Early cancer detection reagent, kit and preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a cancer early-stage detection reagent, a kit and a preparation method and application of the cancer early-stage detection reagent. According to the cancer early detection reagent provided by the invention, through the two-step design of the pretreatment reagent and the FeCl3 chromogenic reagent, the pretreatment reagent can adjust the pH value to a proper range of the reaction of Fe < 3 + > and p-hydroxybenzene-R metabolite, the pH value is 5-6, and ammonia (NH3 + H +-NH4 + is immobilized in an acidic environment, so that the reaction with Fe < 3 + > is avoided); protein in the urine is precipitated, and core interference of ammonia and protein in the urine is effectively eliminated; meanwhile, FeCl3 is used for realizing specific detection on the hydroxybenzene-R metabolite, the operation is simple, the result reporting speed is high, and the method is suitable for laboratories, physical examination screening and family self-inspection. The cancer early detection reagent provided by the invention is simple in preparation method and low in cost, and the cancer early detection reagent is good in application effect and high in accuracy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an early cancer detection reagent, kit, preparation method, and application thereof. Background Technology

[0002] Cancer, a major disease that seriously threatens human life and health worldwide, has a treatment outcome that is closely related to the timing of its detection. However, current clinically used tumor detection methods have significant shortcomings in the crucial step of "early detection," making it difficult to meet the needs of cancer prevention and eradication. Therefore, the research and development and promotion of early cancer detection reagents are of great importance.

[0003] Current clinical cancer detection methods face the challenge of "early detection." The tumor detection methods widely used in clinical practice, such as imaging examinations (CT, MRI, ultrasound, etc.) and routine tumor marker tests, have significant limitations in the early detection of cancer and are difficult to effectively identify in the preclinical stage.

[0004] For example, in imaging examinations: by the time a tumor is "visible," it is no longer in its early stages. Imaging examinations require the tumor to grow to a certain size (usually several millimeters or more in diameter) before it can be clearly identified. When a tumor is discovered through CT scans, ultrasound, or other methods, the cancer cells have often already undergone a long period of proliferation in the body, possibly reaching the middle or late stages, or even causing local invasion or distant metastasis, thus missing the optimal intervention window. For instance, early-stage lung cancer lesions are small, and conventional chest CT scans have a low detection rate for tiny lesions less than 1 centimeter in diameter, while at this point, the cancer cells may already have a certain degree of invasiveness.

[0005] Another example is the use of conventional tumor marker testing, which suffers from insufficient specificity and sensitivity. Common tumor marker tests (such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP)) have issues with low specificity and sensitivity. On the one hand, some benign diseases may cause elevated tumor marker levels, leading to false positive results and unnecessary panic. On the other hand, many early-stage cancer patients do not show significantly abnormal tumor marker levels, resulting in false negatives and missed diagnoses. For instance, only about 20%-30% of early-stage gastric cancer patients show elevated CEA levels, making it difficult to use as a reliable basis for early diagnosis.

[0006] Another example is tissue biopsy: biopsies are invasive and have limitations due to difficulties in sampling. While tissue biopsy is the "gold standard" for tumor diagnosis, it is an invasive procedure that may pose risks such as pain and bleeding to patients. Furthermore, the sampling range is limited, making it difficult to accurately obtain lesion tissue for some hidden or small tumors, thus failing to achieve the goal of early screening.

[0007] Therefore, it is crucial to shift the focus of cancer treatment towards prevention.

[0008] The core pain point of cancer treatment lies in "late stage of detection". Early detection reagents can break through the existing technical bottlenecks and achieve accurate identification in the preclinical stage of cancer (that is, the stage in which tumor cells have already shown abnormal proliferation, but have not yet formed obvious lesions or caused clinical symptoms). This provides the possibility for "preventive" intervention for cancer. Its necessity is mainly reflected in the following aspects.

[0009] First, achieve "preclinical detection" to break the "late-stage dilemma".

[0010] The development of cancer is a long process, often taking years or even decades from cancer cell mutation to the formation of a clinically detectable tumor. Early detection reagents capture trace amounts of tumor-related biomarkers (such as circulating tumor DNA, microRNA, and tumor-related metabolites) in blood and body fluids, enabling the detection of abnormalities in the preclinical stage before the tumor has formed a clear solid lesion and has no clinical symptoms. This significantly advances the detection window and fundamentally changes the passive situation of "late-stage detection."

[0011] Second, it reduces the difficulty of treatment and increases the chances of a cure.

[0012] Clinical studies have shown that early-stage cancers are far less difficult to treat than those in the middle and late stages, and have a much higher cure rate. For example, the 5-year survival rate for early-stage cervical cancer can reach over 90%, while the 5-year survival rate for late-stage cervical cancer is less than 30%. With standardized treatment, the 5-year survival rate for early-stage breast cancer can reach approximately 95%, while the 5-year survival rate for late-stage breast cancer is only 20%-40%. Early detection reagents help patients discover lesions in the early stages of cancer, before the tumor cells have spread. At this stage, simple methods such as surgery, minimally invasive treatment, and traditional Chinese medicine can achieve reversal or radical cure, greatly reducing the difficulty of treatment and the physical and mental suffering of patients.

[0013] Third, reduce the burden of medical care and conserve social resources.

[0014] Treatment of mid-to-late stage cancer often requires a combination of methods, including surgery, chemotherapy, radiotherapy, and targeted therapy. This process is lengthy and expensive, placing a heavy financial burden on patients' families and consuming significant medical resources. Early detection reagents promote widespread cancer screening, enabling more patients to be cured at an early stage. This can significantly reduce the need for treatment of mid-to-late stage cancer, lower overall healthcare expenditures, and conserve valuable medical resources for society.

[0015] Fourth, we will promote the transformation of cancer prevention and control models from "treatment-oriented" to "prevention-oriented".

[0016] Traditional cancer prevention and control focuses on "disease treatment," while the application of early detection reagents can shift the focus of cancer prevention and control to "prevention and early intervention." By proactively screening and identifying high-risk groups and early-stage patients, it can achieve "early detection, early intervention, and early treatment (self-healing)," fundamentally improving the overall effectiveness of cancer prevention and control. This is a key link in building a cancer prevention and control system within the "Healthy China" strategy.

[0017] In conclusion, given the current reality that clinical tumor detection methods are "not early enough," early cancer detection reagents are not only an important supplement and breakthrough to existing detection technologies, but also a core tool for transforming cancer from "incurable" to "preventable and treatable," playing an irreplaceable role in safeguarding human life and health. Therefore, the research, development, and promotion of early cancer detection reagents are extremely urgent and necessary. Summary of the Invention

[0018] Therefore, the technical problem to be solved by the present invention is to overcome the "early detection" dilemma and "detection at an advanced stage" defects of existing clinical tumor detection methods, thereby providing an early cancer detection reagent.

[0019] Therefore, the present invention provides the following technical solution: This invention provides an early cancer detection reagent, which includes a pretreatment reagent and a FeCl3 colorimetric reagent; the pretreatment reagent includes trichloroacetic acid and citric acid.

[0020] To address the core pain point of cancer treatment—"late-stage detection"—and to overcome existing technological bottlenecks, this invention aims to achieve a universal screening method for cancer prevention and to develop an early cancer detection reagent. This invention's early cancer detection reagent is a qualitative, broad-spectrum detection reagent (results are expressed as presence / absence of cancer). The reagent specifically detects urinary p-hydroxybenzene-R metabolites using the ferric chloride (FeCl3) method. The overall design concept of this invention's early cancer detection reagent is as follows: Ammonia (NH3) or ammonium salts (NH4) in urine + ) will be with Fe 3+ The reaction produces a reddish-brown Fe(OH)3 precipitate (especially under neutral / alkaline conditions), directly masking the reaction between the p-hydroxybenzene-R metabolite and Fe. 3+ The blue-purple color development requires the exclusion of interference from phenolic drugs and proteins. Therefore, a two-step reagent method is designed, as follows: (1) Pretreatment reagents: First, adjust the pH of the urine, precipitate urine proteins and fix ammonia to eliminate interference and obtain pretreated urine; (2) Colorimetric reagent: FeCl3 is used to react with pretreated urine to specifically detect p-hydroxybenzene-R metabolites.

[0021] In existing technologies, urine is typically tested directly without pretreatment. However, the reagents in this invention pre-treat the urine. In the pretreatment reagents, TCA is used to precipitate proteins, and citric acid acts as a pH buffer and fixes ammonia. Pretreatment with trichloroacetic acid and citric acid eliminates ammonia, proteins, and adjusts the pH to create a solution system that stabilizes subsequent iron ions and facilitates the formation of complexes for the color reaction. Eliminating ammonia, proteins, and adjusting the pH also reduces interference with the detection of phenolic compounds by ferric chloride, improving the accuracy of the results. Using other reagents to pre-treat urine introduces ions and pH values ​​unsuitable for the chemical reaction system of the reagents in this application, failing to yield the desired results. For example, carboxylic acids of organic acids, such as oxalic acid and tartaric acid, can combine with ammonia to form ammonium salts (eliminating ammonia) and can also break protein peptide bonds through acid hydrolysis (removing proteins; their acidic properties can precisely adjust the pH of the solution to 5-6). However, oxalate ions preferentially combine with ferric chloride ions to form stable complexes, rather than the characteristic reaction products of ferric chloride and the target substance, directly blocking characteristic reactions such as color development and precipitation, leading to detection failure.

[0022] In colorimetric reagents, its core function is to provide free Fe. 3+ It forms a blue-violet coordination compound with the phenolic hydroxyl group of the p-hydroxybenzene-R metabolite.

[0023] Further, the concentration of trichloroacetic acid in the pretreatment reagent is 7 W / V%-10 W / V%, more preferably 10 W / V; the molar concentration of citric acid in the pretreatment reagent is 0.07-0.1 mol / L, more preferably 0.1 mol / L; the concentration of FeCl3 in the FeCl3 colorimetric reagent is 2 W / V%-5 W / V%, and the pH is 1-2; the amount of disodium EDTA solution added per 100 ml of FeCl3 colorimetric reagent is 0.05 ml-0.1 ml. A FeCl3 concentration of 2 W / V%-5 W / V% provides stable colorimetric results. Dilute hydrochloric acid (e.g., 1 mol / L, analytical grade) is used to adjust the pH to 1-2 to inhibit FeCl3 hydrolysis and precipitate formation, without affecting detection. Disodium EDTA (0.01 W / V%-0.05 W / V% disodium ethylenediaminetetraacetate, analytical grade): prevents Fe... 3+ Oxidation or reaction with impurities further improves reagent stability.

[0024] Furthermore, the target of the early cancer detection reagent includes p-hydroxyphenyl-R metabolites in urine; preferably, the hydroxyphenyl-R metabolites include the sum of p-hydroxyphenylalanine and other hydroxyphenyl-R metabolites, that is, the total amount of hydroxyphenyl-R metabolites in urine.

[0025] Compared to detecting p-hydroxyphenylalanine alone, the present invention's detection of p-hydroxyphenyl-R metabolites in urine has significant advantages in cancer detection, such as accuracy and specificity. This is mainly because different types of cancer produce different types of hydroxyphenyl-R metabolites. As a broad-spectrum qualitative detection reagent, it should be able to detect all types of cancer, indicating their presence or absence. Therefore, the reagent of this application will not miss any cases, whereas detecting p-hydroxyphenylalanine alone would lead to serious false negatives.

[0026] Although the target of the early cancer detection reagent of the present invention is not tumor markers, it is the metabolites produced when all cancers occur. Therefore, the early cancer detection reagent of the present invention is a broad-spectrum early cancer detection reagent, which is effective for the detection of all cancers.

[0027] The prior art CN 108152279 A discloses a p-hydroxyphenylalanine detection reagent and preparation method, which is composed of sodium carbonate, sodium hypochlorite, naphthol, and azobisisobutyronitrile. The concentration of naphthol in the p-hydroxyphenylalanine detection reagent is 0.45-2.17 g / L, the concentration of hypochlorous acid is 2.73 g-6.52 g / L, the concentration of sodium carbonate is 0.91-2.17 g / L, and the concentration of azobisisobutyronitrile is 3.64-15.2 mg / L. This reagent is used to qualitatively detect p-hydroxyphenylalanine in urine to determine the presence of malignant tumors. The reagents in the existing technology CN 108152279 A cannot detect p-hydroxyphenylalanine. This reagent combination lacks a specific reaction system for detecting p-hydroxyphenylalanine and also suffers from mutual interference between components and mismatched reaction conditions, as follows: 1. In the reagent combination: sodium carbonate is only an alkaline regulator, sodium hypochlorite is a strong oxidant, naphthol, although a phenol, has no coupling activity, and azobisisobutyronitrile is a free radical initiator. None of these can react with the phenolic hydroxyl group, amino group, etc. of hydroxyphenylalanine to produce characteristic reactions (such as color development and precipitation).

[0028] 2. Severe interference between components: Sodium hypochlorite's strong oxidizing properties can directly oxidize the phenolic hydroxyl and amino groups of p-hydroxyphenylalanine, destroying the target compound's structure; it can also oxidize naphthol, causing it to deteriorate, and may react with sodium carbonate to release carbon dioxide, leading to system instability.

[0029] 3. The reaction conditions are completely mismatched: Azobisisobutyronitrile requires high temperature (60-80℃) to decompose and generate free radicals, while the phenolic hydroxyl and amino groups of p-hydroxyphenylalanine are easily degraded under high temperature and strong oxidizing environment. Moreover, this combination cannot provide the mild reaction environment required for detection, such as suitable pH and no strong oxidizing / reducing properties.

[0030] This invention provides a method for preparing the early cancer detection reagent described in the above technical solution, comprising the following steps: The preparation of the pretreatment reagent includes: weighing trichloroacetic acid and citric acid and dissolving them in an aqueous solution to obtain the pretreatment reagent; The preparation of the FeCl3 colorimetric reagent includes: adding dilute hydrochloric acid and disodium EDTA solution to an aqueous solution of FeCl3 to obtain the FeCl3 colorimetric reagent; the pH of the aqueous solution of FeCl3 after adding dilute hydrochloric acid is 1-2; The pretreatment reagent and the FeCl3 colorimetric reagent are packaged separately.

[0031] Further, the concentration of the disodium EDTA solution is 0.01 W / V%-0.05 W / V; 0.05 ml-0.1 ml of disodium EDTA solution is added to every 100 mL of FeCl3 colorimetric reagent, and the concentration of the added disodium EDTA solution is 0.01 W / V%-0.05 W / V, more preferably 0.02 W / V.

[0032] This invention provides a cancer early detection kit, the active ingredient of which includes a cancer early detection reagent; the cancer early detection reagent is the cancer early detection reagent described in the above technical solution or the cancer early detection reagent prepared by the preparation method described in the above technical solution.

[0033] The present invention provides the application of the early cancer detection reagent described in the above technical solution or the early cancer detection reagent prepared by the preparation method described in the above technical solution or the early cancer detection kit described in the above technical solution in cancer screening; optionally, the sample to be tested includes urine.

[0034] The cancer early detection reagent of this invention is a broad-spectrum detection reagent, theoretically capable of detecting all types of cancer, including but not limited to lung cancer, gastric cardia cancer, colorectal cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, and urinary system cancers. However, in practice, it has been found to be more sensitive to digestive system cancers. Furthermore, the digestive system cancers described in this invention include one or more of the following: gastric cancer, cardia cancer, colorectal cancer, liver cancer, pancreatic cancer, kidney cancer, and bladder cancer.

[0035] This invention provides a method for using an early cancer detection reagent, wherein the early cancer detection reagent is the early cancer detection reagent described in the above technical solution or the early cancer detection reagent prepared by the preparation method described in the above technical solution or the early cancer detection kit described in the above technical solution, comprising: mixing fresh midstream urine with the pretreatment reagent in the first stage, allowing it to stand in the first stage and filtering it to obtain a filtrate; adding the FeCl3 colorimetric reagent to the filtrate in the second stage, allowing it to stand in the second stage, and observing the color change of the solution; If the solution appears blue-purple, light purple, or light green, it indicates the presence of p-hydroxybenzene-R metabolites in the urine, suggesting the growth of cancer cells in the subject's body. The presence of cancer cells in the subject's body includes both preclinical cancer and cancer patients. Preclinical cancer refers to the early stage of cancer development, in its latent period, undetectable clinically, and without typical clinical symptoms; from a clinical perspective, it is considered a disease-free state. Cancer patients are in the clinical stage, meaning they exhibit typical clinical symptoms of cancer upon clinical examination.

[0036] If the solution retains the color of the pretreated filtrate as pale yellow or colorless, without the appearance of blue-purple or green, it is determined that the urine does not contain p-hydroxybenzene-R metabolites and that no cancer cells are growing in the subject's body.

[0037] Only when cancer cells grow and metabolize do they trigger a stress response in the body, leading to increased metabolism of p-hydroxyphenyl-R and a significant increase in p-hydroxyphenyl-R levels in urine. Conversely, when cancer cells are in a dormant or quiescent state, p-hydroxyphenyl-R levels do not rise.

[0038] Further, the first mixing time is 1-5 min, more preferably 1 min, and the first settling time is 10-15 min; the second mixing time is 0.5-1 min, and the second settling time is 0.5-1 min.

[0039] Furthermore, the volume ratio of the fresh midstream urine to the pretreatment reagent is 3-6:1-3, more preferably 5:2; 0.15ml-0.25ml of FeCl3 colorimetric reagent is added per milliliter of filtrate.

[0040] The technical solution of this invention has the following advantages: 1. The early cancer detection reagent provided by this invention employs a two-step design: "pretreatment reagent + FeCl3 colorimetric reagent." The pretreatment reagent can adjust the pH to Fe... 3+ The optimal range for reaction with p-hydroxybenzene-R metabolites (pH 5-6) is used to fix ammonia (NH3+H) in an acidic environment. + →NH4 + Avoid contact with Fe 3+ (Reaction); it can also precipitate proteins in urine, effectively eliminating the core interference of ammonia and proteins in urine; at the same time, it can use FeCl3 to achieve specific detection of hydroxybenzene-R metabolites, which is simple to operate, reports results quickly, and is suitable for laboratory, physical examination screening and home self-testing.

[0041] 2. The preparation method of the early cancer detection reagent provided by the present invention is simple and low in cost, and the application effect and accuracy of the early cancer detection reagent are good.

[0042] 3. The early cancer detection kit provided by this invention has low cost and good application effect.

[0043] In summary, in the face of the current predicament that clinical tumor detection methods are "not early enough", the early cancer detection reagent provided by this invention is not only an important supplement and breakthrough to existing detection technologies, but also a core tool for realizing the transformation of cancer from "difficult to cure" to "preventable and treatable", which has irreplaceable significance for safeguarding human life and health. Attached Figure Description

[0044] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0045] Figure 1 This is a flowchart of a detection operation step according to an embodiment 6 of the present invention. Detailed Implementation

[0046] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0047] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0048] Example 1: Reagent Development 1. Research has found immunosuppression in preclinical cancer. The clinically detectable threshold number of tumors depends primarily on the sensitivity of the detection method and the tumor type, and typically corresponds to a tumor cell count of 10-1. 6 -10 9 When establishing animal tumor models, the routine number of tumors inoculated is 10; 4 -10 7 Between individual cells / animals.

[0049] To investigate the immune status of preclinical cancer, the number of tumor cells inoculated when establishing a tumor model in ordinary mice was 10. 4 -10 6 In the experimental group, mice were subcutaneously inoculated with sarcoma S180, and tumors formed 7 days after inoculation; mice in the control group were subcutaneously injected with physiological saline. The specific experimental method was as follows: Experimental group: 30 adult mice with an average weight of 20g. Method for constructing the S180 sarcoma tumor model: (1) Preparation of tumor source: Select S180 cells in the logarithmic growth phase, dilute with physiological saline to obtain a concentration of 10 6 (2) Inoculation procedure: Use a needle to separate a tiny cavity under the forelimb of each mouse and inject 0.2 mL of cell suspension. Press the needle hole to prevent leakage.

[0050] Control group: 30 adult mice with an average weight of 20g. Construction method: (1) Prepare physiological saline: 0.9% sodium chloride aqueous solution; (2) Injection operation: use a needle to separate a small cavity under the forelimb armpit of the mouse, inject 0.2mL of physiological saline, and press the needle hole to prevent leakage.

[0051] Eighteen hours after inoculation with sarcoma S180, an inflammation test was performed on the base of the rat's tail using inactivated Staphylococcus aureus. The method was as follows: 0.05 mL of inactivated Staphylococcus aureus suspension was subcutaneously injected into the base of the rat's tail. Observation was performed 12 hours after the injection.

[0052] The results showed that, compared with the control group, no purulent inflammation occurred at the base of the tail in the experimental group mice, indicating innate immunosuppression (such as neutrophil and macrophage suppression). The experimental group mice exhibited depressive symptoms such as low mood and reduced activity. In contrast, all mice in the control group developed purulent inflammation at the base of their tails, indicating normal innate immunity, and their activity and mood were normal.

[0053] Conclusions and Discussion: (1) Preclinical cancer patients, i.e. experimental tumor-bearing mice, entered a stress response, with increased and disordered secretion of adrenaline and noradrenaline, which suppressed innate immunity. It was inferred that the metabolism of their precursor substance tyrosine was enhanced, and the urine contained a large amount of adrenaline and noradrenaline as well as the metabolic intermediate of tyrosine (p-hydroxyphenylpyruvic acid), all of which have a common "p-hydroxyphenyl" structural fragment.

[0054] (2) Reasoning for depression: Depression is caused by the disorder of the neurotransmitter hormone system, such as adrenaline and noradrenaline, combined with the disorder of the serotonin system. The disorder of serotonin leads to its increased metabolism, and the metabolite in urine is 5-hydroxyindoleacetic acid.

[0055] 2. Verify that the same metabolic abnormality exists in the urine of human cancer patients, namely, high levels of metabolites containing the "p-hydroxybenzene" structural fragment, abbreviated as: p-hydroxybenzene-R metabolite.

[0056] The specific experimental method is as follows: Experimental group: 100 cancer patients, including 26 lung cancer patients, 18 digestive system tumors such as gastric cancer, 9 cardia cancer, 23 colorectal cancer, 8 liver cancer, 5 pancreatic cancer, 8 kidney cancer, and 3 bladder cancer patients. The cancer patients were provided by Jiangxi Provincial Cancer Hospital.

[0057] Control group: 100 healthy individuals.

[0058] There were no significant differences between patients and healthy individuals in terms of age, gender, and occupation.

[0059] This experiment used reverse high performance liquid chromatography (RP-HPLC) to detect the content of p-hydroxybenzene-R metabolites in the urine of cancer patients and healthy individuals by fluorescence detection (FLD).

[0060] Test results: Analysis of urine p-hydroxyphenyl-R metabolite data from 100 healthy individuals showed that the 95th percentile (P95) was 0.55 mmol / L, meaning that 95% of healthy individuals had p-hydroxyphenyl-R metabolite levels ≤0.55 mmol / L in their urine. In contrast, cancer patients had p-hydroxyphenyl-R metabolite levels greater than 0.82 mmol / L in their urine, indicating that cancer patients have high levels of p-hydroxyphenyl-R metabolite in their urine.

[0061] Analysis of significant differences between the cancer group and the healthy group: 1. Premise Assumptions Data distribution: The metabolite contents of both groups follow a normal distribution, and the variances of the two groups are homogeneous. Null hypothesis (H0): There is no difference in metabolite levels between cancer patients and healthy individuals (μ1=μ2); Alternative hypothesis (H1): The levels of metabolites in cancer patients are significantly higher than those in healthy individuals (μ1>μ2), and a one-sided test is used (since the levels in cancer patients are known to be >0.82). 2. Choose appropriate statistical methods Based on the characteristics of "two independent samples and continuous quantitative data (metabolite content is a continuous value)," the independent samples t-test is the preferred choice. 3. Specific statistical analysis steps (using independent samples t-test) (1) Data (healthy group and cancer patient group) Healthy individuals: Given P95 = 0.55 mmol / L, assume its concentration follows a normal distribution N(μ0, σ0). 2 The mean (μ0) and standard deviation (σ0) can be estimated in the following ways: In a normal distribution, P95 corresponds to "mean + 1.645 × standard deviation" (the upper limit of the 95th percentile for one-sided distributions), that is: μ0 + 1.645 × σ0 = 0.55 Based on the physiological rationality of metabolite content in healthy individuals (mean μ0 = 0.35 mmol / L), we can calculate σ0≈(0.55-0.35) / 1.645≈0.1216 mmol / L.

[0062] Cancer patient group: It is known that the content of all patients is >0.82mmol / L, and the measured mean μ1=1.0mmol / L, σ1=0.15mmol / L.

[0063] (2) Calculate the t-value (independent samples t-test formula) The calculation yields: t ≈ 24.07; (3) Determine the P-value and judge the result. Degrees of freedom (df): df=128; Referring to the t-critical value table: In a one-tailed test, when df=128, t0. 05 (128)≈1.657 (i.e., the critical t value corresponding to P=0.05); Result judgment: t=24.07>t0. 05 (128) = 1.657, therefore P <0.05, reject H0, accept H1, proving that the metabolite content in cancer patients is significantly higher than that in healthy individuals.

[0064] Example 2 This embodiment provides an early cancer detection reagent, which consists of an individually packaged pretreatment reagent and a FeCl3 colorimetric reagent.

[0065] Pretreatment reagents: Trichloroacetic acid concentration is 10 W / V%, citric acid molar concentration is 0.1 mol / L, and the remainder is water. Preparation method for 100 mL of pretreatment reagents: 1) Weigh 10g of trichloroacetic acid (TCA, analytical grade) and 2.1g of citric acid (analytical grade) and put them into a 100mL beaker; 2) Add about 50 mL of deionized water and stir with a glass rod until completely dissolved (the dissolution of trichloroacetic acid releases a little heat, which is normal); 3) Transfer the solution to a 100mL volumetric flask, add deionized water to the mark, and shake well; 4) Dispense into reagent bottles, label them "Urine Pretreatment Reagent (containing 10W / V% TCA + 0.1mol / L Citric Acid)", and store at room temperature.

[0066] FeCl3 colorimetric reagent: The concentration of FeCl3 is 2 w / v, the concentration of the added disodium EDTA solution is 0.02 w / v, the pH is 1-2, and the remainder is water. Preparation method, taking 100 mL as an example: 1) Weigh 2g of anhydrous FeCl3 (analytical grade), put it into a 100mL beaker, add about 30mL of deionized water, stir, and let it dissolve initially; 2) Add 1 drop of 1 mol / L dilute hydrochloric acid (analytical grade), and continue stirring until the solution is clear. If it is still turbid, add 1 drop of dilute hydrochloric acid until the solution pH is 1-2. 3) Transfer the solution to a 100mL volumetric flask, add deionized water to bring it to the mark on the volumetric flask, and shake well; 4) Add 1 drop (i.e., 0.05 mL) of EDTA disodium solution with a mass concentration of 0.02 W / V; 5) Dispense the obtained solution into dropper bottles, label them "2W / V% FeCl3 colorimetric reagent", seal them, protect them from light, and store them at room temperature (seal them for 18 months).

[0067] Example 3 This embodiment provides an early cancer detection reagent, which consists of an individually packaged pretreatment reagent and a FeCl3 colorimetric reagent.

[0068] 1. Pretreatment reagents: Trichloroacetic acid concentration is 10 W / V%, citric acid molar concentration is 0.1 mol / L, and the remainder is water. Preparation method, taking 100 mL as an example: 1) Weigh 10g of trichloroacetic acid (TCA, analytical grade) and 2.1g of citric acid (analytical grade) and put them into a 100mL beaker; 2) Add about 50 mL of deionized water and stir with a glass rod until completely dissolved (the dissolution of trichloroacetic acid releases a little heat, which is normal); 3) Transfer the solution to a 100mL volumetric flask, add deionized water to the mark, and shake well; 4) Dispense into reagent bottles, label them "Urine Pretreatment Reagent (containing 10W / V% TCA + 0.1mol / L Citric Acid)", and store at room temperature.

[0069] 2. FeCl3 colorimetric reagent: The concentration of FeCl3 is 3 w / v, the concentration of the added disodium EDTA solution is 0.04 w / v, the pH is 1-2, and the remainder is water. Preparation method, taking 100 mL as an example: 1) Weigh 3g of anhydrous FeCl3 (analytical grade), put it into a 100mL beaker, add about 30mL of deionized water, stir, and let it dissolve initially; 2) Add 1 drop (i.e. 0.05 mL) of 1 mol / L dilute hydrochloric acid (1 mol / L, analytical grade), and continue stirring until the solution is clear. If it is still turbid, add 1 drop of dilute hydrochloric acid until the solution pH is 1-2. 3) Transfer the solution to a 100mL volumetric flask, add deionized water to bring it to the mark on the volumetric flask, and shake well; 4) Add 1 drop (i.e., 0.05 mL) of 0.04 W / V % EDTA disodium solution; 5) Dispense the obtained solution into dropper bottles, label them "3W / V% FeCl3 colorimetric reagent", seal them, protect them from light, and store them at room temperature (seal them for 18 months).

[0070] Example 4 This embodiment provides an early cancer detection reagent, which consists of an individually packaged pretreatment reagent and a FeCl3 colorimetric reagent.

[0071] 1. Pretreatment reagents: Trichloroacetic acid concentration is 10 W / V%, citric acid molar concentration is 0.1 mol / L, and the remainder is water. Preparation method, taking 100 mL as an example: 1) Weigh 10g of trichloroacetic acid (TCA, analytical grade) and 2.1g of citric acid (analytical grade) and put them into a 100mL beaker; 2) Add about 50 mL of deionized water and stir with a glass rod until completely dissolved (the dissolution of trichloroacetic acid releases a little heat, which is normal); 3) Transfer the solution to a 100mL volumetric flask, add deionized water to the mark, and shake well; 4) Dispense into reagent bottles, label them "Urine Pretreatment Reagent (containing 10W / V% TCA + 0.1mol / L Citric Acid)", and store at room temperature.

[0072] 2. FeCl3 colorimetric reagent: The concentration of FeCl3 is 4% (w / v), the concentration of disodium EDTA solution added is 0.03% (w / v), the pH is 1-2, and the remainder is water. Preparation method, taking 100mL as an example: 1) Weigh 4g of anhydrous FeCl3 (analytical grade), put it into a 100mL beaker, add about 30mL of deionized water, stir, and let it dissolve initially; 2) Add 2 drops (i.e. 0.1 mL) of 1 mol / L dilute hydrochloric acid (1 mol / L, analytical grade), and continue stirring until the solution is clear. If it is still turbid, add 1 drop of dilute hydrochloric acid until the solution pH is 1-2. 3) Transfer the solution to a 100mL volumetric flask, add deionized water to bring it to the mark on the volumetric flask, and shake well; 4) Add 1 drop (i.e., 0.05 mL) of EDTA disodium solution with a mass concentration of 0.03 W / V; 5) Dispense the obtained solution into dropper bottles, label them "4W / V% FeCl3 colorimetric reagent", seal them, protect them from light, and store them at room temperature (seal them for 18 months).

[0073] Example 5 This embodiment provides a method for using the detection reagent of Example 2, the steps of which are as follows: 1. Urine pretreatment (eliminating interference) Take 5 mL of fresh midstream urine and add it to a test tube; Add 2 mL of the pretreatment reagent from the early cancer detection kit to the test tube, gently shake for 1 minute, and let stand for 10 minutes (during which time the protein will precipitate and the ammonia will be fixed as NH4). + ); After standing for 10 minutes, filter the solution using a small funnel filter paper to obtain a filtrate (approximately 2 mL, which has been free of ammonia and protein interference, with a pH of approximately 5-6), which will be used for subsequent colorimetric detection.

[0074] 2. Colorimetric detection of p-hydroxybenzene-R metabolites Take 1 mL of the above filtrate and transfer it to a new test tube; Add 5 drops (0.25 ml) of FeCl3 colorimetric reagent using a dropper, shake to mix for 0.5 min, and let stand for 1 min; Observe the color change and judge the result.

[0075] 3. Result Judgment and Key Explanations (1) Result judgment If the solution turns blue-purple, light purple, or light green (the color varies depending on the concentration, and all are specific reactions), it indicates that the urine contains the p-hydroxybenzene-R metabolite, which is positive. This means that the subject has cancer cells growing in their body, i.e., they are preclinical cancer and / or cancer patients. If the solution retains the color of the pretreated filtrate as pale yellow or colorless, without the appearance of blue-purple, pale purple, or light green, then the urine is considered negative as it does not contain p-hydroxybenzene-R metabolites, and no cancer cells are growing in the subject's body.

[0076] (2) Key Explanation If you have taken phenolic medications (such as acetaminophen) before the test, you need to stop taking the medication for 24 hours before the test, because exogenous phenols in urine may still interfere, and the pretreatment reagent cannot completely eliminate them.

[0077] The cancer early detection reagent of this invention adopts a two-step design of "pretreatment reagent + FeCl3 colorimetric reagent", which can effectively eliminate the core interference of ammonia and protein in urine. At the same time, it uses FeCl3 to achieve specific detection of hydroxybenzene-R metabolites. It is simple to operate, reports results quickly, and can be packaged into a kit, which is suitable for laboratory, physical examination screening and home self-testing.

[0078] Example 6 This embodiment provides a method for using the detection reagent of Example 3, the steps of which are as follows: 1. Urine pretreatment (eliminating interference) Take 5 mL of fresh midstream urine and add it to a test tube; Add 2 mL of the pretreatment reagent from the early cancer detection kit to the test tube, gently shake for 1 minute, and let stand for 10 minutes (during which time the protein will precipitate and the ammonia will be fixed as NH4). + ); After standing for 10 minutes, filter the solution using a small funnel filter paper to obtain a filtrate (approximately 2 mL, which has been free of ammonia and protein interference, with a pH of approximately 5-6), which will be used for subsequent colorimetric detection.

[0079] 2. Colorimetric detection of p-hydroxybenzene-R metabolites Take 1 mL of the above filtrate and transfer it to a new test tube; Add 3 drops (0.15 ml) of FeCl3 colorimetric reagent using a dropper, shake to mix for 0.5 min, and let stand for 0.5 min. Observe the color change and judge the result; if the solution turns blue-purple, light purple or light green (the color varies with different concentrations, all of which are specific reactions), then the urine contains the p-hydroxybenzene-R metabolite, which is a positive result; If the solution maintains the pretreated filtrate's pale yellow or colorless color, without any blue-purple, pale purple, or light green hues, then the urine is considered negative as it does not contain the p-hydroxybenzene-R metabolite. See the flowchart below. Figure 1 .

[0080] Example 7 This embodiment provides a method for using the detection reagent of Example 4, the steps of which are as follows: 1. Urine pretreatment (eliminating interference) Take 5 mL of fresh midstream urine and add it to a test tube; Add 2 mL of the pretreatment reagent from the early cancer detection kit to the test tube, gently shake for 0.5 min, and let stand for 10 min (at this time, the protein will precipitate, and the ammonia will be fixed as NH4). + ); After standing for 10 minutes, filter the solution using a small funnel filter paper to obtain a filtrate (approximately 2 mL, which has been free of ammonia and protein interference, with a pH of approximately 5-6), which will be used for subsequent colorimetric detection.

[0081] 2. Colorimetric detection of p-hydroxybenzene-R metabolites Take 1 mL of the above filtrate and transfer it to a new test tube; Add 3 drops (0.15 mL) of FeCl3 colorimetric reagent using a dropper, shake to mix for 0.5 min, and let stand for 1 min. Observe the color change and judge the result; if the solution turns blue-purple, light purple or light green (the color varies with different concentrations, all of which are specific reactions), then the urine contains the p-hydroxybenzene-R metabolite, which is a positive result; If the solution retains the color of the pretreated filtrate as pale yellow or colorless, without the appearance of blue-purple, pale purple, or light green, then the urine is considered negative as it does not contain the p-hydroxybenzene-R metabolite.

[0082] Example 8: Prospective Study of Early Cancer Detection Reagents (July 2005 - July 2014) In July 2005, 11,753 individuals aged 25-65 years from 10 randomly selected counties and cities in Jiangxi Province, including civil servants, teachers, enterprise employees, and farmers, had their urine collected for testing. The complete reagent preparation method and the complete testing procedure for the early cancer detection reagent were the same as in Example 6. The reagent detected 171 positive cases, with a positive rate of 1.45%. The early cancer detection reagent of this invention has a set sensitivity, i.e., a minimum detection limit (0.83 mmol / L); values ​​exceeding this detection limit are considered positive.

[0083] Every July, positive cases are retested using early cancer detection reagents, and the number of confirmed cancer cases is counted. The cancer incidence prediction rate over the 9-year period is 85.4% (146 / 171=85.4%, this is only the statistical data for 9 years, and the prediction rate will increase if the process continues). The detailed test results are shown in Table 1 below. It can be seen that the early cancer detection reagent of this invention can detect preclinical cancer with high accuracy.

[0084] Table 1 Test Results

[0085] Note: The number of confirmed cases and the number of people who tested negative each year from 2007 to 2014 will be dynamically reduced. The total number is 171. For example, the number in 2007 = 137 + 15 + 4 + 13 + 2 = 171.

[0086] Example 9: Application of Early Cancer Detection Reagent This example includes 200 cancer patients (including lung cancer, digestive system cancer, urinary system cancer, leukemia, and lymphoma, provided by the Department of Oncology, Second Affiliated Hospital of Nanjing Medical University) and 100 healthy individuals (provided by the Health Examination Center of Jiangxi University of Traditional Chinese Medicine). There were no significant differences in baseline characteristics such as age and gender between the cancer patients and the healthy individuals.

[0087] The preparation method of the complete reagent and the complete detection procedure of the early cancer detection reagent are the same as in Example 6. The detection results are as follows: Table 2 Detection Results

[0088] Note: Accuracy = Number of samples with disease (cancer) detected / Total number of samples with disease (cancer), calculated based on the number of cancer cases; Specificity = Number of samples without disease (cancer) detected / Total number of samples without disease (cancer), calculated based on the number of healthy individuals.

[0089] The components of the p-hydroxyphenylalanine detection reagent from Jiangxi Gelang Biotechnology Co., Ltd. are: mercury, nitric acid, mercuric sulfate, nickel nitrate, sulfuric acid, and pure water.

[0090] Example 10: Qualitative Cancer Early Screening Reagent ROC Curve Analysis Report The reagent for early cancer detection in this application was used to detect p-hydroxybenzene-R metabolites in urine. The limit of detection (LOD) of the reagent was determined by ROC curve analysis to verify the suitability of the reagent for clinical screening.

[0091] The subjects tested were simulated clinical sample test data, including 100 negative samples and 70 positive gradient samples.

[0092] The 100 negative samples were urine samples from healthy individuals (without a history of cancer, benign tumors, or chronic inflammation), and were confirmed by clinical examination to have a concentration of the target marker hydroxybenzene-R metabolite <0.1 mg / ml (i.e., 0.55 mmol / L). Seventy positive gradient samples: Seven concentration gradients (0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.18 mg / mL, 0.2 mg / mL) were prepared using p-hydroxyphenylalanine standard, with 10 replicates for each gradient, for a total of 70 samples (true state was marked as positive); The early cancer detection reagent prepared in Example 3 was used to test all samples according to the method in Example 6. Positive / negative results were recorded, and the positive rate for each concentration gradient was calculated. The data were processed using Python (matplotlib + scikit-learn). The results are as follows: 1. ROC curve analysis process (1) Data preprocessing Using the sample's "true state" (positive = 1, negative = 0) as the dependent variable and "detection concentration" as the independent variable, we iterated through all concentration gradients as the cutoff values ​​(≥ this concentration is judged as potential positive, < this concentration is judged as potential negative). We calculated the sensitivity (true positive rate, TPR), specificity (true negative rate, TNR), and false positive rate (1-specificity, FPR) corresponding to each cutoff value. (2) Construction of ROC curve ROC curves were plotted with the false positive rate (FPR) on the horizontal axis and the sensitivity (TPR) on the vertical axis; the area under the curve (AUC) was calculated using the trapezoidal method to evaluate the reagent's ability to distinguish between positive and negative samples.

[0093] (3) Calculation of key parameters The core ROC parameters corresponding to each critical concentration are shown in Table 3 below: Table 3. Core ROC parameters corresponding to each critical concentration.

[0094] 2. Analysis Results (1) Core indicators of ROC curve ROC curve area under curve (AUC): 0.916; critical threshold: when the false positive rate is 4% (corresponding to specificity = 96%), the sensitivity is 95%, and the corresponding critical concentration is 0.15 mg / ml (i.e. 0.83 mmol / L).

[0095] (2) Determination of the limit of detection (LOD) 1) Pre-set clinical standards: Cancer screening reagents must meet the following requirements: specificity ≥96% (false positive rate ≤4%). 2) LOD confirmation: Based on ROC curve analysis, when the critical concentration is 0.15 mg / ml (i.e. 0.83 mmol / L), the reagent specificity is 96% (meets the preset standard), the sensitivity is 95%, and the positive rate of samples at this concentration gradient is 95% (close to the industry's ≥95% validation standard). In summary, the limit of detection for early cancer detection reagent of the present invention is 0.15 mg / ml (0.83 mmol / L), sensitivity is 95%, false positive rate is 4%, specificity is ≥96%, and the detection results are accurate and reliable.

[0096] Comparative Example 1 A cancer early detection reagent, consisting of individually packaged pretreatment reagent and FeCl3 colorimetric reagent.

[0097] 1. Pretreatment reagents: 0.63 W / V oxalic acid solution and 0.9 W / V tartaric acid solution. Preparation method, taking 100 mL as an example: To prepare 0.63 W / V oxalic acid, weigh 0.63 g of oxalic acid (analytical grade), place it in a 100 ml beaker, dissolve it in 30 ml of deionized water, then transfer it to a 100 ml volumetric flask and add deionized water to bring the volume to 100 ml.

[0098] To prepare 0.9 W / V tartaric acid, weigh 0.9 g of tartaric acid (analytical grade), place it in a 100 ml beaker, dissolve it in 30 ml of deionized water, then transfer it to a 100 ml volumetric flask and add deionized water to bring the volume to 100 ml.

[0099] 2. FeCl3 colorimetric reagent: The concentration of FeCl3 is 3 W / V, the concentration of the added disodium EDTA solution is 0.04 W / V, the pH is 1-2, and the remainder is water. Preparation method, taking 100 mL as an example: 1) Weigh 3g of anhydrous FeCl3 (analytical grade), put it into a 100mL beaker, add about 30mL of deionized water, stir, and let it dissolve initially; 2) Add 1 drop (i.e. 0.05 mL) of 1 mol / L dilute hydrochloric acid (1 mol / L, analytical grade), and continue stirring until the solution is clear. If it is still turbid, add 1 drop of dilute hydrochloric acid until the solution pH is 1-2. 3) Transfer the solution to a 100mL volumetric flask, add deionized water to bring it to the mark on the volumetric flask, and shake well; 4) Add 1 drop of 0.04 W / V % EDTA disodium solution; 5) Dispense the obtained solution into dropper bottles, label them "3W / V% FeCl3 colorimetric reagent", seal them, protect them from light, and store them at room temperature (seal them for 18 months).

[0100] The detection method and steps are as follows: 1. Urine pretreatment (eliminating interference) Take 5 mL of fresh midstream urine and add it to a test tube; Add 1 mL of oxalic acid solution and 1 mL of tartaric acid solution, totaling 2 mL, to a test tube. Gently shake for 1 min and let stand for 10 min. After standing for 10 minutes, filter the solution using a small funnel filter paper to obtain a filtrate (approximately 2 mL, which has been free of ammonia and protein interference, with a pH of approximately 5-6), which will be used for subsequent colorimetric detection.

[0101] 2. Colorimetric detection of p-hydroxybenzene-R metabolites Take 1 mL of the above filtrate and transfer it to a new test tube; Add 3 drops of FeCl3 colorimetric reagent using a dropper, shake to mix for 0.5 min, and let stand for 0.5 min. No color reaction occurred; the test failed.

[0102] The reasons for the detection failure are analyzed as follows: Carboxylic acids of organic acids, such as oxalic acid and tartaric acid, can combine with ammonia to form ammonium salts (eliminating ammonia) and can also break protein peptide bonds through acid hydrolysis (removing proteins; their acidic properties can precisely adjust the pH of the system to 5-6). However, oxalate ions preferentially combine with ferric chloride ions to form stable complexes, rather than the characteristic reaction products of ferric chloride and the target substance, directly blocking characteristic reactions such as color development and precipitation, leading to detection failure.

[0103] Comparative Example 2 The reagent for detecting p-hydroxyphenylalanine consists of an aqueous solution of sodium carbonate, sodium hypochlorite, naphthol, and azobisisobutyronitrile. The concentration of naphthol is 0.45 g / L, the concentration of hypochlorous acid is 4.55 g / L, the concentration of sodium carbonate is 0.91 g / L, and the concentration of azobisisobutyronitrile is 3.64 mg / L.

[0104] The testing steps are as follows: 1. Place 3 mL of fresh urine in a test tube; 2. Use a dropper to add 1 mL of the p-hydroxyphenylalanine test reagent to the urine test tube, shake the test tube, let it stand, and observe. No color reaction should occur. The test reagent consists of sodium carbonate, sodium hypochlorite, naphthol, and azobisisobutyronitrile. Those skilled in the art are aware that this test is impossible to perform for p-hydroxyphenylalanine; this combination of reagents cannot be used to detect p-hydroxyphenylalanine.

[0105] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A reagent for early cancer detection, characterized in that, The early cancer detection reagent includes a pretreatment reagent and a FeCl3 colorimetric reagent; the pretreatment reagent includes trichloroacetic acid and citric acid.

2. The early cancer detection reagent according to claim 1, characterized in that, The concentration of trichloroacetic acid in the pretreatment reagent is 7 W / V%-10 W / V; the molar concentration of citric acid in the pretreatment reagent is 0.07-0.1 mol / L; the concentration of FeCl3 in the FeCl3 colorimetric reagent is 2 W / V%-5 W / V, and the pH is 1-2.

3. The early cancer detection reagent according to claim 1, characterized in that, The target of the early cancer detection reagent includes p-hydroxyphenyl-R metabolites in urine; preferably, the p-hydroxyphenyl-R metabolites include p-hydroxyphenylpyruvic acid.

4. A method for preparing the early cancer detection reagent according to any one of claims 1-3, characterized in that, Includes the following steps: The preparation of the pretreatment reagent includes: weighing trichloroacetic acid and citric acid and dissolving them in an aqueous solution to obtain the pretreatment reagent; The preparation of the FeCl3 colorimetric reagent includes: adding dilute hydrochloric acid and disodium EDTA solution to an aqueous solution of FeCl3 to obtain the FeCl3 colorimetric reagent; the pH of the aqueous solution of FeCl3 after adding dilute hydrochloric acid is 1-2; The pretreatment reagent and the FeCl3 colorimetric reagent are packaged separately.

5. The preparation method according to claim 4, characterized in that, The amount of EDTA disodium solution added to each 100ml FeCl3 colorimetric reagent is 0.05mL-0.1mL, and the concentration of the added EDTA disodium solution is 0.01 W / V%-0.05W / V.

6. A cancer early detection kit, characterized in that, The active ingredient includes an early cancer detection reagent; the early cancer detection reagent is any one of the early cancer detection reagents described in claims 1-3 or the early cancer detection reagent prepared by the preparation method described in claim 4 or 5.

7. The application of the early cancer detection reagent according to any one of claims 1-3, or the early cancer detection reagent prepared by the preparation method according to claim 4 or 5, or the early cancer detection kit according to claim 6, in cancer screening; optionally, the sample to be tested includes urine.

8. A method of using an early cancer detection reagent, characterized in that, The early cancer detection reagent is any one of the early cancer detection reagents of claims 1-3 or the early cancer detection reagent prepared by the preparation method of claim 4 or 5 or the early cancer detection kit of claim 6, comprising: mixing fresh midstream urine with the pretreatment reagent in a first mixing, a first standing and filtration to obtain a filtrate; adding the FeCl3 colorimetric reagent to the filtrate in a second mixing and a second standing, and observing the color change of the solution. If the solution turns blue-purple, light purple, or light green, it indicates that the urine contains p-hydroxybenzene-R metabolites, and that cancer cells are growing in the subject's body. If the solution retains the color of the pretreated filtrate as pale yellow or colorless, without the appearance of blue-purple or green, it is determined that the urine does not contain p-hydroxybenzene-R metabolites and that no cancer cells are growing in the subject's body.

9. The method of use according to claim 8, characterized in that, The first mixing time is 1-5 min, and the first settling time is 10-15 min; the second mixing time is 0.5-1 min, and the second settling time is 0.5-1 min.

10. The method of use according to claim 8, characterized in that, The volume ratio of the fresh midstream urine to the pretreatment reagent is 3-6:1-3; 0.15-0.25 mL of FeCl3 colorimetric reagent is added per milliliter of filtrate.

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