技术领域Technical field
本发明属于生物诊断技术领域，具体涉及一种基因检测试剂盒及其制备方法和应用。The invention belongs to the field of biological diagnosis technology, and specifically relates to a gene detection kit and its preparation method and application.
背景技术Background technique
犬乳腺肿瘤(Canine mammary tumor)是犬的常见的一类疾病，其成因复杂，发病机理尚不明确。乳腺肿瘤是未绝育母犬最常见的肿瘤疾病，占所有肿瘤疾病50％-70％；犬乳腺肿瘤中约50％为恶性，50％为良性；一些良性乳腺肿瘤，随着病程发展，可能会成为恶性肿瘤；年龄越大，肿瘤风险越高；大型犬的发病年龄可能比小型犬更小；肿物越大，恶性几率越高，医治难度越大，动物越疼！Canine mammary tumor is a common disease in dogs. Its causes are complex and its pathogenesis is still unclear. Mammary gland tumors are the most common tumor disease in unneutered female dogs, accounting for 50%-70% of all tumor diseases. About 50% of canine breast tumors are malignant and 50% are benign. Some benign breast tumors may develop as the disease progresses. It can become a malignant tumor; the older you are, the higher the risk of tumors; the age of onset of large dogs may be younger than that of small dogs; the larger the tumor, the higher the chance of malignancy, the more difficult it is to treat, and the more pain the animal will suffer!
目前针对犬乳腺肿瘤的诊断主要有5种方法。一是乳区触诊，对狗狗所有的乳区进行触诊检查，确认肿物的数量和大小；二是细胞学检查，确认长在乳区的肿物的确是乳腺肿瘤，而不是其他更恶性的肿瘤；三是血液学检查和尿常规检查，老龄动物需要全面的血检和尿检来评估动物体况；四是腹部B超、胸部X线检查：可以在术前评估腹腔和胸腔主要脏器的形态，有无明显转移和异常；五是CT，相比胸部X线检查，可以评估有无更小的转移性结节。这些诊断方法成本较高，人员要求高。因此，急需开发一种常规检测犬乳腺肿瘤的方法。Currently, there are five main methods for diagnosing canine mammary tumors. The first is mammary area palpation, which involves palpating and inspecting all the dog's mammary areas to confirm the number and size of the tumors; the second is cytological examination, which confirms that the tumors growing in the mammary area are indeed mammary tumors and not other more specific tumors. Malignant tumors; third, hematological examination and routine urine examination. Older animals need comprehensive blood tests and urine tests to evaluate the animal's physical condition; fourth, abdominal B-ultrasound and chest X-ray examination: the main organs of the abdominal cavity and chest can be evaluated before surgery. The morphology of the organ, whether there are obvious metastases and abnormalities; fifth, CT, compared with chest X-ray examination, can evaluate whether there are smaller metastatic nodules. These diagnostic methods are costly and require high personnel. Therefore, there is an urgent need to develop a method for routine detection of canine mammary tumors.
发明内容Contents of the invention
本发明要解决的技术问题一是筛选合适可用于犬乳腺肿瘤诊断的标志物；二是在此基础上，开发一种用于检测犬乳腺肿瘤的基因试剂盒。The technical problems to be solved by the present invention are: first, screening suitable markers that can be used for the diagnosis of canine mammary gland tumors; second, on this basis, developing a gene kit for detecting canine mammary gland tumors.
因此，一方面，本发明提供了一种犬乳腺肿瘤诊断标志物，所述的标志物为CLCP1和CPRDX1。Therefore, on the one hand, the present invention provides a canine mammary tumor diagnostic marker, and the markers are CLCP1 and CPRDX1.
再一方面，本发明还提供了一种犬乳腺肿瘤诊断试剂盒，所述试剂盒包括检测CLCP1和CPRDX1表达水平的试剂。In another aspect, the present invention also provides a canine breast tumor diagnostic kit, which includes reagents for detecting the expression levels of CLCP1 and CPRDX1.
优选地，本发明所述的试剂包括特异性扩增CLCP1和CPRDX1的引物。Preferably, the reagents of the invention include primers that specifically amplify CLCP1 and CPRDX1.
优选地，本发明所述的特异性扩增CLCP1和CPRDX1的引物如下所示：Preferably, the primers of the present invention that specifically amplify CLCP1 and CPRDX1 are as follows:
CLCP1-F：5’-cttggtacgagctcggatcc-3’；CLCP1-F: 5’-cttggtacgagctcggatcc-3’;
CLCP1-R：5’-cgtacacccttgctccgatt-3’；CLCP1-R: 5’-cgtacacccttgctccgatt-3’;
CPRDX1-F：5’-ttgctttcagtgacagggca-3’；CPRDX1-F: 5’-ttgctttcagtgacagggca-3’;
CPRDX1-R：5’-acagagcgaccaacaggaag-3’。CPRDX1-R: 5’-acagagcgaccaacaggaag-3’.
优选地，本发明所述的PCR反应液还包括内参基因β-actin的特异性扩增引物，所述引物和探针的序列如下：Preferably, the PCR reaction solution of the present invention also includes specific amplification primers for the internal reference gene β-actin. The sequences of the primers and probes are as follows:
β-actin-F：5’-aagtaccccattgagcacgg-3’；β-actin-F: 5’-aagtaccccattgagcacgg-3’;
β-actin-R：5’-catacagggacaggacagcc-3’。β-actin-R: 5’-catacagggacaggacagcc-3’.
再一方面，本发明还提供一种所述的犬乳腺肿瘤诊断标志物在制备犬乳腺肿瘤诊断试剂中的应用。In yet another aspect, the present invention also provides an application of the canine breast tumor diagnostic marker in preparing a canine breast tumor diagnostic reagent.
本发明通过生物信息学和现有的分子生物学的技术手段，筛选用于犬乳腺肿瘤诊断的标志物。通过大量的筛选，从中选择CLCP1和CPRDX1作为标记物。在此基础上，本发明提供了检测CLCP1和CPRDX1的表达水平在制备诊断犬乳腺肿瘤的产品中的应用以及相关的检测试剂盒。The present invention uses bioinformatics and existing molecular biology technical means to screen markers for canine breast tumor diagnosis. Through extensive screening, CLCP1 and CPRDX1 were selected as markers. On this basis, the present invention provides the application of detecting the expression levels of CLCP1 and CPRDX1 in preparing products for diagnosing canine mammary tumors and related detection kits.
本发明的试剂盒能够作为犬乳腺肿瘤诊断的手段之一，相比于现有的犬乳腺肿瘤的检测手段，本试剂盒具有检测快速方便、检测灵敏度高、特异度好、成本低，可以满足绝大多数的检测需求，应用范围广等。The kit of the present invention can be used as one of the means of diagnosing canine mammary tumors. Compared with the existing detection means of canine mammary tumors, the kit has the advantages of rapid and convenient detection, high detection sensitivity, good specificity and low cost, and can meet the needs of Most detection needs, wide range of applications, etc.
附图说明Description of drawings
图1CLCP1和CPRDX1在犬乳腺肿瘤中表达量检测结果。Figure 1 Detection results of expression levels of CLCP1 and CPRDX1 in canine mammary tumors.
具体实施方式Detailed ways
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，以下实施例所用试剂和材料均为市购。The present invention will be further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.
本发明人经过广泛而深入的研究，通过生物信息学技术和分子生物学技术，检测犬乳腺肿瘤中相关基因的表达水平，发现其中具有明显表达差异的基因，探讨其与犬乳腺肿瘤的发生之间的关系，从而为犬乳腺肿瘤的检测及靶向治疗寻找更好的途径和方法。通过大量的研究和筛选，本发明筛选到2个与人中淋巴细胞胞浆蛋白1(lymphocytecytosolic protein 1，LCP1)(GenBank:DR107736.1，如SEQ ID NO.1所示)和过氧化物酶1(peroxiredoxin 1，PRDX1)(GenBank:DR103789.1，如SEQ ID NO.2所示)相似的蛋白。在本研究中将其命名为犬淋巴细胞胞浆蛋白1(Canine lymphocyte cytosolic protein 1，CLCP1)和犬过氧化物酶1(Canine peroxiredoxin 1，CPRDX1)。After extensive and in-depth research, the inventors used bioinformatics technology and molecular biology technology to detect the expression levels of related genes in canine mammary gland tumors, found genes with obvious expression differences, and explored their relationship with the occurrence of canine mammary gland tumors. relationship, thereby finding better ways and methods for the detection and targeted treatment of canine mammary tumors. Through extensive research and screening, the present invention screened out two proteins related to human lymphocyte cytosolic protein 1 (LCP1) (GenBank: DR107736.1, as shown in SEQ ID NO. 1) and peroxidase 1 (peroxiredoxin 1, PRDX1) (GenBank: DR103789.1, as shown in SEQ ID NO. 2) similar proteins. In this study, they were named canine lymphocyte cytosolic protein 1 (CLCP1) and canine peroxidase 1 (Canine peroxiredoxin 1, CPRDX1).
通过检测分析，CLCP1和CPRDX1在犬乳腺肿瘤中高表达。进一步的实验证明，通过调节CLCP1和CPRDX1的表达水平可以影响犬乳腺肿瘤的生长，提示CLCP1和CPRDX1可以作为药物靶标用于犬乳腺肿瘤的诊断或治疗。Through detection and analysis, CLCP1 and CPRDX1 are highly expressed in canine mammary tumors. Further experiments demonstrated that the growth of canine mammary tumors can be affected by regulating the expression levels of CLCP1 and CPRDX1, suggesting that CLCP1 and CPRDX1 can be used as drug targets for the diagnosis or treatment of canine mammary tumors.
“生物标志物”与“标志物”可以等同替代，指代具有特异性生物学特性、生物化学特征或者方面的分子指示物，其可用于确定存在或不存在特定疾病或状况和/或特定疾病或状况的严重程度。"Biomarker" and "marker" are used interchangeably and refer to a molecular indicator with a specific biological property, biochemical characteristic or aspect that can be used to determine the presence or absence of a specific disease or condition and/or a specific disease or the severity of the condition.
实施例1犬乳腺肿瘤中CLCP1和CPRDX1检测Example 1 Detection of CLCP1 and CPRDX1 in canine mammary tumors
1.1样本收集1.1 Sample collection
选用在广州市某动物医院进行治疗并经病理确诊的犬乳腺肿瘤病例，无菌操作留取部分癌组织和癌旁正常组织(距离癌组织2～5cm左右)分装于冻存管，立即放入液氮保存。所有患畜均经资深病理医生的病理诊断，纳入本研究前未患其它肿瘤，且未经放射或化学药物治疗。选择其中5例恶性肿瘤组织和癌旁组织进行本研究。Cases of canine mammary gland tumors that were treated in an animal hospital in Guangzhou and confirmed pathologically were selected. Part of the cancer tissue and adjacent normal tissue (about 2 to 5 cm away from the cancer tissue) were collected aseptically and packed into cryopreservation tubes, and placed immediately. Store in liquid nitrogen. All animals were pathologically diagnosed by senior pathologists, did not suffer from other tumors before being included in this study, and had not been treated with radiation or chemical drugs. Five cases of malignant tumor tissue and adjacent tissue were selected for this study.
1.2组织总RNA的提取1.2 Extraction of total tissue RNA
(1)每50～100mg组织用1ml Trizol试剂(Takara公司)对组织进行裂解；(1) Use 1 ml Trizol reagent (Takara Company) for every 50 to 100 mg of tissue to lyse the tissue;
(2)将上述组织的Trizol裂解液转入EP管中，在室温下放置5分钟；(2) Transfer the Trizol lysis solution of the above tissues into an EP tube and place it at room temperature for 5 minutes;
(3)在上述EP管中，按照每1ml Trizol后加0.2ml氯仿的量加入氯仿，盖上EP管盖子，在手中用力震荡15秒，在室温下放置2～3分钟后，12000g(2～8℃)离心15分钟；(3) In the above EP tube, add 0.2 ml of chloroform per 1 ml of Trizol, cover the EP tube, shake vigorously in your hand for 15 seconds, and leave it at room temperature for 2 to 3 minutes. 12000g (2~ 8℃) centrifuge for 15 minutes;
(4)去上层水相置于新EP管中，按照每1ml Trizol加0.5ml异丙醇的量加入异丙醇，在室温下放置10分钟后，12000g(2～8℃)离心10分钟；(4) Remove the upper aqueous phase and place it in a new EP tube. Add 0.5 ml of isopropyl alcohol per 1 ml of Trizol. After leaving it at room temperature for 10 minutes, centrifuge at 12000g (2-8°C) for 10 minutes;
(5)弃上清，按照每1ml Trizol加1ml 75％乙醇进行洗涤，涡旋混合，12000g(2～8℃)离心5分钟，弃上清；(5) Discard the supernatant, add 1 ml of 75% ethanol per 1 ml of Trizol for washing, vortex to mix, centrifuge at 12000g (2-8°C) for 5 minutes, and discard the supernatant;
(6)让沉淀的RNA在室温在自然干燥；(6) Let the precipitated RNA dry naturally at room temperature;
(7)用Rnase-free water溶解RNA沉淀；(7) Dissolve the RNA pellet with RNase-free water;
(8)提取的RNA样本测定浓度和OD260/OD280的比值以控制样本质量，选择OD260/OD280的比值在1.8～2.0之间的样本用于后续试验。(8) Measure the concentration and OD260/OD280 ratio of the extracted RNA sample to control the sample quality, and select samples with an OD260/OD280 ratio between 1.8 and 2.0 for subsequent tests.
1.3引物设计1.3 Primer design
根据的CLCP1(SEQ ID NO.1)和CPRDX1(SEQ ID NO.2)序列，运用Primer Primer5.0软件进行引物的设计。得到多对特异性引物，经过比对筛选，最终分别确定一组最佳引物。具体如下：Primer Primer5.0 software was used to design primers based on the CLCP1 (SEQ ID NO.1) and CPRDX1 (SEQ ID NO.2) sequences. Multiple pairs of specific primers were obtained, and after comparison and screening, a set of optimal primers were finally determined. details as follows:
CLCP1-F：5’-cttggtacgagctcggatcc-3’；CLCP1-F: 5’-cttggtacgagctcggatcc-3’;
CLCP1-R：5’-cgtacacccttgctccgatt-3’；CLCP1-R: 5’-cgtacacccttgctccgatt-3’;
CPRDX1-F：5’-ttgctttcagtgacagggca-3’；CPRDX1-F: 5’-ttgctttcagtgacagggca-3’;
CPRDX1-R：5’-acagagcgaccaacaggaag-3’；CPRDX1-R: 5’-acagagcgaccaacaggaag-3’;
β-actin-F：5’-aagtaccccattgagcacgg-3’；β-actin-F: 5’-aagtaccccattgagcacgg-3’;
β-actin-R：5’-catacagggacaggacagcc-3’。β-actin-R: 5’-catacagggacaggacagcc-3’.
1.4逆转录反应1.4 Reverse transcription reaction
逆转录实验体系20μl，参照EasyScript First-Strand cDNA SynthesisSuperMix(目录号AE301-02，北京全式金生物技术有限公司)说明书进行。The reverse transcription experimental system was 20 μl, and was carried out according to the instructions of EasyScript First-Strand cDNA SynthesisSuperMix (catalog number AE301-02, Beijing Quanshijin Biotechnology Co., Ltd.).
1.5荧光定量PCR反应1.5 Fluorescent quantitative PCR reaction
取逆转录反应进行实时荧光定量PCR操作，反应体系为20μl：10μl SYBR Premix、cDNA模板2μl，上下游引物各0.6μl及DEPC水6.8μl。应用美国ABI公司的7500型荧光定量PCR进行实验，反应设定条件为：95℃预变性30s，95℃变性30s，60℃退火20s，72℃延伸30s，40个循环。Take the reverse transcription reaction and perform real-time fluorescence quantitative PCR operation. The reaction system is 20 μl: 10 μl SYBR Premix, 2 μl cDNA template, 0.6 μl each of the upstream and downstream primers, and 6.8 μl DEPC water. The 7500 fluorescent quantitative PCR model of the American ABI Company was used for the experiment. The reaction setting conditions were: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 30 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s, 40 cycles.
其中2-△△Ct表示实验组与对照组目的基因表达的倍比关系，公式如下：△Ct＝CT(目的基因)－CT(内参)、△△Ct＝△Ct实验组－△Ct对照组。Ct为反应的实时荧光强度达到设定的阈值时所经过的扩散循环数，此时扩散是呈对数期增长。计算CLCP1和CPRDX1的表达量。其中内参为β-actin(如SEQ ID NO.3所示)。Among them, 2-△△Ct represents the fold ratio of the expression of the target gene between the experimental group and the control group. The formula is as follows: △Ct=CT (target gene)-CT (internal reference), △△Ct=△Ctexperimental group- △Ctcontrol group . Ct is the number of diffusion cycles that pass when the real-time fluorescence intensity of the reaction reaches the set threshold. At this time, the diffusion increases in a logarithmic phase. Calculate the expression levels of CLCP1 and CPRDX1. The internal reference is β-actin (as shown in SEQ ID NO. 3).
1.6检测结果1.6 Test results
具体结果见图1。从图中可以看出，CLCP1和CPRDX1在犬乳腺肿瘤中表达量显著上调。The specific results are shown in Figure 1. As can be seen from the figure, the expression levels of CLCP1 and CPRDX1 are significantly up-regulated in canine mammary tumors.
实施例2试剂盒对临床样品的检测Example 2 Detection of clinical samples by kit
采用实施例2的检测试剂盒，对30例犬乳腺肿瘤及30例健康犬血清中的CLCP1和CPRDX1的表达量进行检测。检测结果使用origin软件进行回归分析，其中CLCP1和CPRDX1单个检测指标结果、两个指标联合及三个指标的联合检测结果见表1所示。The detection kit of Example 2 was used to detect the expression levels of CLCP1 and CPRDX1 in the serum of 30 cases of canine mammary tumors and 30 cases of healthy dogs. The detection results were regression analyzed using origin software. The results of single detection indicators of CLCP1 and CPRDX1, the combined detection results of two indicators and the combined detection results of three indicators are shown in Table 1.
结果显示，各指标及联合指标P值均<0.05，说明各检测指标均与犬乳腺肿瘤预测显著相关。CLCP1和CPRDX1的联合预测准确率达到96％(ROC曲线下面积)，高于单个检测指标。因此，CLCP1和CPRDX1的联合检测在犬乳腺肿瘤中具有明显的优势。The results showed that the P values of each index and the combined index were all <0.05, indicating that each detection index was significantly related to the prediction of canine breast tumors. The joint prediction accuracy of CLCP1 and CPRDX1 reaches 96% (area under the ROC curve), which is higher than the single detection index. Therefore, the combined detection of CLCP1 and CPRDX1 has obvious advantages in canine mammary tumors.
表1检测结果分析结果Table 1 Test results analysis results
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.
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| CN202310287150.4ACN116083590B (en) | 2023-03-23 | 2023-03-23 | Gene detection kit and preparation method and application thereof |
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| CN202310287150.4ACN116083590B (en) | 2023-03-23 | 2023-03-23 | Gene detection kit and preparation method and application thereof |
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| CN116083590A CN116083590A (en) | 2023-05-09 |
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| CN202310287150.4AActiveCN116083590B (en) | 2023-03-23 | 2023-03-23 | Gene detection kit and preparation method and application thereof |
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| CN (1) | CN116083590B (en) |
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| Publication number | Publication date |
|---|---|
| CN116083590A (en) | 2023-05-09 |
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