WO2021019944A1 - 食道癌バイオマーカー及びその用途 - Google Patents

食道癌バイオマーカー及びその用途 Download PDF

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WO2021019944A1
WO2021019944A1 PCT/JP2020/024027 JP2020024027W WO2021019944A1 WO 2021019944 A1 WO2021019944 A1 WO 2021019944A1 JP 2020024027 W JP2020024027 W JP 2020024027W WO 2021019944 A1 WO2021019944 A1 WO 2021019944A1
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mir
esophageal cancer
hsa
urinary
biomarker
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French (fr)
Japanese (ja)
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貴也 志村
悠介 奥田
弘靖 岩崎
洋望 片岡
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Nagoya City University
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Nagoya City University
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Priority to JP2021536655A priority patent/JP7531916B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to an index useful for testing / diagnosing esophageal cancer and its use. Specifically, the present invention relates to an esophageal cancer biomarker and a test method using the biomarker.
  • esophageal cancer diagnosis is upper gastrointestinal endoscopy and pathological diagnosis using biopsy tissue, but it is extremely advanced by endoscopy at the time of onset of symptoms because symptoms do not occur unless it progresses and causes passage obstruction. It is found in a state of illness and has a high risk of metastasis. Therefore, esophageal cancer is one of the diseases with a poor prognosis.
  • upper gastrointestinal angiography performed in gastric cancer screening only one or two pictures of the esophagus are usually taken, and it is difficult to detect unless the tumor is in the advanced stage that causes passage obstruction.
  • Serum markers such as SCC, CYFRA21-1, and anti-p53 antibody are used in blood tests, but it is hard to say that the sensitivity and specificity are sufficient.
  • biomarkers specific to esophageal cancer for example, Patent Documents 1 to 3
  • Patent Documents 1 to 3 there are no examples of clinical application.
  • Non-Patent Document 1 it has been reported that a combination of specific microRNAs in serum is useful for diagnosing esophageal cancer.
  • an object of the present invention is to provide a highly practical biomarker specific to esophageal cancer and minimally invasive.
  • the present inventors have proceeded with research aiming at the creation of biomarkers specific to esophageal cancer, with an emphasis on enabling minimally invasive and simple examinations. Specifically, we attempted to identify biomarkers specific to esophageal cancer from urine. As a result of detailed and comprehensive analysis using more than 350 samples, it was found that specific microRNAs enable the determination of esophageal cancer with high specificity and sensitivity. That is, we succeeded in identifying a biomarker with excellent diagnostic ability for esophageal cancer. Importantly, it was suggested that each of the identified microRNAs alone would be useful in diagnosing esophageal cancer.
  • microRNAs are useful for the detection and diagnosis of early stage (stage I) esophageal cancer.
  • stage I early stage esophageal cancer.
  • four of the five microRNAs identified (urinary hsa-miR-619-5p, urinary hsa-miR-1273f, urinary hsa-miR-4327 and urinary hsa-miR) -150-3p) is highly useful for early detection and diagnosis of esophageal cancer, and the remaining one (urinary hsa-miR-3135b) is also highly useful for early detection and diagnosis of esophageal cancer. It was suggested that it could be used for.
  • a method for testing early-stage esophageal cancer which comprises using the expression level of the esophageal cancer biomarker according to [3] or [4] as an index.
  • step (1) the expression level of the esophageal cancer biomarker is determined by standardization using hsa-miR-4669 and hsa-miR-6756-5p as standardizing factors, according to [6] or [7].
  • the method described. [9] The item according to any one of [6] to [8], wherein the esophageal cancer biomarker detected in step (1) is urinary hsa-miR-619-5p or urinary hsa-miR-1273f. Method. [10] The method according to any one of [6] to [9], wherein the esophageal cancer biomarker is detected by the qRT-PCR method.
  • An esophageal cancer test kit containing the reagent for detecting the esophageal cancer biomarker according to [1] and an instruction manual.
  • An esophageal cancer test kit containing the reagent for detecting the esophageal cancer biomarker according to [3] or [4] and an instruction manual.
  • the reagents are urinary hsa-miR-619-5p detection reagent, urinary hsa-miR-1273f detection reagent, urinary hsa-miR-3135b detection reagent, and urinary hsa-miR-4327 detection.
  • the kit for esophageal cancer test according to [11] or [12], which is a reagent for use and / or a reagent for detecting hsa-miR-150-3p in urine. [14] Further comprising one or more elements selected from the group consisting of reagents for microRNA extraction, standardizing factor-specific primers, DNA polymerase, reverse transcriptase, dNTPs, urine sample collection vessels, reactors and detectors [14].
  • the kit for detecting esophageal cancer according to any one of [13].
  • Het-1A Human esophageal normal squamous cell carcinoma cell line
  • TE-1 Human esophageal squamous cell carcinoma cell line
  • T.T Human esophageal squamous cell carcinoma cell line
  • KYSE-30 Human esophageal squamous cell carcinoma cell line.
  • the first aspect of the present invention relates to an esophageal cancer biomarker.
  • esophageal cancer biomarker refers to a biomolecule that is an indicator of esophageal cancer morbidity.
  • the biomarker of the present invention is useful for detecting esophageal cancer (understanding that the patient has esophageal cancer), particularly for early detection of esophageal cancer.
  • the esophagus is a tubular organ that connects the pharynx and stomach, and is roughly divided into the cervical esophagus, thoracic esophagus, and abdominal esophagus.
  • the wall of the esophagus is composed of mucosa (mucosal epithelium, lamina intestinal, muscularis mucosae), submucosa, lamina intestinal and outer membrane.
  • mucosa mucosal epithelium, lamina intestinal, muscularis mucosae
  • esophageal cancer is often found near the center of the esophagus. Esophageal cancer is characterized by easy lymph node metastasis, and when lymph node metastasis occurs, metastasis is often found in a wide range.
  • Esophageal cancer is subdivided according to histological type, stage, etc. Most early-stage esophageal cancers have no subjective symptoms, and it is difficult to detect esophageal cancer at an early stage.
  • blood test, esophageal contrast agent test, endoscopy, CT test, MRI test, PET test, ultrasonography, etc. are performed.
  • the "biomolecule” means a molecule (compound) found in a living body.
  • microRNA which is a biomolecule
  • the molecule in a sample (sample) separated from the living body is used.
  • Urine is used as the sample. Therefore, specific microRNAs in urine are biomarkers of the present invention.
  • MicroRNA is a small RNA of about 22 bases existing in the living body. MicroRNA suppresses protein production by binding to a target gene having a partially complementary sequence in the 3'untranslated region (3'UTR) and suppressing mRNA destabilization and translation. This microRNA function constitutes part of an important mechanism for post-transcriptional expression regulation of genes.
  • the biomarker of the present invention consists of specific microRNAs, namely hsa-miR-619-5p, hsa-miR-1273f, hsa-miR-3135b, hsa-miR-4327 or hsa-miR-150-3p.
  • hsa-miR-619-5p and hsa-miR-1273f are highly useful in that they bring about higher diagnostic accuracy.
  • the biomarkers of the present invention are also useful for the detection of early stage esophageal cancer, and in particular, the usefulness of hsa-miR-619-5p, hsa-miR-1273f, hsa-miR-4327 and hsa-miR-150-3p. Is expensive. Also in this case, hsa-miR-619-5p and hsa-miR-1273f are highly useful.
  • the stages of esophageal cancer are broadly divided into stages I, II, III and IV. Unless otherwise specified, "early esophageal cancer" in the present invention is stage I (stage I) esophageal cancer.
  • hsa-miR-619-5p, hsa-miR-1273f, hsa-miR-3135b, hsa-miR-4327 and hsa-miR-150-3p are microRNAs consisting of the following sequences, respectively.
  • hsa-miR-619-5p GCUGGGAUUACAGGCAUGAGCC (SEQ ID NO: 1) hsa-miR-1273f: GGAGAUGGAGGUUGCAGUG (SEQ ID NO: 2) hsa-miR-3135b: GGCUGGAGCGAGUGCAGUGGUG (SEQ ID NO: 3) hsa-miR-4327: GGCUUGCAUGGGGGGACUGG (SEQ ID NO: 4) hsa-miR-150-3p: CUGGUACAGGCCUGGGGGACAG (SEQ ID NO: 5)
  • hsa- may be omitted from the names of microRNAs in the present specification.
  • the second aspect of the present invention provides an esophageal cancer examination method (hereinafter, also referred to as “the examination method of the present invention”) with respect to the use of the biomarker of the present invention.
  • the test method of the present invention is useful as a means for easily detecting esophageal cancer. According to the test method of the present invention, an objective basis that enables determination of whether or not a person has esophageal cancer (presence or absence of esophageal cancer) is provided.
  • test method of the present invention is useful for detecting early-stage esophageal cancer, and in the case of this aspect, an objective basis useful for determining whether or not the patient has early-stage esophageal cancer can be obtained.
  • the information (test result) provided by the test method of the present invention is based on an objective index called a biomarker (biomolecule) that reflects the state or change in the living body, and enables the determination of esophageal cancer by itself. However, it may be used as a supplement to make a final decision (typically a definitive diagnosis) in consideration of other indicators as necessary.
  • the examination method of the present invention can also be referred to as "a method of assisting an esophageal cancer examination".
  • the present invention provides useful information for the diagnosis of esophageal cancer by using an objective (index) regardless of the judgment of a doctor.
  • the expression level of the biomarker of the present invention in a sample derived from a subject is used as an index.
  • the “level” here typically means “amount” or “concentration”.
  • the term “level” is also used to indicate whether or not the molecule to be detected can be detected (that is, whether or not it has an apparent presence) in accordance with customs and common general technical knowledge.
  • the inspection method of the present invention performs the following steps (1) and (2).
  • Step (1) a sample collected from a subject is prepared, the biomarker of the present invention (esophageal cancer biomarker) is detected, and the expression level is determined.
  • Hsa-miR-619-5p, hsa-miR-1273f, hsa-miR-3135b, hsa-miR-4327 or hsa-miR-150-3p are used as biomarkers for the purpose of early detection of esophageal cancer.
  • hsa-miR-619-5p or hsa-miR-1273f is adopted.
  • the subject is not particularly limited. That is, the present invention can be widely applied to a person who needs to determine the presence or absence of esophageal cancer. For example, those who may or may have esophageal cancer, such as those who have signs of esophageal cancer or those who have symptoms characteristic of esophageal cancer, as well as those who have no subjective symptoms (including healthy people) are also examined. Can be a person.
  • the sample is collected prior to the implementation of the present invention.
  • Urine is used as a sample.
  • the sample may be collected and prepared by a conventional method.
  • the biomarker in the sample is detected, but it is not essential to strictly quantify the expression level of the biomarker. That is, it suffices if the biomarker can be detected to the extent that the esophageal cancer can be determined in the subsequent step (2). For example, detection can be performed so that it can be determined whether or not the level of the biomarker in the sample exceeds a predetermined reference value.
  • the biomarker detection method is not particularly limited.
  • it can be detected by a microarray, which is a method using a nucleic acid amplification reaction represented by the qRT-PCR method.
  • Nucleic acid amplification reactions include the PCR (Polymerase chain reaction) method or a modified method thereof, as well as the LAMP (Loop-Mediated Isothermal Amplification) method (Tsugunori Notomi et al. Nucleic Acids Research, Vol.28, No.12, e63, 2000. ; Kentaro Nagamine, Keiko Watanabe et al.
  • the expression level of the biomarker is determined from the detection result.
  • standardization is usually performed in order to improve accuracy, objectivity, and the like.
  • the detection results of microRNAs with constitutive expression may be used.
  • hsa-miR-4669 and hsa-miR-6756-5p are used as standardization factors. That is, the detection result of the biomarker is corrected by the detection results of hsa-miR-4669 and hsa-miR-6756-5p to obtain the expression level used for the determination in step (2).
  • RNU6B which is generalized as a standardization factor for tissues and cell lines, may be used as a standardization factor.
  • the Ct value (Threshold Cycle) should be used to determine the expression level.
  • the value ( ⁇ Ct value) obtained by subtracting the Ct value of the standardization factor (standardization factor Ct value) from the Ct value of the biomarker is obtained, and the ⁇ Ct value is used as the expression level used for the determination in step (2).
  • Step (2) the prevalence of esophageal cancer is determined based on the expression level determined in step (1). If the purpose is to detect early esophageal cancer, the prevalence of early esophageal cancer will be determined.
  • the expression level obtained in step (1) is compared with the expression level of the control (control sample), or in light of the criteria set based on the expression level of the control. It is good to judge.
  • Controls include, for example, the biomarker expression level of healthy subjects and / or the biomarker expression level of esophageal cancer patients, or the biomarker expression level of early (stage I) esophageal cancer patients (the purpose is to detect early esophageal cancer). Case) can be used.
  • hsa-miR-619-5p, hsa-miR-1273f, hsa-miR-3135b, hsa-miR-4327 and hsa-miR-150-3p which are biomarkers of the present invention.
  • the expression level of the biomarker of the present invention shows a positive correlation with the morbidity of esophageal cancer. Therefore, regarding the expression level of biomarkers, a high level is a basic index (judgment standard) for suffering from esophageal cancer.
  • hsa-miR-619-5p, hsa-miR-1273f, hsa-miR-4327 and hsa-miR-150-3p showed a significant increase in expression in early (stage I) esophageal cancer cases.
  • the expression of hsa-miR-3135b was also significantly increased in early esophageal cancer cases compared with healthy subjects. Therefore, the high expression level of these biomarkers is an index (judgment criterion) for suffering from early-stage esophageal cancer.
  • the determination in step (2) may be qualitative, semi-quantitative, or quantitative. Examples of qualitative and quantitative judgments are shown below.
  • the following example is an example of determining the morbidity of esophageal cancer, but the same applies to the determination of morbidity of early esophageal cancer.
  • the number of determination categories, the expression level of the biomarker associated with each determination category, the determination result, etc. can be arbitrarily set through preliminary experiments and the like without being bound by the following examples.
  • the reference value and the cutoff value used for the determination may be set while considering, for example, the sample to be used and the required accuracy (reliability). When setting the reference value or cutoff value, it is advisable to use statistical analysis using a large number of samples. As is clear from the judgment criteria, the judgment here can be performed automatically or mechanically without the judgment of a person having specialized knowledge such as a doctor or a laboratory technician.
  • biomarker expression level is higher than the reference value A, it is determined that the patient has esophageal cancer, and when the biomarker expression level is equal to or lower than the reference value A, it is determined that the patient does not have esophageal cancer. judge.
  • ⁇ Expression level More than 80% chance of having esophageal cancer
  • ⁇ Expression level ⁇ a 20% -80% chance of having esophageal cancer
  • Expression level ⁇ b Less than 20% chance of having esophageal cancer
  • the biomarker expression level is preferably a value obtained by subtracting the standardizing factor Ct value from the Ct value of the biomarker ( ⁇ Ct value).
  • the determination in step (2) is performed. For example, if the 2- ⁇ Ct value is equal to or higher than the reference value (cutoff value), it is determined that the patient has esophageal cancer or is likely to have esophageal cancer, and the 2- ⁇ Ct value is high. If it is lower than the reference value, it is determined that "there is no esophageal cancer" or "the possibility of having esophageal cancer is low".
  • the reference value here (the value of 2- ⁇ Ct value, which is the boundary between the high and low possibility of suffering from esophageal cancer) is set for each microRNA used as a biomarker. As described above, the reference value may be determined by utilizing statistical analysis using a large number of samples.
  • step (2) becomes useful information for diagnosing esophageal cancer, and not only for diagnosing esophageal cancer, but also for early detection of esophageal cancer, determination of a more appropriate treatment policy (selection of effective treatment method, etc.) Useful. It can also be an opportunity to undergo a detailed examination (for example, a secondary examination).
  • the present invention also provides a kit for testing esophageal cancer.
  • the kit of the present invention contains a reagent for detecting the biomarker of the present invention (biomarker detection reagent) as an essential element.
  • biomarker detection reagent an example of a reagent for detecting a biomarker is a biomarker-specific primer.
  • reagents for detecting hsa-miR-4669 and hsa-miR-6756-5p as standardizing factors, that is, reagents for detecting hsa-miR-4669 (eg, hsa-miR-4669-specific primers) and hsa.
  • reagents for detecting hsa-miR-4669 eg, hsa-miR-4669-specific primers
  • -MiR-6756-5p detection reagents eg hsa-miR-6756-5p-specific primers
  • kits of other reagents microRNA extraction reagents, DNA polymerase, reverse transcriptase, dNTP, etc.
  • devices or instruments urine sample collection container, reaction device, detection device, etc. used when performing esophageal cancer testing methods May be included in.
  • a miRNA array analysis was performed using 9 cases in the comprehensive analysis cohort, and multiple miRNAs abnormally expressed in the urine of esophageal cancer cases were identified.
  • each miRNA extracted in (i) was measured by the qPCR method to identify significant esophageal cancer biomarkers.
  • RNA extraction / cDNA synthesis MiRNA was extracted from 200 ⁇ l of urine sample cryopreserved at -80 ° C immediately after collection using the miRNeasy Serum / Plasma Kit (Qiagen, Valencia, CA) according to the protocol. Using the extracted miRNA as a template, cDNA synthesis was performed according to the protocol using the TaqMan Advanced MicroRNA cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, USA).
  • miR-4669 SEQ ID NO: 6
  • miR-6756-5p SEQ ID NO: 7
  • the Ct values of miR-619-5p, miR-1273f, miR-3135b, miR-4327 and miR-150-3p were measured by qRT-PCR, and the standardized Ct values (miR-) were measured from each.
  • the ⁇ Ct value was calculated by subtracting the average Ct value of 4669 and miR-6756-5p).
  • miRNA array analysis was performed using 9 comprehensive analysis cohorts to identify multiple miRNAs that were significantly elevated or decreased in the urine of esophageal cancer cases.
  • expression analysis was performed in the biomarker establishment cohort. Specifically, in (ii) 192 biomarker establishment cohorts, each miRNA extracted in (i) was measured by the qPCR method. As a result, in univariate analysis, miR-619-5p, miR-3135b, miR-1273f, Significantly high expression of miR-4327 and miR-150-3p was observed in the urine of patients with esophageal cancer.
  • each of the five types of miRNA could be a biomarker for esophageal cancer alone (Fig. 2).
  • the AUCs for determining esophageal cancer were miR-619-5p: 0.747, miR-3135b: 0.642, miR-1273f: 0.767, miR-4327: 0.623, and miR-150-3p: 0.670, all of which were significantly esophageal. Cancer could be determined, but among them, urinary miR-619-5p and urinary miR-1273f made it possible to determine with particularly high accuracy (Fig. 3).
  • the sensitivity / specificity of esophageal cancer determination was 81.3% / 54.2% for urinary miR-619-5p and 81.3% / 52.1% for urinary miR-1273f.
  • the cutoff value of each biomarker was set as follows. miR-619-5p: 0.265 miR-3135b: 1.15 miR-1273f: 0.035 miR-4327: 0.32 miR-150-3p: 0.0062
  • stage I esophageal cancer 17 cases, miR-619-5p, miR-1273f, miR-4327 and miR-150-3p were also healthy subjects.
  • a significantly higher expression was found in the urine of patients with stage I esophageal cancer (Fig. 4).
  • the AUCs for determining stage I esophageal cancer are miR-619-5p: 0.775, miR-1273f: 0.794, miR-4327: 0.707, and miR-150-3p: 0.673, all of which can significantly determine stage I esophageal cancer.
  • urinary miR-619-5p and urinary miR-1273f enabled particularly high-precision judgment (that is, high-precision early diagnosis of esophageal cancer) (Fig. 5).
  • the sensitivity / specificity of stage I esophageal cancer judgment when the cutoff value was determined by ROC analysis was 88.2% / 63.2% for urinary miR-619-5p and 88.2% / 59.7% for urinary miR-1273f. there were.
  • the cutoff value of each biomarker reference 2 - ⁇ CT value
  • urinary miRNAs (urinary miR-1273f, urinary miR-619-5p, urinary miR-150-3p and urinary miR-4327) have early esophageal tract. It confirms that it is a biomarker that is extremely excellent in detecting or identifying cancer.
  • urinary miR-3135b the median of patients with stage I esophageal cancer was about twice the median of healthy subjects, suggesting that it could be used to detect or identify early-stage esophageal cancer.
  • MiRNAs were obtained from the culture media of normal human esophageal squamous epithelial cell line (Het-1A) and three types of human esophageal squamous cell line (TE-1, TT, KYSE-30). Expression analysis of the extracted and identified 5 types of miRNA was performed. In the expression analysis, RNU6B was used as a standardizing factor. The analysis result is shown in FIG. Compared with normal cell lines, all five miRNAs were significantly highly expressed in the culture medium of human esophageal cancer cell lines.
  • urinary miRNAs As a result, 5 types of urinary miRNAs (urinary miR-1273f, urinary miR-619-5p, urinary miR-150-3p, urinary miR-3135b and urinary miR-4327) detected esophageal cancer. Or prove that it is useful for identification.
  • MiR-4327 was significantly more highly expressed in esophageal cancer tissues than in normal tissues, and miR-1273f, miR-619-5p, and miR-150-3p were also highly expressed.
  • miR-3135b showed no significant difference between the two groups. This suggests that the number of cases is small and the detection power may be insufficient (false negative), and that miR-3135b may be less useful than the other four miRNAs in detecting early-stage cancer.
  • the present invention provides a biomarker useful for diagnosing esophageal cancer.
  • the biomarker of the present invention can be detected in a urine sample, enabling a minimally invasive and simple test. Since urine is used as a sample, examination / diagnosis can be performed "non-invasively”, “easily”, and “at home”, which is extremely useful in the field of primary screening, for example. Moreover, if the biomarker of the present invention is used, highly accurate inspection becomes possible.
  • the present invention can be implemented as one item of urinalysis in a health examination performed in, for example, a hospital, a health center, a medical examination vehicle, or the like.
  • a health examination performed in, for example, a hospital, a health center, a medical examination vehicle, or the like.
  • urine is used as a sample, a new style of testing is possible, in which urine collected at home is sent to a testing institution for testing.

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CN112877434A (zh) * 2021-02-22 2021-06-01 南充市中心医院 一组用于检测食管癌组织的circRNA标志物、引物及其应用和含有其的试剂盒
CN114854855A (zh) * 2022-02-22 2022-08-05 武汉艾米森生命科技有限公司 食管癌的生物标志物、核酸产品和试剂盒

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CN112813169A (zh) * 2021-02-26 2021-05-18 南充市中心医院 一种用于检测食管癌组织的jam-a生物标志物、引物及其应用和含有其的试剂盒

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