针；其中，PCA3反应液中的前引物序列如SEQ ID NO:3所示，后引物序列如SEQ ID NO:4所示；TMPRSS2-ERG反应液中的前引物序列如SEQ ID NO:5所示，后引物序列如SEQ ID NO:6所示；SPDEF反应液中的前引物序列如SEQ ID NO:7所示，后引物序列如SEQID NO:8所示；HAAH反应液中的前引物序列如SEQ ID NO:9所示，后引物序列如SEQ ID NO:10所示；AMACR反应液中的前引物序列如SEQ ID NO:11所示，后引物序列如SEQ ID NO:12所示。Specifically, the reaction solution for detecting five markers includes: PCR buffer, MgCl2, dNTP, taq polymerase, front primer, back primer, probe; wherein, the front primer sequence in the PCA3 reaction solution is as SEQ ID NO:3 As shown, the back primer sequence is shown in SEQ ID NO:4; the front primer sequence in the TMPRSS2-ERG reaction solution is shown in SEQ ID NO:5, and the back primer sequence is shown in SEQ ID NO:6; in the SPDEF reaction solution The front primer sequence is shown in SEQ ID NO:7, and the back primer sequence is shown in SEQ ID NO:8; the front primer sequence in the HAAH reaction solution is shown in SEQ ID NO:9, and the back primer sequence is shown in SEQ ID NO:10 Shown; The front primer sequence in the AMACR reaction solution is shown in SEQ ID NO:11, and the back primer sequence is shown in SEQ ID NO:12.

进一步的，检测五种标记物的反应液均包括：1X的PCR buffer，2mM的MgCl₂，0.2mM的dNTP，0.03U/ul的taq聚合酶，前引物 0.2μM，后引物0.2 μM，探针0.08 μM，余量为去离子水。Further, the reaction solution for detecting the five markers includes: 1X PCR buffer, 2mM MgCl₂ , 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2 μM front primer, 0.2 μM back primer, probe 0.08 μM and the balance in deionized water.

进一步的，β-actin反应液包括：PCR buffer，MgCl₂，0.2mM的dNTP，taq聚合酶，β-actin的前引物 0.2μM，β-actin的后引物0.2 μM，β-actin探针。Further, the β-actin reaction solution includes: PCR buffer, MgCl₂ , 0.2mM dNTP, taq polymerase, 0.2 μM of β-actin pre-primer, 0.2 μM of β-actin post-primer, and β-actin probe.

进一步的，β-actin反应液包括：终体系1X的PCR buffer，2mM的MgCl₂，0.2mM的dNTP，0.03U/ul的taq聚合酶，β-actin前引物 0.2μM，β-actin后引物0.2 μM，β-actin探针0.05 μM，余量为去离子水。Further, the β-actin reaction solution includes: final system 1X PCR buffer, 2mM MgCl₂ , 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2 μM of β-actin pre-primer, 0.2 μM of β-actin post-primer μM, β-actin probe 0.05 μM, the balance is deionized water.

进一步的，所述试剂盒还包括阳性对照和阴性对照，所述阳性对照为质粒DNA；所述阴性对照为tris-HCl。Further, the kit also includes a positive control and a negative control, the positive control is plasmid DNA; the negative control is tris-HCl.

进一步的，所述试剂盒还包括外泌体提取试剂，所述外泌体提取试剂按体积百分比包括：18% PEG6000和10% PEG800，余量为PBS缓冲液。Further, the kit also includes an exosome extraction reagent, the exosome extraction reagent includes by volume percentage: 18% PEG6000 and 10% PEG800, and the balance is PBS buffer.

所述的试剂盒的使用方法，所述使用方法包括以下步骤：The using method of described test kit, described using method comprises the following steps:

（1）收集尿液，提取尿液外泌体；(1) Collect urine and extract urine exosomes;

（2）尿液外泌体中加入外泌体裂解液，提取mRNA；(2) Add exosome lysate to urine exosomes to extract mRNA;

（3）mRNA反转录成cDNA；(3) reverse transcription of mRNA into cDNA;

（4）通过逆转录聚合酶链反应，检测PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR表达。(4) The expressions of PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR were detected by reverse transcription polymerase chain reaction.

进一步的，步骤（1）具体为：Further, step (1) is specifically:

第一步：收集尿液，备用；The first step: collect urine and reserve;

第二步：将尿液进行离心，上清液转入新的离心管，加外泌体提取试剂 5ml，4℃过夜；外泌体提取试剂的组分包括体积百分比的18% PEG6000、10% PEG800和PBS ；Step 2: Centrifuge the urine, transfer the supernatant to a new centrifuge tube, add 5ml of exosome extraction reagent, and keep overnight at 4°C; the components of the exosome extraction reagent include 18% PEG6000, 10% by volume PEG800 and PBS;

第三步: 将所述上清液进行第二次离心，弃上清，收集外泌体沉淀，-80℃保存。Step 3: Centrifuge the supernatant for the second time, discard the supernatant, collect the exosome pellet, and store it at -80°C.

进一步的，步骤（4）具体为：Further, step (4) is specifically:

按反应布局，向96孔板中加入20 ul的反应液；外泌体cDNA稀释10倍，每个反应孔内加入10ul 的稀释后的cDNA ；阳性对照用每反应1000拷贝的质粒DNA，阴性对照用tris-HCl；在ABI PCR仪上进行PCR反应。According to the reaction layout, add 20 ul of reaction solution to the 96-well plate; dilute exosome cDNA 10 times, add 10 ul of diluted cDNA to each reaction well; use 1000 copies of plasmid DNA per reaction for positive control, negative control Use tris-HCl; perform PCR reaction on ABI PCR machine.

HAAH的PCR程序：95℃taq酶激活 15min，95℃变性15S，58℃退火延伸30S，共50个循环；HAAH的PCR产物长度为138bp；β-actin的PCR产物长度为165bp。HAAH PCR program: 95°C taq enzyme activation for 15min, 95°C denaturation for 15S, 58°C annealing extension for 30S, a total of 50 cycles; the length of the PCR product of HAAH is 138bp; the length of the PCR product of β-actin is 165bp.

PCA3、TMPRSS2-ERG、SPDEF和AMACR的PCR程序中退火温度与HAAH不同，分别为53℃、57℃、59℃和52℃。The annealing temperatures in the PCR programs of PCA3, TMPRSS2-ERG, SPDEF and AMACR were different from those of HAAH, which were 53°C, 57°C, 59°C and 52°C, respectively.

HAAH的PCR产物长度为138bp；PCA3的PCR产物长度为233bp；TMPRSS2-ERG的PCR产物长度为118bp；SPDEF的PCR产物长度为100bp；AMACR的PCR产物长度为188bp；β-actin的PCR产物长度为165bp。The length of the PCR product of HAAH is 138bp; the length of the PCR product of PCA3 is 233bp; the length of the PCR product of TMPRSS2-ERG is 118bp; the length of the PCR product of SPDEF is 100bp; the length of the PCR product of AMACR is 188bp; 165 bp.

前列腺癌抗原3（PCA3或DD3）是前列腺特异性基因，其具有高度的特异性且与前列腺的其它疾病和前列腺体积无关。PCA3是目前研究和应用最为广泛的非编码ＲＮＡ肿瘤标记物，尿液PCA3检测具有简便、重复性高及敏感性强的特点。1996 年，PCA3基因被发现在90%的前列腺癌组织中高表达。2002年，发现前列腺癌细胞会脱落到尿液中，从而“液体活检”的概念诞生。与血清PSA相比，PCA3具有较低的敏感性，但具有较高的特异性和较好的阳性和阴性预测值。PCA3是目前最有可能替代PSA进行前列腺癌筛查的生物标志物。Prostate cancer antigen 3 (PCA3 or DD3) is a prostate-specific gene that is highly specific and independent of other diseases of the prostate and prostate volume. PCA3 is currently the most widely studied and applied non-coding RNA tumor marker, and urine PCA3 detection has the characteristics of simplicity, high reproducibility and strong sensitivity. In 1996, the PCA3 gene was found to be highly expressed in 90% of prostate cancer tissues. In 2002, it was discovered that prostate cancer cells would shed into the urine, thus the concept of "liquid biopsy" was born. Compared with serum PSA, PCA3 has lower sensitivity, but higher specificity and better positive and negative predictive value. PCA3 is currently the most likely biomarker to replace PSA for prostate cancer screening.

人类天冬胺酰基β－羟化酶(Human Aspartyl/Asparaginyl β-hydroxylase，HAAH) 是胚胎期即存在于细胞内的一种酶，属于依赖α-酮戊二酸双加氧酶家族，可催化特定蛋白中表皮生长因子(EGF)受体样结构域的天冬氨酸或天冬酰胺残基上的β－碳原子羟基化。研究表明HAAH在肿瘤细胞表面的高表达，是一种新型恶性肿瘤高特异性的分子标记物。本团队的研究证实HAAH是前列腺癌的理想标记物。Human Aspartyl/Asparaginyl β-hydroxylase (HAAH) is an enzyme present in cells during the embryonic period, which belongs to the α-ketoglutarate dioxygenase family and can catalyze Hydroxylation of the β-carbon atom on an aspartic acid or asparagine residue of the epidermal growth factor (EGF) receptor-like domain of a specific protein. Studies have shown that the high expression of HAAH on the surface of tumor cells is a new type of molecular marker with high specificity for malignant tumors. Our team's research confirmed that HAAH is an ideal marker for prostate cancer.

α甲酰辅酶Ａ消旋酶（AMACR）由Ｐ５０４Ｓ基因编码，主要参与支链脂肪酸的β氧化。利用尿液中ＰＳＡ ｍＲＮＡ校准过的AMACR评分可被用于诊断前列腺癌，其敏感性约为70％。而在近期的研究中，Ｏｕｙａｎｇ等则将AMACR和PAC３评分相结合，将诊断敏感性提高到81％。α-formyl-CoA racemase (AMACR), encoded by the P504S gene, is mainly involved in the β-oxidation of branched-chain fatty acids. The AMACR score calibrated by PSA mRNA in urine can be used to diagnose prostate cancer with a sensitivity of about 70%. In a recent study, Ouyang et al. combined the AMACR and PAC3 scores to increase the diagnostic sensitivity to 81%.

TMPRSS2-ERG基因融合是前列腺癌的主要分子亚型。TMPRSS2是雄激素调节的跨膜丝氨酸蛋白酶，其在正常前列腺上皮中表达，在前列腺癌组织中的表达增加。尽管TMPRSS2-ERG的假阳性很低，但其在很多种癌症中都存在高表达，因此需要联合其它指标进行检测来提高其特异性。目前研究较多的是结合TMPRSS2-ERG和PCA3的联合检测来进行早期前列腺癌诊断。TMPRSS2-ERG gene fusion is the major molecular subtype of prostate cancer. TMPRSS2 is an androgen-regulated transmembrane serine protease expressed in normal prostate epithelium and increased in prostate cancer tissues. Although the false positive rate of TMPRSS2-ERG is very low, it is highly expressed in many cancers, so it needs to be tested in combination with other indicators to improve its specificity. At present, more researches are combined with the combined detection of TMPRSS2-ERG and PCA3 to diagnose early prostate cancer.

本发明与现有技术相比，有益效果为：Compared with the prior art, the present invention has beneficial effects as follows:

（1）本发明的试剂盒采用逆转录聚合酶链反应检测PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR五个标记物，5个标记物结合使用，大大提高了诊断的敏感性和特异性。本试剂盒同时检测尿外泌体中的PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR，这在国内外还没有报导，完全属于首创产品。(1) The kit of the present invention uses reverse transcription polymerase chain reaction to detect five markers, PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR, and the combined use of the five markers greatly improves the sensitivity and specificity of diagnosis. This kit simultaneously detects PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR in urinary exosomes, which has not been reported at home and abroad, and is completely a pioneering product.

（2）本发明的试剂盒既可用于前列腺癌诊断又可监控术后复发的新技术。(2) The kit of the present invention is a new technology that can be used not only for prostate cancer diagnosis but also for postoperative recurrence monitoring.

（3）本发明在对50例前列腺癌的尿外泌体的RT-qPcr PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR的检测显示，ROC 特征曲线显示，本发明的试剂盒诊断前列腺癌的灵敏度为91.43%，特异性达到了89.66%；此结果优于市场其他产品。(3) The detection of RT-qPcr PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR of urinary exosomes of 50 cases of prostate cancer by the present invention shows that the ROC characteristic curve shows that the sensitivity of the kit of the present invention in diagnosing prostate cancer It was 91.43%, and the specificity reached 89.66%. This result is better than other products in the market.

（4）通过尿液检测，方便、无痛、成本低，病人容易接受；(4) Through urine testing, it is convenient, painless, low-cost, and easy for patients to accept;

（5）此外，本发明对尿外泌体的提取方法进行深入研究，探索出了能适合临床运用的外泌体提取方法。(5) In addition, the present invention conducted in-depth research on the extraction method of urinary exosomes, and explored an exosome extraction method suitable for clinical use.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

图1显示采用本发明的试剂盒对已经确定诊断结果的前列腺癌患者、前列腺良性肿块患者、正常人的尿液外泌体检测结果；Figure 1 shows the test results of urine exosomes of prostate cancer patients, patients with benign prostatic tumors, and normal people whose diagnostic results have been determined using the kit of the present invention;

图2为ROC分析结果。Figure 2 shows the results of ROC analysis.

具体实施方式Detailed ways

为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other implementations obtained by persons of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

在一些较为具体的实施例中，用于前列腺癌筛查的尿液外泌体标记物为PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR。用于前列腺癌筛查的试剂盒，包含检测五种标记物的反应液和β-actin反应液；β-actin反应液中的前引物序列如SEQ ID NO:1所示，后引物序列如SEQ ID NO:2所示；通过逆转录聚合酶链反应检测尿外泌体中的PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR表达水平。In some more specific embodiments, the urine exosome markers used for prostate cancer screening are PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR. A kit for prostate cancer screening, including a reaction solution for detecting five markers and a β-actin reaction solution; the front primer sequence in the β-actin reaction solution is shown in SEQ ID NO:1, and the back primer sequence is shown in SEQ ID NO:1 ID NO: 2; PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR expression levels in urinary exosomes were detected by reverse transcription polymerase chain reaction.

进一步的，五种标记物的反应液均包括：PCR buffer，MgCl2，dNTP，taq聚合酶，前引物，后引物，探针。Further, the reaction solutions of the five markers include: PCR buffer, MgCl2, dNTP, taq polymerase, front primer, back primer, and probe.

进一步的，β-actin反应液包括：PCR buffer，MgCl2，0.2mM的dNTP，taq聚合酶，β-actin的前引物 0.2μM，β-actin的后引物0.2 μM，β-actin探针。Further, the β-actin reaction solution includes: PCR buffer, MgCl2, 0.2mM dNTP, taq polymerase, 0.2 μM of the front primer of β-actin, 0.2 μM of the back primer of β-actin, and β-actin probe.

进一步的，β-actin反应液包括：终体系1X的PCR buffer，2mM的MgCl2，0.2mM的dNTP，0.03U/ul的taq聚合酶，β-actin前引物 0.2μM，β-actin后引物0.2 μM，β-actin探针0.05 μM，去离子水补充至20ul。Further, the β-actin reaction solution includes: final system 1X PCR buffer, 2mM MgCl2, 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2 μM of β-actin pre-primer, 0.2 μM of β-actin post-primer , β-actin probe 0.05 μM, supplemented with deionized water to 20ul.

进一步的，所述试剂盒还包括阳性对照和阴性对照，所述阳性对照为质粒DNA；所述阴性对照为tris-HCl；所述试剂盒还包括外泌体提取试剂，所述外泌体提取试剂按体积百分比包括：18% PEG6000和10% PEG800，余量为PBS缓冲液。Further, the kit also includes a positive control and a negative control, the positive control is plasmid DNA; the negative control is tris-HCl; the kit also includes exosome extraction reagents, the exosome extraction The reagents include by volume percentage: 18% PEG6000 and 10% PEG800, and the balance is PBS buffer.

实施例1Example 1

本实施例提供了一种用于前列腺癌筛查的试剂盒，所述试剂盒包含五种标记物的反应液和β-actin反应液；β-actin反应液中的前引物序列如SEQ ID NO:1所示（TGGCATTGCCGACAGGAT），后引物序列如SEQ ID NO:2所示（ATACTCCTGCTTGCTGATCCACAT）；通过逆转录聚合酶链反应检测尿外泌体中的PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR表达水平。The present embodiment provides a kind of test kit that is used for prostate cancer screening, and described test kit comprises the reaction solution of five kinds of markers and β-actin reaction solution; The front primer sequence in β-actin reaction solution is as SEQ ID NO : 1 (TGGCATTGCCGACAGGAT), the back primer sequence is shown in SEQ ID NO: 2 (ATACTCCTGCTTGCTGATCCACAT); the expression levels of PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR in urinary exosomes were detected by reverse transcription polymerase chain reaction .

五种标记物的反应液均为：1X的PCR buffer，2mM的MgCl2，0.2mM的dNTP，0.03U/ul的taq聚合酶，前引物 0.2μM，后引物0.2 μM，探针0.08 μM，去离子水补充至20ul。The reaction solutions for the five markers are: 1X PCR buffer, 2mM MgCl2, 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2μM front primer, 0.2 μM back primer, 0.08 μM probe, deionized Water was added to 20ul.

检测PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR的反应液所使用的试剂相同，只是引物不同。The reagents used in the reaction solutions for detecting PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR are the same, but the primers are different.

具体的，PCA3反应液中的前引物序列如SEQ ID NO:3所示，后引物序列如SEQ IDNO:4所示；TMPRSS2-ERG反应液中的前引物序列如SEQ ID NO:5所示，后引物序列如SEQ IDNO:6所示；SPDEF反应液中的前引物序列如SEQ ID NO:7所示，后引物序列如SEQ ID NO:8所示；HAAH反应液中的前引物序列如SEQ ID NO:9所示，后引物序列如SEQ ID NO:10所示；AMACR反应液中的前引物序列如SEQ ID NO:11所示，后引物序列如SEQ ID NO:12所示。Specifically, the front primer sequence in the PCA3 reaction solution is shown in SEQ ID NO:3, and the back primer sequence is shown in SEQ ID NO:4; the front primer sequence in the TMPRSS2-ERG reaction solution is shown in SEQ ID NO:5, The back primer sequence is shown in SEQ ID NO:6; The front primer sequence in the SPDEF reaction solution is shown in SEQ ID NO:7, and the back primer sequence is shown in SEQ ID NO:8; The front primer sequence in the HAAH reaction solution is shown in SEQ ID NO:8. As shown in ID NO:9, the back primer sequence is shown in SEQ ID NO:10; the front primer sequence in the AMACR reaction solution is shown in SEQ ID NO:11, and the back primer sequence is shown in SEQ ID NO:12.

β-actin反应液包括：终体系1X的PCR buffer，2mM的MgCl2，0.2mM的dNTP，0.03U/ul的taq聚合酶，β-actin前引物 0.2μM，β-actin后引物0.2 μM，β-actin探针0.05 μM，去离子水补充至20ul。The β-actin reaction solution includes: final system 1X PCR buffer, 2mM MgCl2, 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2μM primer for β-actin, 0.2μM primer for β-actin, β-actin Actin probe 0.05 μM, supplemented with deionized water to 20ul.

所述试剂盒还包括阳性对照和阴性对照，所述阳性对照为质粒DNA；所述阴性对照为tris-HCl。The kit also includes a positive control and a negative control, the positive control is plasmid DNA; the negative control is tris-HCl.

实施例2 使用方法Embodiment 2 usage method

第一步：收集病人尿液标本50ml，4度保存不超过24小时。Step 1: Collect 50ml of the patient's urine sample and store it at 4°C for no more than 24 hours.

第二步：3000g 离心30分钟，上清转入新的离心管，加外泌体提取试剂 5ml，4℃过夜。外泌体提取试剂的组分包括18% PEG6000、10% PEG800和PBS。使用此试剂可以提取大量高质量的外泌体，而且使得检测成本更低。Step 2: Centrifuge at 3000g for 30 minutes, transfer the supernatant to a new centrifuge tube, add 5ml of exosome extraction reagent, and overnight at 4°C. The components of the exosome extraction reagent include 18% PEG6000, 10% PEG800 and PBS. Using this reagent can extract a large number of high-quality exosomes, and makes the detection cost lower.

第三步: 3000g 离心30分钟，弃上清。收集外泌体沉淀-80℃保存。Step 3: Centrifuge at 3000g for 30 minutes, discard the supernatant. The exosome pellets were collected and stored at -80°C.

第四步: 外泌体沉淀中加入350ul外泌体裂解液，提取mRNA。Step 4: Add 350ul of exosome lysate to the exosome precipitation to extract mRNA.

第五步: mRNA反转录成cDNA。Step 5: Reverse transcription of mRNA into cDNA.

第六步: RT-qPCR检测PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR表达。Step 6: RT-qPCR detection of PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR expression.

qPCR方案如下：The qPCR protocol is as follows:

1.配制检测标记物的反应液：终体系为1X的PCR buffer，2mM的MgCl2，0.2mM的dNTP，0.03U/ul的taq聚合酶，前引物 0.2μM，后引物0.2 μM，探针0.08 μM；去离子水补充至20ul。1. Prepare the reaction solution for detecting markers: the final system is 1X PCR buffer, 2mM MgCl2, 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2μM front primer, 0.2 μM back primer, and 0.08 μM probe ; Add deionized water to 20ul.

2.配制β-actin反应液：终体系1X的PCR buffer，2mM的MgCl2，0.2mM的dNTP，0.03U/ul的taq聚合酶，β-actin前引物 0.2μM，β-actin后引物0.2 μM，β-actin探针0.05 μM。去离子水补充至20ul。2. Prepare β-actin reaction solution: final system 1X PCR buffer, 2mM MgCl2, 0.2mM dNTP, 0.03U/ul taq polymerase, 0.2μM primer for β-actin, 0.2μM primer for β-actin, β-actin probe 0.05 μM. Deionized water was added to 20ul.

3.3.

PCA3引物：PCA3 primers:

前引物序列：CGCCCTTATTGTCTCCTC ， TM52.5℃；Front primer sequence: CGCCCTTATTGTCTCCTC , TM52.5℃;

后引物序列：GCCTTTCTTATTTCTCACCC ；TM53.5℃；Back primer sequence: GCCTTTTCTTATTTCTCACCC; TM53.5℃;

产物条带：233bpProduct band: 233bp

TMPRSS2-ERG引物：TMPRSS2-ERG primers:

前引物序列：AGTGCCCAAAACTGAAGACC，TM 56.6℃；Front primer sequence: AGTGCCCAAAACTGAAGACC, TM 56.6°C;

后引物序列：CAGGAGGAACTGCCAAAGC；TM 58.0℃；Back primer sequence: CAGGAGGAACTGCCAAAGC; TM 58.0°C;

产物条带：118bpProduct band: 118bp

SPDEF引物：SPDEF primers:

前引物序列：AACGAGCCAGACTTGACCTG，TM 59.97℃；Front primer sequence: AACGAGCCAGACTTGACCTG, TM 59.97°C;

后引物序列：ATGCTTCCGGCTCGTATGTT；TM 59.82℃；Back primer sequence: ATGCTTCCGGCTCGTATGTT; TM 59.82°C;

产物条带：100bpProduct band: 100bp

HAAH引物：HAAH primers:

前引物序列：AGC GGT AGC ACG AGT GC，TM 58℃；Front primer sequence: AGC GGT AGC ACG AGT GC, TM 58°C;

后引物序列：GCCCAGCAATGCAATCAC；TM 58℃；Back primer sequence: GCCCAGCAATGCAATCAC; TM 58°C;

产物条带：138bp；Product band: 138bp;

AMACR引物：AMACR primers:

前引物序列：TTGGCTTTGTCAGGTGG，TM52.0℃；Front primer sequence: TTGGCTTTGTCAGGTGG, TM52.0℃;

后引物序列：GCTCGTAGAACTGGGGTT；TM51.9℃；Back primer sequence: GCTCGTAGAACTGGGGTT; TM51.9℃;

产物条带：188bp。Product band: 188bp.

β-actin的前引物序列:TGGCATTGCCGACAGGAT，后引物序列：ATACTCCTGCTTGCTGATCCACAT，PCR产物长度165bp。The front primer sequence of β-actin: TGGCATTGCCGACAGGAT, the back primer sequence: ATACTCCTGCTTGCTGATCCACAT, the length of PCR product is 165bp.

4.按反应布局，向96孔板中加入20 ul的反应液4. According to the reaction layout, add 20 ul of reaction solution to the 96-well plate

5.每个反应孔内加入10ul 的模板cDNA（外泌体cDNA稀释10倍）5. Add 10ul template cDNA to each reaction well (exosome cDNA is diluted 10 times)

6.阳性对照用每反应1000拷贝的质粒DNA，阴性对照用tris-HCl6. Use 1000 copies of plasmid DNA per reaction for positive control and tris-HCl for negative control

7.在ABI PCR仪上进行PCR，PCR程序：95℃taq酶激活 15min，95℃变性15S，退火延伸30S，共50个循环；退火温度见引物标注。7. Perform PCR on the ABI PCR instrument, PCR program: activate taq enzyme at 95°C for 15min, denature at 95°C for 15S, anneal and extend for 30S, a total of 50 cycles; see the primer label for the annealing temperature.

临床验证试验clinical validation test

对已经确定诊断结果的前列腺癌患者、前列腺良性肿块患者、正常人的尿液外泌体检测结果进行分析，结果如图1所示；The urine exosome detection results of prostate cancer patients, benign prostatic tumor patients, and normal people whose diagnosis results have been confirmed are analyzed, and the results are shown in Figure 1;

从图1可以看出前列腺癌患者的德达Ct值仅0.75，前列腺良性肿块患者的德达Ct值则是6.54，两者有显著性差异（P《0.01），而正常人的德达Ct值高达9.24。It can be seen from Figure 1 that the Deda Ct value of prostate cancer patients is only 0.75, and the Deda Ct value of patients with benign prostatic masses is 6.54. There is a significant difference between the two (P<0.01), while the Deda Ct value of normal people Up to 9.24.

对检测结果进行ROC分析，结果如图2所示；从图中可以看出，ROC 特征曲线显示，本发明的使用方法对前列腺癌诊断的准确率超过了90%。The detection result is carried out ROC analysis, and the result is as shown in Figure 2; As can be seen from the figure, the ROC characteristic curve shows that the accuracy rate of the using method of the present invention exceeds 90% to prostate cancer diagnosis.

采用本发明的试剂盒在对50例前列腺癌的尿外泌体的RT-qPcr PCA3、TMPRSS2-ERG、SPDEF、HAAH和AMACR的检测显示，ROC 特征曲线显示，本发明的试剂盒诊断前列腺癌的灵敏度为91.43%，特异性达到了89.66%；此结果优于市场其他产品。对于患者而言，实现了癌症病变的早确诊早治疗。相对于传统的检测手段而言，该技术不仅将癌变发现期大幅度提前，而且检验成本低，对病人零伤害，这应是人类的“福音”。由于本试剂盒比物理检查便宜许多，为社会及患者节省了大量的检查费用。本项目的成功研发将大大提高前列腺癌的筛查率，即可节约庞大的治疗开支，又可大幅度降低前列腺癌的死亡率，必将产生良好的社会效益。The detection of RT-qPcr PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR in urinary exosomes of 50 cases of prostate cancer using the kit of the present invention shows that the ROC characteristic curve shows that the kit of the present invention is effective in diagnosing prostate cancer. The sensitivity is 91.43%, and the specificity is 89.66%; this result is better than other products in the market. For patients, early diagnosis and early treatment of cancer lesions have been realized. Compared with traditional detection methods, this technology not only greatly advances the detection period of cancer, but also has low inspection costs and zero harm to patients. This should be a "good news" for mankind. Because the test kit is much cheaper than physical examination, it saves a lot of examination expenses for the society and patients. The successful research and development of this project will greatly increase the screening rate of prostate cancer, which can not only save huge treatment expenses, but also greatly reduce the mortality rate of prostate cancer, which will definitely produce good social benefits.

以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (10)

1. A urine exosome marker for prostate cancer screening, wherein the urine exosome marker is PCA3, TMPRSS2-ERG, SPDEF, HAAH, and AMACR.

2. A kit for prostate cancer screening, characterized in that the kit comprises a reaction solution of five markers and a beta-actin reaction solution; the sequence of a front primer in the beta-actin reaction liquid is shown as SEQ ID NO. 1, and the sequence of a rear primer is shown as SEQ ID NO. 2; PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR expression levels in urinary exosomes were detected by reverse transcription polymerase chain reaction.

3. The kit of claim 1, wherein the reaction solutions of the five markers each comprise: PCR buffer, mgCl₂ dNTP, taq polymerase, pre-primer, post-primer, probe; wherein, the front primer sequence in the PCA3 reaction liquid is shown as SEQ ID NO. 3, and the rear primer sequence is shown as SEQ ID NO. 4; the sequence of a front primer in TMPRSS2-ERG reaction liquid is shown as SEQ ID NO. 5, and the sequence of a rear primer is shown as SEQ ID NO. 6; the sequence of a front primer in the SPDEF reaction liquid is shown as SEQ ID NO. 7, and the sequence of a rear primer is shown as SEQ ID NO. 8; the sequence of the front primer in the HAAH reaction liquid is shown as SEQ ID NO. 9, and the sequence of the rear primer is shown as SEQ ID NO. 10; the sequence of the front primer in the AMACR reaction liquid is shown as SEQ ID NO. 11, and the sequence of the rear primer is shown as SEQ ID NO. 12.

4. The kit according to claim 2, wherein the reaction liquid for detecting the five markers comprises: 1 XPCR buffer,2mM MgCl₂ 0.2mM dNTP,0.03U/ul taq polymerase, 0.2. Mu.M front primer, 0.2. Mu.M rear primer, 0.08. Mu.M probe, the balance deionized water.

5. The kit of claim 1, wherein the beta-actin reaction solution comprises: PCR buffer, mgCl2,0.2mM dNTP, taq polymerase, 0.2. Mu.M of beta-actin pre-primer, 0.2. Mu.M of beta-actin post-primer, beta-actin probe.

6. The kit according to claim 4, wherein the beta-actin reaction solution comprises: PCR buffer of final System 1X, mgCl 2mM₂ 0.2mM dNTP,0.03U/ul taq polymerase, 0.2. Mu.M beta-actin pre-primer, 0.2. Mu.M beta-actin post-primer, 0.05. Mu.M beta-actin probe, deionized water was added to 20ul.

7. The kit of claim 1, further comprising a positive control and a negative control, the positive control being plasmid DNA; the negative control is tris-HCl; the kit also comprises an exosome extraction reagent, wherein the exosome extraction reagent comprises the following components in percentage by volume: 18% PEG6000 and 10% PEG800, the balance PBS buffer.

8. A method of using the kit of claim 1, comprising the steps of:

(1) Collecting urine and extracting urine exosomes;

(2) Adding exosome lysate into urine exosome to extract mRNA;

(3) Reverse transcription of mRNA into cDNA;

(4) PCA3, TMPRSS2-ERG, SPDEF, HAAH and AMACR expression were detected by reverse transcription polymerase chain reaction.

9. The method of claim 8, wherein step (1) is specifically:

the first step: collecting urine for later use;

and a second step of: centrifuging urine, transferring supernatant into a new centrifuge tube, adding exosome extraction reagent, and standing at 4deg.C overnight;

thirdly, centrifuging the supernatant for the second time, discarding the supernatant, collecting exosome sediment and preserving at-80 ℃;

and fourthly, adding 350ul exosome lysate into the exosome sediment to extract mRNA.

10. The method of claim 1, wherein step (4) is specifically:

adding 20ul of reaction solution into a 96-well plate according to the reaction layout; diluting exosome cDNA by 10 times, and adding 10ul of diluted cDNA into each reaction hole; the positive control used 1000 copies of plasmid DNA per reaction and the negative control used tris-HCl; PCR reactions were performed on an ABI PCR instrument.

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