CN116716405A - Diagnostic markers associated with esophageal squamous cell carcinoma and uses thereof - Google Patents
Diagnostic markers associated with esophageal squamous cell carcinoma and uses thereof Download PDFInfo
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Abstract
The application belongs to the technical field of molecular biology, and particularly relates to a diagnosis marker related to esophageal squamous cell carcinoma and application thereof. Diagnostic markers associated with esophageal squamous cell carcinoma include: hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p. The diagnosis marker expression level can be used for diagnosing esophageal squamous cell carcinoma by in vitro detection, and has good sensitivity and specificity, so that the kit has good application in the diagnosis field of esophageal squamous cell carcinoma.
Description
Technical Field
The application belongs to the technical field of molecular biology, and particularly relates to a diagnosis marker related to esophageal squamous cell carcinoma and application thereof.
Background
Esophageal cancer (esophageal carcinoma) is a common tumor of the digestive tract with a high prevalence in specific geographic locations and in certain ethnicities. Esophageal squamous cell carcinoma (esophageal squamous cell carcinoma, ESCC) accounts for about 80% of global cases of esophageal cancer, with a particularly high incidence in regions of east asia and africa. ESCC has an average 5-year survival rate of between 10 and 41%, and this poor prognosis results from the presence of extensive lymphatic networks in the oesophagus, coupled with the lack of protective serosa, leading to early regional tumor progression and metastasis. Furthermore, ESCC patients are often asymptomatic at an early stage, which can easily lead to a delay in diagnosis.
Currently, diagnostic methods for ESCC include endoscopic and non-endoscopic detection. Endoscopy includes conventional white light endoscopy, pigment endoscopy, endoscopy and narrow band imaging, transnasal endoscopy, endocytosis, and the like. The detection conditions of different endoscopy are different, and the greatest disadvantage of the endoscopy is that the method can only be used for detecting late differentiated esophageal cancer, and the judgment benefit of early esophageal cancer is poor. Non-endoscopic detection methods include screening for autoantibodies in the blood, circulating Tumor Cells (CTCs), and the like. Evidence based on esophageal squamous dysplasia is more limited and thus autoantibodies for screening for esophageal cancers have not been used clinically far, and the presence of circulating tumor cells is predictive of advanced cancer disease, and thus circulating tumor cell detection is not a suitable screening modality for early cancers.
Micrornas (mirnas) are a small class of non-coding RNAs, about 20-25 nucleotides in length, that regulate gene expression by transcription interfering with translational inhibition of downstream target gene mRNA. mirnas are involved in most biological events, including tumorigenesis of human cancers such as ESCC. Due to the stability and high abundance of mirnas in body fluids, as well as unique expression profiles under various biological conditions, circulating mirnas are becoming attractive candidates for non-invasive "liquid biopsy" methods.
Disclosure of Invention
The application aims to provide a diagnosis marker related to esophageal squamous cell carcinoma and application thereof, and aims to solve the technical problem of how to better improve the sensitivity and specificity of esophageal squamous cell carcinoma detection.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the application provides a diagnostic marker associated with esophageal squamous cell carcinoma, comprising hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p.
The embodiment of the application provides a group of diagnosis markers for esophageal squamous cell carcinoma, in particular to serum microRNA, which comprises hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p. The diagnosis marker expression level can be detected in vitro, so that the diagnosis marker can diagnose esophageal squamous cell carcinoma, has good sensitivity and specificity, and has good application in the diagnosis field of esophageal squamous cell carcinoma.
In a second aspect, the present embodiment provides an application of a reagent for detecting the diagnostic marker of the first aspect of the present embodiment in preparing a diagnosis product for esophageal squamous cell carcinoma.
Based on the in vitro detection of the expression level of the diagnosis markers including hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p, esophageal squamous cell carcinoma can be diagnosed, and the reagent for detecting the diagnosis markers has good sensitivity and specificity, so that the reagent can be used for preparing an esophageal squamous cell carcinoma diagnosis product.
In some embodiments, the reagent comprises:
detecting a first reverse transcription primer of hsa-miR-26a-5p, a first amplification primer pair and a first probe;
detecting a second reverse transcription primer of hsa-miR-151a-3p, a second amplification primer pair and a second probe;
detecting a third reverse transcription primer of hsa-miR-181a-5p, a third amplification primer pair and a third probe;
and detecting a fourth reverse transcription primer of hsa-miR-320a-3p, and a fourth amplification primer pair and a fourth probe.
And designing corresponding reverse transcription primers, amplification primers and probes aiming at four serum microRNAs related to esophageal squamous cell carcinoma, so as to realize detection.
In some embodiments, the reagent further comprises: positive quality control and negative quality control.
Positive quality control detection results are positive, and negative quality control detection results are negative; the negative quality control can be used for eliminating false positive results caused by reagent or environmental pollution, and the positive quality control can be used for monitoring whether the reagent performance or personnel operation are normal or not and eliminating false negative results.
In some embodiments, the esophageal squamous cell carcinoma diagnostic product comprises a diagnostic kit.
The diagnosis kit can detect samples such as human body fluid, blood and the like, thereby realizing the detection of the diagnosis marker related to the esophageal squamous cell carcinoma.
In a third aspect, embodiments of the present application provide a diagnostic product for esophageal squamous cell carcinoma comprising reagents for detecting the diagnostic markers of the first aspect of embodiments of the present application.
Based on the in vitro detection of the expression levels of diagnostic markers including hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p, esophageal squamous cell carcinoma can be diagnosed, and the diagnostic products including the reagents for detecting the diagnostic markers can be used for esophageal squamous cell carcinoma with good sensitivity and specificity.
In some embodiments, the reagent comprises:
detecting a first reverse transcription primer of hsa-miR-26a-5p, a first amplification primer pair and a first probe;
detecting a second reverse transcription primer of hsa-miR-151a-3p, a second amplification primer pair and a second probe;
detecting a third reverse transcription primer of hsa-miR-181a-5p, a third amplification primer pair and a third probe;
and detecting a fourth reverse transcription primer of hsa-miR-320a-3p, and a fourth amplification primer pair and a fourth probe.
For four serum microRNAs related to esophageal squamous cell carcinoma, the diagnosis product comprises the design of corresponding reverse transcription primers, amplification primers and probes, so that detection is realized.
In some embodiments, the reagent further comprises: positive quality control and negative quality control.
Positive quality control detection results are positive, and negative quality control detection results are negative; the negative quality control can be used for eliminating false positive results caused by reagent or environmental pollution, and the positive quality control can be used for monitoring whether the reagent performance or personnel operation are normal or not and eliminating false negative results.
In some embodiments, the diagnostic product comprises a diagnostic kit.
The diagnosis kit can detect samples such as human body fluid, blood and the like, thereby realizing the detection of the diagnosis marker related to the esophageal squamous cell carcinoma.
In some embodiments, the diagnostic kit further comprises a regression model: logit (P) =k1× (Ct (hsa-miR-181a-5p) －Ct (hsa-miR-151a-3p) )+k2×(Ct (hsa-miR-26a-5p) －Ct (hsa-miR-320a-3p) )+k3。
Based on the regression model described above in the diagnostic kit, it can be obtained that the greater the logic (P), the greater the risk of esophageal squamous cell carcinoma.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a box plot of Ct values detected for a combination of diagnostic markers, i.e., miRNAs, in example 1 of the present application;
FIG. 2 is a risk assessment graph of 105 clinical samples (including 54 ESCCs and 51 Healthy samples) tested in example 1 of the present application;
fig. 3 is a scatter plot and ROC plot of 105 clinical samples (including 54 ESCCs and 51 healhs) tested in example 1 of the present application, with AUC value= 0.9009.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s).
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
Embodiments of the application relate to determining the expression level of a miRNA. In some embodiments, the level is compared to a control to determine whether the expression level or activity of the miRNA is increased as compared to the level in the non-cancerous biological sample. The control may be non-cancerous esophageal tissue, or it may be cancerous esophageal tissue. If the control is cancerous tissue, an elevated level of miRNA in the sample can be determined because the levels in the control and patient samples are similar, e.g., within at least or up to 1, 2, 3, or 4 standard deviations of each other (or any range derivable therein). In some embodiments, the control is a level of the one or more mirnas in a non-ESCC patient sample. In some embodiments, the control sample is a non-cancerous biological sample. In some embodiments, the control is from a particular group described herein. It is contemplated that one or more controls may be measured simultaneously with the test sample, or it may be a normalized value collected from a plurality of control samples. In some embodiments, the patient sample and/or the control sample is a tissue sample. Alternatively, the patient sample and/or the control sample is a serum sample.
An embodiment of the application provides a diagnosis marker related to esophageal squamous cell carcinoma, comprising hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p.
Wherein the sequences of the above 4 diagnostic markers of blood circulation are:
hsa-miR-26a-5p(SEQ ID NO.1)：5’-UUCAAGUAAUCCAGGAUAGGCU-3’；hsa-miR-151a-3p(SEQ ID NO.2)：5’-CUAGACUGAAGCUCCUUGAGG-3’；hsa-miR-181a-5p(SEQ ID NO.3)：5’-AACAUUCAACGCUGUCGGUGAGU-3’；hsa-miR-320a-3p(SEQ ID NO.4)：5’-AAAAGCUGGGUUGAGAGGGCGA-3’。
the embodiment of the application provides a group of diagnosis markers for esophageal squamous cell carcinoma, in particular to serum microRNA comprising hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p. The diagnosis marker expression level can be detected in vitro, so that the diagnosis marker can diagnose esophageal squamous cell carcinoma, has good sensitivity and specificity, and has good application in the diagnosis field of esophageal squamous cell carcinoma.
In a second aspect, the embodiment of the application provides an application of a reagent for detecting the diagnosis marker in the first aspect of the embodiment of the application in preparing an esophageal squamous cell carcinoma diagnosis product.
Based on the in vitro detection of the expression level of the diagnosis markers including hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p, esophageal squamous cell carcinoma can be diagnosed, and the reagent for detecting the diagnosis markers has good sensitivity and specificity, so that the reagent can be used for preparing an esophageal squamous cell carcinoma diagnosis product.
In some embodiments, the reagent comprises:
(1) Detecting a first reverse transcription primer of hsa-miR-26a-5p, a first amplification primer pair and a first probe; wherein, the specific sequence is as follows:
first reverse transcription primer sequence (SEQ ID NO. 5):
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA GCCTATC-3’；
first amplification primer pair sequences:
a specific forward primer (SEQ ID NO. 6) 5'-GCGCGCTTCAAGTAATCC-3',
5'-GTGCAGGGTCCGAGGT-3' universal reverse primer (SEQ ID NO. 7);
first probe sequence (SEQ ID NO. 8):
5'-FAM-TGGATACGACAGCCTAT-MGB-3', the 5 end of the sequence of which is connected with a fluorescent group FAM, and the 3 end is connected with a quenching group MGB.
(2) Detecting a second reverse transcription primer of hsa-miR-151a-3p, a second amplification primer pair and a second probe; wherein, the specific sequence is as follows:
second reverse transcription primer sequence (SEQ ID NO. 9):
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACC CTCAAG-3’；
second amplification primer pair sequences:
a specific forward primer (SEQ ID NO. 10) 5'-GCGCCTAGACTGAAGCTCCT-3',
5'-GTGCAGGGTCCGAGGT-3' universal reverse primer (SEQ ID NO. 7);
second probe sequence (SEQ ID NO. 11):
5'-FAM-TGGATACGACCCTCAAGG-MGB-3', the 5 end of the sequence of which is connected with a fluorescent group FAM, and the 3 end is connected with a quenching group MGB.
(3) Detecting a third reverse transcription primer of hsa-miR-181a-5p, a third amplification primer pair and a third probe; wherein, the specific sequence is as follows:
third reverse transcription primer sequence (SEQ ID NO. 12):
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA CTCACCG-3’；
third amplification primer pair sequence:
a specific forward primer (SEQ ID NO. 13) 5'-GCGCAACATTCAACGCTG-3',
5'-GTGCAGGGTCCGAGGT-3' universal reverse primer (SEQ ID NO. 7);
third probe sequence (SEQ ID NO. 14):
5' -FAM-TGGATACGACACTCACC-MGB-3", the 5 end of the sequence of which is connected with a fluorescent group FAM and the 3 end is connected with a quenching group MGB.
(4) And detecting a fourth reverse transcription primer of hsa-miR-320a-3p, and a fourth amplification primer pair and a fourth probe. Wherein, the specific sequence is as follows:
fourth reverse transcription primer sequence (SEQ ID NO. 15):
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACT CGCCCTC-3’；
fourth amplification primer pair sequence:
a specific forward primer (SEQ ID NO. 16) 5'-TGCCGAAAAGCTGGGTTGAGA-3',
5'-GTGCAGGGTCCGAGGT-3' universal reverse primer (SEQ ID NO. 7);
fourth probe sequence (SEQ ID NO. 17):
5'-FAM-CTGGATACGACTCGCC-MGB-3', the 5 end of the sequence of which is connected with a fluorescent group FAM, and the 3 end is connected with a quenching group MGB.
Designing corresponding reverse transcription primers, amplification primers and probes aiming at four serum microRNAs related to esophageal squamous cell carcinoma, specifically, the reverse transcription primers are reverse transcription stem loop primers, the reverse transcription stem loop primers reversely transcribe a target template into cDNA, and then the cDNA products are amplified and detected by using specific forward primers, specific TaqMan probes and universal reverse primers so as to realize detection. Therefore, the reagent contains the reverse transcription primer of serum microRNA, and the amplification primer and probe of fluorescent PCR, so that the reagent has good sensitivity and specificity in detection, and can assist in diagnosis of esophageal squamous cell carcinoma in vitro.
In some embodiments, the reagent further comprises: positive quality control and negative quality control.
Positive quality control detection results are positive, and negative quality control detection results are negative; the negative quality control can be used for eliminating false positive results caused by reagent or environmental pollution, and the positive quality control can be used for monitoring whether the reagent performance or personnel operation are normal or not and eliminating false negative results.
In some embodiments, the esophageal squamous cell carcinoma diagnostic product comprises a diagnostic kit.
The diagnosis kit can detect samples such as human body fluid, blood and the like, thereby realizing the detection of the diagnosis marker related to the esophageal squamous cell carcinoma.
A third aspect of embodiments of the application provides a diagnostic product for esophageal squamous cell carcinoma comprising reagents for detecting a diagnostic marker of the first aspect of embodiments of the application.
Based on the in vitro detection of the expression levels of diagnostic markers including hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p, esophageal squamous cell carcinoma can be diagnosed, and the diagnostic products including the reagents for detecting the diagnostic markers can be used for esophageal squamous cell carcinoma with good sensitivity and specificity.
In one embodiment, the detection reagent of the diagnostic marker may be a reagent suitable for the following method: real-time fluorescent quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry.
In some embodiments, the reagent comprises: detecting a first reverse transcription primer of hsa-miR-26a-5p, a first amplification primer pair and a first probe; detecting a second reverse transcription primer of hsa-miR-151a-3p, a second amplification primer pair and a second probe; detecting a third reverse transcription primer of hsa-miR-181a-5p, a third amplification primer pair and a third probe; and detecting a fourth reverse transcription primer of hsa-miR-320a-3p, and a fourth amplification primer pair and a fourth probe. See above for specific sequences. For four serum microRNAs related to esophageal squamous cell carcinoma, the diagnosis product comprises the design of corresponding reverse transcription primers, amplification primers and probes, so that detection is realized.
In some embodiments, the diagnostic product comprises a diagnostic kit. The diagnosis kit can detect samples such as human body fluid, blood and the like, thereby realizing the detection of the diagnosis marker related to the esophageal squamous cell carcinoma. In particular, the diagnosis kit is a kit for in vitro auxiliary diagnosis of esophageal squamous cell carcinoma.
In some embodiments, the reagent further comprises: positive quality control and negative quality control. Positive quality control detection results are positive, and negative quality control detection results are negative; the negative quality control can be used for eliminating false positive results caused by reagent or environmental pollution, and the positive quality control can be used for monitoring whether the reagent performance or personnel operation are normal or not and eliminating false negative results.
In some embodiments, the diagnostic kit further comprises non-human RNA solutions of different copy numbers of hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p as standard substances, and artificial synthetic product mixtures of specific copy numbers of hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p and internal control are used as positive quality control substances, and artificial synthetic product mixtures of specific copy numbers of hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p and internal control substances are used as negative quality control substances. The copy number ranges for the positive and negative quality controls hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3p are shown in Table 1.
TABLE 1
The positive quality control product and the negative quality control product can better exclude false positives and false negatives.
In one embodiment, the diagnostic kit comprises reverse transcription primers, fluorescent quantitative PCR amplification primer pairs and probes for detecting four miRNA markers; a positive quality control and a negative quality control; reverse transcriptase, dNTPs, RT buffer, DEPC water, buffer for DNA polymerase, mgCl 2 And DNA polymerase. In particular, the composition of the diagnostic kit preferably includes the components shown in table 2.
TABLE 2
In some embodiments, the diagnostic kit further comprises a regression model: logit (P) =k1× (Ct (hsa-miR-181a-5p) －Ct (hsa-miR-151a-3p) )+k2×(Ct (hsa-miR-26a-5p) －Ct (hsa-miR-320a-3p) ) +k3. Wherein the value of k1 is-1.0-4.0, the value of k2 is-1.0-2.0, and the value of k3 is 30-50. The model reference value logic (P) is determined to be high risk of esophageal squamous cell carcinoma when the model reference value logic (P) is larger than a positive determination value, and is determined to be low risk of esophageal squamous cell carcinoma when the model reference value logic (P) is smaller than or equal to the positive determination value. The positive determination value may take a value of about 28 to 32.
Based on the regression model described above in the diagnostic kit, it can be obtained that the greater the logic (P), the greater the risk of esophageal squamous cell carcinoma.
In the embodiment of the application, the applicable sample for detection is serum, the dosage is only 0.2ml, and the sample collection is convenient and easy to implement. Through the verification of 105 cases of clinical samples, the in-vitro detection is carried out on the expression level of the diagnostic marker, the detection sensitivity can reach more than 85%, the specificity can reach more than 92%, the total coincidence rate is more than 88%, and the detection performance of the diagnostic marker is obviously better than that of the common marker.
The following description is made with reference to specific embodiments.
Example 1
The embodiment establishes a kit for in vitro detection of 4 micro ribonucleic acids (hsa-miR-26 a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p and hsa-miR-320a-3 p) related to esophageal cancer in human serum and application thereof in diagnosis of esophageal squamous cell carcinoma, and the result is a risk score, and a high risk result indicates that esophageal cancer may exist. Wherein, the specific sequences of the 4 kinds of micro ribonucleic acids are SEQ ID NO.1-4.
In this example, reverse transcription stem-loop primer and TaqMan MGB probe technology are adopted, firstly, RNA template is reverse transcribed into cDNA by reverse transcription stem-loop primer, then cDNA product is amplified and detected by specific forward primer, specific TaqMan MGB probe and general reverse primer (specific sequence is SEQ ID NO.5-17 mentioned above), and the detected Ct value passes through regression model: logit (P) =2.2× (Ct) (hsa-miR-181a-5p) －Ct (hsa-miR-151a-3p) )+1.0×(Ct (hsa-miR-26a-5p) －Ct (hsa-miR-320a-3p) ) +38.95; and calculating a reference value to judge a result. The positive determination value was 40.
This example was carried out in the form of a kit whose main components are shown in table 3 below:
TABLE 3 Table 3
The kit can be stored at-20+/-5 ℃ in a dark place, the effective period is 12 months, the repeated freezing and thawing times are not more than 8 times, and the unsealed product is recommended to be used up within 3 months. The kit is applicable to an ABI 7500 real-time fluorescence quantitative PCR instrument.
Sample requirements of the kit: peripheral blood samples (5 ml) of the project participants were collected at room temperature, and after blood collection, blood vessels were reversely procoagulated for 8-9 times, and the samples were uniformly mixed. The procoagulant vessel was placed upright for 60 minutes to allow clot formation. At the end of the clotting time, the procoagulant vessel of the blood-containing sample was centrifuged at 1900g for 10 minutes at room temperature. The supernatant Serum (0.2 ml) is taken and packed into a 1.5ml centrifuge tube, and RNA extraction can be performed using nucleic acid extraction reagent miR50 (record number: guangdong deep mechanical equipment 20211225) or MIRNeasy Serum/Plasma Kit (Kaiji, cat number: 217184) of Shenzhen Co., ltd. The RT reaction can be performed immediately after RNA extraction, and the storage time is not longer than 4 hours. The positive control and the negative control are simultaneously extracted with the serum to be tested.
The detection method of the kit comprises the following steps: taking out the components of the kit, fully thawing, reversing and uniformly mixing, and rapidly centrifuging for 15 seconds by a palm centrifuge for later use; preparing 4 RT reaction systems (1.5 microlitres of RT reaction liquid, 0.5 microlitres of RT enzyme and 3.0 microlitres of RNA sample to be tested) respectively according to a proportion; placing the prepared RT reaction tube in an ABI 7500 real-time fluorescence quantitative PCR instrument for reaction, wherein the reaction procedure is as follows: (1) 5 minutes at 16 ℃; (2) 42 ℃ for 30 minutes; (3) 5min at 85 ℃. Preparing 4 PCR reaction systems (8.9 microliter of PCR reaction liquid, 0.1 microliter of PCR enzyme and 1.0 microliter of RT product) respectively according to the proportion; placing the prepared PCR reaction tube in an ABI 7500 real-time fluorescence quantitative PCR instrument for reaction, wherein the reaction procedure is as follows: (1) 95 ℃ for 5 minutes; (2) 95℃for 15 seconds and 60℃for 30 seconds (FAM fluorescence signal was collected), 45 cycles. After the reaction, data analysis was performed, and an appropriate baseline and threshold line were set according to the fluorescence PCR instrument manual. Baseline setting should ensure that the baseline of all amplification curves is stable and at the same level; the threshold line setting needs to be slightly above baseline and at the signal doubling period. Each amplification curve is examined and if an abnormality occurs in the baseline of the individual amplification curve, manual correction should be performed. The Ct value is derived by regression model: logit (P) =2.2× (Ct) (hsa-miR-181a-5p) －Ct (hsa-miR-151a-3p) )+1.0×(Ct (hsa-miR-26a-5p) －Ct (hsa-miR-320a-3p) ) +38.95 calculates a reference value to determine the result. The sample to be tested detects a reference logic (P)>40, determining that the esophageal squamous cell carcinoma is at high risk; otherwise, the low risk of esophageal squamous cell carcinoma is judged.
The kit completes the verification of 105 clinical samples, including 54 Esophageal Squamous Cell Carcinoma (ESCC) and 51 normal persons (Healthy), and performs in vitro detection on the expression levels of the 4 diagnostic markers.
As shown in FIG. 1, the box graph of the Ct values detected by the four miRNAs is that the Ct value detected by the miRNA151a (namely hsa-miR-151a-3 p) of the health group is slightly larger than the Ct value detected by the miRNA151a of the ESCC group, the Ct value detected by the miRNA181a (namely hsa-miR-181a-5 p) of the health group is slightly smaller than the Ct value detected by the miRNA181a of the ESCC group, the Ct value detected by the miRNA26a (namely hsa-miR-26a-5 p) of the health group is slightly smaller than the Ct value detected by the miRNA26a of the ESCC group, and the Ct value detected by the miRNA320a (namely hsa-miR-320a-3 p) of the health group is slightly equivalent to the Ct value detected by the miRNA320a of the ESCC group.
The risk assessment results for 105 clinical specimens are shown in fig. 2. A scatter plot and ROC plot of 105 clinical samples is shown in fig. 3. The detection sensitivity can reach 85.19%, the specificity is 92.16%, and the total coincidence rate is 88.57%, which is obviously better than the detection performance of a general marker.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (10)
1. A diagnostic marker associated with esophageal squamous cell carcinoma, comprising hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-miR-181a-5p, and hsa-miR-320a-3p.
2. Use of an agent for detecting a diagnostic marker according to claim 1 for the preparation of a diagnostic product for esophageal squamous cell carcinoma.
3. The use of claim 2, wherein the agent comprises:
detecting a first reverse transcription primer of hsa-miR-26a-5p, a first amplification primer pair and a first probe;
detecting a second reverse transcription primer of hsa-miR-151a-3p, a second amplification primer pair and a second probe;
detecting a third reverse transcription primer of hsa-miR-181a-5p, a third amplification primer pair and a third probe;
and detecting a fourth reverse transcription primer of hsa-miR-320a-3p, and a fourth amplification primer pair and a fourth probe.
4. The use of claim 2, wherein the agent further comprises: positive quality control and negative quality control.
5. The use of any one of claims 2-4, wherein the esophageal squamous cell carcinoma diagnostic product comprises a diagnostic kit.
6. A diagnostic product for esophageal squamous cell carcinoma comprising reagents for detecting the diagnostic marker of claim 1.
7. The diagnostic product of claim 6, wherein the reagent comprises:
detecting a first reverse transcription primer of hsa-miR-26a-5p, a first amplification primer pair and a first probe;
detecting a second reverse transcription primer of hsa-miR-151a-3p, a second amplification primer pair and a second probe;
detecting a third reverse transcription primer of hsa-miR-181a-5p, a third amplification primer pair and a third probe;
and detecting a fourth reverse transcription primer of hsa-miR-320a-3p, and a fourth amplification primer pair and a fourth probe.
8. The diagnostic product of claim 6, wherein the reagent further comprises: positive quality control and negative quality control.
9. The diagnostic product of any one of claims 6-8, wherein the diagnostic product comprises a diagnostic kit.
10. The diagnostic product of claim 9, wherein the diagnostic kit further comprises a regression model: logit (P) =k1× (Ct (hsa-miR-181a-5p) －Ct (hsa-miR-151a-3p) )+k2×(Ct (hsa-miR-26a-5p) －Ct (hsa-miR-320a-3p) ) +k3; wherein, the value of k1 is-1.0-4.0, the value of k2 is-1.0-2.0, and the value of k3 is 30-50.
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