Composition for identifying oral squamous cell carcinoma based on saliva sample detection and application

Abstract

The invention relates to a composition for identifying oral squamous cell carcinoma based on saliva sample detection and application, and belongs to the technical field of molecular biology. The composition provided by the invention is used for identifying the oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in a saliva sample; the composition comprises a specific reverse transcription stem-loop primer, a specific detection forward primer, a specific detection probe and a universal reverse primer. According to the composition disclosed by the invention, the expression of SNORD52 and / or SNORD65 in a saliva sample is detected, and a binomial logistic regression model is combined to perform auxiliary judgment on the oral squamous cell carcinoma, so that the composition has relatively high sensitivity and specificity, and the identification accuracy of the oral squamous cell carcinoma can be improved. The invention further provides a kit containing the composition, a detection object of the kit is a saliva sample, and the kit has the advantages of being small in sample dosage, noninvasive in sampling, convenient, rapid, small in risk and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a composition for identifying oral squamous cell carcinoma based on saliva sample detection and its use. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in the head and neck, accounting for more than 90% of oral cancers. The global annual incidence is about 300,000 cases, mainly in middle-aged and elderly men, but the proportion of young patients has increased in recent years. The incidence of oral squamous cell carcinoma is closely related to smoking, alcoholism, human papillomavirus (HPV) infection (especially HPV16 / 18 type), and long-term chronic oral irritation (such as ill-fitting dentures, sharp tooth roots). The survival rate of oral squamous cell carcinoma is closely related to the stage at diagnosis. For early-stage (I-II stage) patients, the 5-year survival rate can reach 60%-80% through surgery combined with radiotherapy. However, most patients are diagnosed at an advanced stage (III-IV stage), and the 5-year survival rate drops sharply to less than 30%. Patients with lymph node metastasis, positive resection margins, or distant metastasis have a very poor prognosis, and the recurrence rate is as high as 50%-60%, often occurring within 2 years after treatment. Moreover, the oral cavity is located at the starting position of the digestive and respiratory tracts, with dense important organs and located on the face. Therefore, in clinical practice, radical resection cannot be performed on patients with advanced tumors and recurrence and metastasis according to the "tumor-free principle". Therefore, seeking a molecular biological marker with less trauma to patients, convenient detection, high specificity and sensitivity for the auxiliary diagnosis of oral squamous cell carcinoma has important clinical significance for its early diagnosis and follow-up detection.

[0003] Liquid biopsy is usually regarded as an extension of in vitro molecular diagnosis. It extends the sample from relevant tissues to liquids such as blood, saliva, and cerebrospinal fluid, with the characteristics of non-invasive sampling, convenience, speed, and less risk. Moreover, liquid biopsy can achieve continuous detection, which is of great significance for the early diagnosis of cancer, disease progression monitoring, and efficacy evaluation during cancer treatment.

[0004] Small nucleolar RNA (snoRNA) belongs to short-chain non-coding RNA (ncRNA), which is widely present in the nucleolus of eukaryotes, with a size of 60-300 nucleotides, and participates in the modification and maturation of ribosomal RNA (rRNA). More and more studies have shown that there are abnormal expressions of snoRNA in tumor tissues, cells, and body fluids, which participate in the regulation of tumor biological behavior. Therefore, if obvious differentially expressed snoRNAs can be screened in the saliva of OSCC patients and reagents for detecting the differentially expressed snoRNAs in saliva samples are developed for the auxiliary diagnosis of oral squamous cell carcinoma, it has important significance. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a composition for identifying oral squamous cell carcinoma based on saliva sample detection and its use.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: A composition for identifying oral squamous cell carcinoma based on saliva sample detection, which identifies oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in saliva samples; The composition includes: a specific reverse transcription stem-loop primer, a specific detection forward primer, a specific detection probe, and a universal reverse primer; When used to detect the expression of SNORD52 in saliva samples: The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.1; The nucleotide sequence of the specific detection forward primer is as shown in SEQ ID NO.4; The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.5; The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10; When used to detect the expression of SNORD65 in saliva samples: The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.2; The nucleotide sequence of the specific detection forward primer is as shown in SEQ ID NO.6; The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.7; The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10.

[0007] In the above technical solution, the composition further includes a positive control product and a negative control product.

[0008] In the above technical solution, the positive control product is a mixture of artificially synthesized single-stranded SNORD52 mimics, artificially synthesized single-stranded SNORD65 mimics, and artificially synthesized RNA mimics of the internal reference gene U68; The nucleotide sequence of the artificially synthesized single-stranded SNORD52 mimics is as shown in SEQ ID NO.11; The nucleotide sequence of the artificially synthesized single-stranded SNORD65 mimics is as shown in SEQ ID NO.12; The nucleotide sequence of the artificially synthesized RNA mimics of the internal reference gene U68 is as shown in SEQ ID NO.13.

[0009] In the above technical solution, the negative control product is nuclease-free water.

[0010] In the above technical solution, when detecting the artificially synthesized reference gene U68: The nucleotide sequence of the corresponding specific reverse transcription stem-loop primer is as shown in SEQ ID NO.3; The nucleotide sequence of the corresponding specific detection forward primer is as shown in SEQ ID NO.8; The nucleotide sequence of the corresponding specific detection probe is as shown in SEQ ID NO.9; The nucleotide sequence of the corresponding universal reverse primer is as shown in SEQ ID NO.10.

[0011] In the above technical solution, the composition performs auxiliary judgment on oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in saliva samples and combining with a binomial logistic regression model.

[0012] A kit for identifying oral squamous cell carcinoma, the kit includes the above composition.

[0013] In the above technical solution, the kit further includes: a snoRNA reverse transcription reaction solution, and the components of the snoRNA reverse transcription reaction solution include: RNA reverse transcriptase, 5× buffer, 0.1M DTT, dNTPs, and RNase inhibitor.

[0014] In the above technical solution, the kit further includes: a PCR reaction solution, and the components of the PCR reaction solution include DNA polymerase, KCl, MgCl2, Tris-HCl, and dNTPs.

[0015] In the above technical solution, the kit further includes: 50×ROX Reference Dye and nuclease-free water.

[0016] The beneficial effects of the present invention are: (1) The composition of the present invention can detect the changes in the expression levels of SNORD52 and / or SNORD65 in saliva samples, and the changes in the expression levels are closely related to oral squamous cell carcinoma. Therefore, by detecting the expression of SNORD52 and / or SNORD65 in saliva samples and combining with a binomial logistic regression model, auxiliary judgment of oral squamous cell carcinoma is performed, which has high sensitivity and specificity and can improve the accuracy of identifying oral squamous cell carcinoma.

[0017] (2)For the composition of the present invention, by real-time fluorescence quantitative PCR detection, the difference in the number of detection cycles (Ct value) between SNORD52, SNORD65 and the internal reference gene U68 is calculated to obtain the relative quantitative value of each oral squamous cell carcinoma-related marker, and the comprehensive evaluation value is calculated. Oral squamous cell carcinoma is judged by comparing the comprehensive evaluation value with the critical value. This detection method does not require operation steps such as electrophoresis and hybridization, reduces false positives, reduces contamination, and is simple to operate.

[0018] (3)The composition of the present invention is provided with the internal reference gene U68, which can effectively avoid missed detection and determine the quality of the sample; it is provided with a positive control product. If the positive control product shows a positive result in the detection, it means that the detection system is working properly and there will be no false negative results and missed detections; it is provided with a negative control product. If the negative control detection shows a negative result, it means that the detection system is working properly. Such a design makes the detection method rigorous and effectively avoids the possibility of missed detection and misdetection.

[0019] (4)The kit containing the composition of the present invention has the characteristics of small sample volume, non-invasive sampling, convenience, quickness, and low risk for saliva samples as the detection object. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0021] Figure 1 It is a diagram showing the expression differences of SNORD52 and SNORD65 in the saliva of healthy people and oral squamous cell carcinoma patients screened by high-throughput sequencing method; Figure 2 It is a diagram showing the expression differences of SNORD52 and SNORD65 detected by fluorescence quantitative PCR method; Figure 3 It is an ROC curve for the identification of oral squamous cell carcinoma using the expression levels of SNORD52 and SNORD65 as detection markers. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The inventive concept of the present invention is as follows: By performing snoRNA expression profile sequencing on saliva samples from oral squamous cell carcinoma patients and healthy individuals, snoRNAs with differential expression are screened out. The screened snoRNAs with differential expression are verified by fluorescence quantitative PCR, and it is confirmed that snoRNA SNORD52 and / or SNORD65 have significant differential expression in the saliva of oral squamous cell carcinoma patients and that of healthy individuals. The test results of saliva samples show that a specific combination of snoRNAs, including SNORD52 and / or SNORD65, has excellent performance in the differential diagnosis of oral squamous cell carcinoma patients. In summary, the specific combination of snoRNAs of the present invention, including SNORD52 and / or SNORD65, can be applied in the preparation of reagents or kits for the differential diagnosis of oral squamous cell carcinoma. The present invention also provides a method for detecting the expression levels of SNORD52 and / or SNORD65 in saliva samples, a composition capable of detecting the expression levels of SNORD52 and / or SNORD65, and a kit containing the composition, and a specific reverse transcription reaction system and a fluorescence quantitative PCR reaction system are constructed. Using the kit and detection method of the present invention, oral squamous cell carcinoma can be quickly and accurately differentiated by detecting saliva samples, which can save medical resources for the country and has a very broad clinical application prospect.

[0023] The present invention provides a composition for differentiating oral squamous cell carcinoma based on saliva sample detection. By detecting the expression of SNORD52 and / or SNORD65 in saliva samples and combining with a binary logistic regression model, it can assist in the judgment of oral squamous cell carcinoma, effectively improving the sensitivity and specificity of the auxiliary diagnosis of oral squamous cell carcinoma; The composition includes: A specific reverse transcription stem-loop primer, a specific detection forward primer, a specific detection probe, and a universal reverse primer; When detecting the expression of SNORD52 in saliva samples: The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.1; The nucleotide sequence of the specific detection forward primer is as shown in SEQ ID NO.4; The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.5; The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10; When detecting the expression of SNORD65 in saliva samples: The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.2; The nucleotide sequence of the specific detection forward primer is as shown in SEQ ID NO.6; The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.7; The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10.

[0024] Furthermore, the composition further comprises a positive control and a negative control.

[0025] The positive control is a mixture of a synthetic single-stranded SNORD52 mimics with the nucleotide sequence as shown in SEQ ID NO.11, a synthetic single-stranded SNORD65 mimics with the nucleotide sequence as shown in SEQ ID NO.12, and an RNA mimics of the reference gene U68 with the nucleotide sequence as shown in SEQ ID NO.13; the negative control is nuclease-free water.

[0026] Furthermore, when detecting the reference gene U68: The nucleotide sequence of the corresponding specific reverse transcription stem-loop primer is as shown in SEQ ID NO.3; The nucleotide sequence of the corresponding specific detection forward primer is as shown in SEQ ID NO.8; The nucleotide sequence of the corresponding specific detection probe is as shown in SEQ ID NO.9; The nucleotide sequence of the corresponding universal reverse primer is as shown in SEQ ID NO.10.

[0027] Furthermore, the specific reverse transcription stem-loop primers with the nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 are mixed in equal proportions to form a reverse transcription stem-loop primer pool.

[0028] Furthermore, the synthetic single-stranded SNORD52 mimics with the nucleotide sequence as shown in SEQ ID NO.11, the synthetic single-stranded SNORD65 mimics with the nucleotide sequence as shown in SEQ ID NO.12, and the RNA mimics of the reference gene U68 with the nucleotide sequence as shown in SEQ ID NO.13 are mixed in equal proportions to form the positive control.

[0029] The nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.13 are as follows:

[0030] The present invention also provides a kit for identifying oral squamous cell carcinoma, and the kit comprises the above composition.

[0031] Further, the kit further comprises a snoRNA reverse transcription reaction solution, the main components of which are RNA reverse transcriptase, 5× buffer, 0.1M DTT, dNTPs and RNase inhibitor.

[0032] Further, the kit further comprises a PCR reaction solution, the main components of which are DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs.

[0033] Further, the kit further comprises 50× ROX Reference Dye and nuclease-free water.

[0034] The kit for identifying oral squamous cell carcinoma according to the present invention has a saliva sample as the detection object, and the usage method comprises the following steps: (1) Use an extraction kit to extract snoRNA (SNORD52 or SNORD6) from the saliva sample. The extracted snoRNA should be used immediately. If it is not to be used temporarily, it should be stored at -70°C or below. (2) Using the snoRNA extracted in step (1) as a template, and positive and negative control products as templates, use a reverse transcription stem-loop primer pool as primers to reverse transcribe and prepare cDNA. Specifically, the reverse transcription is carried out according to the following reaction system and amplification conditions:

[0035] Amplification program settings:

[0036] Use the obtained cDNA as a template for the amplification reaction for standby.

[0037] (3) Perform quantitative PCR detection on the prepared cDNA. Using the prepared cDNA as a template, perform SNORD52 fluorescence quantitative PCR amplification with a specific detection forward primer with a nucleotide sequence such as SEQ ID NO.4, a specific detection probe with a nucleotide sequence such as SEQ ID NO.5, and a universal reverse primer with a nucleotide sequence such as SEQ ID NO.10; perform SNORD65 fluorescence quantitative PCR amplification with a specific detection forward primer with a nucleotide sequence such as SEQ ID NO.6, a specific detection probe with a nucleotide sequence such as SEQ ID NO.7, and a universal reverse primer with a nucleotide sequence such as SEQ ID NO.10; perform internal reference gene U68 fluorescence quantitative PCR amplification with a specific detection forward primer with a nucleotide sequence such as SEQ ID NO.8, a specific detection probe with a nucleotide sequence such as SEQ ID NO.9, and a universal reverse primer with a nucleotide sequence such as SEQ ID NO.10. The SNORD52 quantitative PCR detection system is prepared according to the following reaction system:

[0038] The SNORD65 quantitative PCR detection system is prepared according to the following reaction system:

[0039] The internal reference gene U68 quantitative PCR detection system is prepared according to the following reaction system:

[0040] PCR amplification is carried out according to the following amplification conditions:

[0041] (4)Judgment of detection results.

[0042] Specifically, the judgment of detection results includes the following steps: ① Judge whether the detection system is qualified: the Ct values of SNORD52 detection, SNORD65 detection and internal reference gene U68 detection in the positive control are all ≤ 30; there is no detection signal or the Ct values of SNORD52, SNORD65 and internal reference gene U68 in the negative control are all > 43; judge that the detection system is qualified; ② Judge whether the sample meets the requirements: if the Ct value of the internal reference gene U68 detection in the sample is ≤ 30, the sample is judged to be qualified; ③ Calculate the difference dCt in the detection cycle numbers (Ct values) of SNORD52, SNORD65 and internal reference gene U68 respectively to obtain the relative quantitative value of each oral squamous cell carcinoma-related marker. They are respectively marked as: dCt 基因1 、dCt 基因2 ; then calculate the comprehensive evaluation value according to the formula Logit = 9.478 - 0.536 * dCt 基因1 -0.673 * dCt 基因2 ; ④ If the comprehensive evaluation value ≥ the critical value, it is judged that the probability of the sample being oral squamous cell carcinoma increases; if the comprehensive evaluation value < the critical value, it is judged that the probability of the sample being oral squamous cell carcinoma decreases.

[0043] Furthermore, the critical value is set according to the expression levels of SNORD52 and SNORD65 and the expression level of the internal reference gene U68 in the saliva samples of oral squamous cell carcinoma patients and healthy population saliva samples, combined with the binomial logistic regression model, and is used to distinguish whether it is oral squamous cell carcinoma. The critical value is 0.7375.

[0044] To more thoroughly and comprehensively understand the content of the present invention, the present invention will be described in detail below with reference to the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Generally, those of ordinary skill in the technical field of the present invention can understand these descriptions and may make several non-unexpected improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as within the protection scope of the present invention.

[0045] Example 1 Perform whole-snoRNA expression profiling sequencing on saliva samples to screen for differentially expressed snoRNAs. By performing high-throughput sequencing of the whole-snoRNA expression profiles on saliva samples from 16 patients with oral squamous cell carcinoma and 9 healthy individuals determined by clinical pathology (sample information is shown in Table 1), differentially expressed snoRNAs were screened. The screening results are as Figure 1 shown: As Figure 1 can be seen, it was found that SNORD52 and SNORD65 had significant differences in expression between the two groups of saliva samples.

[0046] Table 1. Sample information for high-throughput sequencing of whole-snoRNA expression profiles

[0047] Example 2 Verify the expression levels of the differentially expressed snoRNAs screened by high-throughput sequencing using fluorescence quantitative PCR method. For the differentially expressed snoRNAs in saliva samples from patients with oral squamous cell carcinoma and healthy individuals screened by high-throughput sequencing, specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes, and universal reverse primers were designed, and fluorescence quantitative PCR detection method was used to verify the expression levels of the differentially expressed snoRNAs screened by high-throughput sequencing.

[0048] The research results showed that using the fluorescence quantitative PCR detection method, it was found that SNORD52 and SNORD65 had significant differences in expression between the saliva samples of 26 patients with oral squamous cell carcinoma determined by clinical pathology and 25 healthy individuals (sample information is shown in Table 2), as Figure 2 shown.

[0049] Table 2. Sample information for screening differentially expressed snoRNAs by fluorescence quantitative PCR

[0050] Continued Table 2. Sample information for screening differentially expressed snoRNAs by fluorescence quantitative PCR

[0051] Example 3 Design specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes, and universal reverse primers used in the present invention; The sequences of SNORD52, SNORD65, and the internal reference gene U68 involved in this example are from the GeneBank database. Specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes, and universal reverse primers are designed based on the relevant sequences. The designed nucleotide sequences are detailed in the specific implementation part.

[0052] The specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes, and universal reverse primers have been repeatedly verified and have the advantages of strong detection specificity, stable product structure, and few dimer structures.

[0053] Specifically, the specific reverse transcription stem-loop primers in this example have good reverse transcription specificity for the target snoRNA and high reverse transcription efficiency; the specific detection forward primers and specific detection probes show good specificity and stability during fluorescence quantitative PCR amplification, ensuring the specificity of PCR amplification while guaranteeing its amplification efficiency.

[0054] Example 4 Evaluation of the detection performance for differentiating oral squamous cell carcinoma with SNORD52 alone as the detection target; Using SNORD52 alone as the detection target, detect the expression level of the target in clinical saliva samples to differentiate oral squamous cell carcinoma. Taking the clinical pathological detection result as the "gold standard" for the diagnosis of oral squamous cell carcinoma, evaluate the detection performance of using SNORD52 as the detection target to differentiate oral squamous cell carcinoma.

[0055] The specific steps are as follows: Step 1. Sample collection: Same as Example 2.

[0056] Step 2. Extract snoRNA (SNORD52) from saliva samples: Use the Tiangen miRcute Serum / Plasma miRNA isolation kit to extract snoRNA from saliva samples. The specific steps are as follows: (1) Add 900 μL of lysis buffer MZA to every 200 μL of saliva, mix well by shaking on a shaker for 30 s until completely homogenized, and invert to mix. (2) Let it stand at room temperature for 5 min to completely separate the nucleic acid-protein complex. (3) Add 200 μL of chloroform, mix well by shaking on a shaker for 15 s, and let it stand at room temperature for 5 min. (4)Centrifuge at 12,000 rpm for 15 minutes at 4°C, take the supernatant, and transfer it to a new Rnase-free centrifuge tube; (5)Add 2 volumes of absolute ethanol, mix well, transfer the resulting solution and precipitate together into the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 30 s at room temperature, and discard the waste liquid; (6)Add 700 μL of protein removal solution MRD to the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid; (7)Add 500 μL of washing solution RW to the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid; (8)Add 500 μL of washing solution RW to the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid; (9)Place the adsorption column miRelute into the collection tube, centrifuge at 12,000 rpm for 1 min to remove the residual liquid; (10)Place the adsorption column miRelute on the ultra-clean bench for a moment to ventilate and dry it thoroughly; (11)Transfer the adsorption column miRelute into a new 1.5 mL centrifuge tube, add 30 μL of Rnase-free ddH2O, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min; (12)Detect the concentration and purity of the snoRNA solution obtained by centrifugation with Nanodrop; (13)Store the qualified snoRNA solution in a -20°C refrigerator for standby.

[0057] Step 3: Reverse transcription to prepare cDNA: Using the snoRNA extracted in Step 2 as a template, reverse transcribe to prepare cDNA with the reverse transcription stem-loop primer pool as primers; Perform reverse transcription according to the following reaction system and amplification conditions:

[0058] Amplification program settings:

[0059] Use the obtained cDNA as a template for the amplification reaction for standby.

[0060] Step 4: Perform quantitative PCR detection on the prepared cDNA: The SNORD52 quantitative PCR detection system is prepared according to the following reaction system:

[0061] The internal reference gene U68 quantitative PCR detection system was prepared according to the following reaction system:

[0062] PCR amplification was carried out according to the following amplification conditions:

[0063] As Figure 2 shown in the left figure in [reference], calculate the difference dCt between the detection cycle number (Ct value) of SNORD52 and the internal reference gene U68 to obtain the relative quantitative value of the SNORD52 marker. Using t The test analysis results showed that there were significant differences in the expression of SNORD52 in the saliva samples of oral squamous cell carcinoma patients and healthy people, p <0.0001.

[0064] Step Five: Judgment of Detection Results Specifically, the judgment of detection results includes the following steps: ① Calculate the difference dCt between the detection cycle number (Ct value) of SNORD52 and the internal reference gene U68 to obtain the relative quantitative value of the SNORD52 marker. Marked as: dCt 基因1 ; ④ If dCt 基因1 ≤ critical value, it is determined that the probability of the sample being oral squamous cell carcinoma may increase; if dCt 基因1 > critical value, it is determined that the probability of the sample being oral squamous cell carcinoma may decrease; The critical value is determined according to the expression levels of SNORD52 and the internal reference gene U68 in the saliva samples of oral squamous cell carcinoma patients and healthy people, combined with the Receiver Operating Characteristic Curve (ROC), according to the maximum value of the Youden index. The critical value is 5.715.

[0065] Step Six: Compare the SNORD52 expression level detection results with the clinicopathological results to evaluate the detection performance of using SNORD52 alone as a detection target to identify oral squamous cell carcinoma: Compare the identification results of oral squamous cell carcinoma obtained by using SNORD52 alone as a detection target with the clinicopathological detection results of the samples to be tested, as shown in Table 3 specifically: Table 3. Evaluation results of the detection performance of detecting actual clinical samples with SNORD52 as a detection target

[0066] Continued Table 3. Evaluation results of the detection performance for detecting actual clinical samples with SNORD52 as the detection target

[0067] For these 51 actual clinical samples, the differential diagnosis results of oral squamous cell carcinoma obtained using SNORD52 as the detection target were compared with the clinicopathological detection results of the samples examined. By applying statistical methods, it was calculated that: the sensitivity using SNORD52 as the detection target reached 76.92%, the specificity reached 96.00%, the positive predictive value reached 95.24%, the negative predictive value reached 80.00%, and the accuracy reached 86.27%.

[0068] Example 5 Evaluation of the detection performance for differentiating oral squamous cell carcinoma with SNORD65 as the sole detection target: Using SNORD65 as the sole detection target, the expression level of the target in clinical saliva samples was detected to differentiate oral squamous cell carcinoma. With the clinicopathological detection results as the "gold standard" for the diagnosis of oral squamous cell carcinoma, the detection performance of SNORD65 as the detection target for differentiating oral squamous cell carcinoma was evaluated.

[0069] The specific steps are as follows: Step 1. Sample collection: Same as Example 2.

[0070] Step 2. Extract snoRNA (SNORD65) from saliva samples: Same as Example 4.

[0071] Step 3. Reverse transcription to prepare cDNA: Same as Example 4.

[0072] Step 4. Perform quantitative PCR detection on the prepared cDNA: The quantitative PCR detection system for SNORD65 was prepared according to the following reaction system:

[0073] The quantitative PCR detection system for the internal reference gene U68 was prepared according to the following reaction system:

[0074] PCR amplification was performed according to the following amplification conditions:

[0075] As Figure 2 shown in the right figure int The test analysis results show that there are significant differences in the expression of SNORD65 in the saliva samples of patients with oral squamous cell carcinoma and healthy people. p <0.0001.

[0076] Step Five: Judgment of test results: Specifically, the judgment of test results includes the following steps: ① Calculate the difference dCt between the detection cycle number (Ct value) of SNORD65 and the internal reference gene U68 to obtain the relative quantitative value of the SNORD65 marker. Marked as: dCt 基因2 ; ④ If dCt 基因2 ≤ critical value, it is determined that the probability of this sample being oral squamous cell carcinoma may increase; if dCt 基因2 > critical value, it is determined that the probability of this sample being oral squamous cell carcinoma may decrease; The critical value is determined according to the expression levels of SNORD65 and the internal reference gene U68 in the saliva samples of patients with oral squamous cell carcinoma and healthy people, combined with the Receiver Operating Characteristic Curve (ROC), and based on the maximum value of the Youden index. The critical value is 8.125.

[0077] Step Six: Compare the detection results of the SNORD65 expression level with the clinical pathological results to evaluate the detection performance of using SNORD65 alone as a detection target for differentiating oral squamous cell carcinoma: Compare the identification results of oral squamous cell carcinoma obtained by using SNORD65 alone as a detection target with the clinical pathological detection results of the samples to be tested, as shown in Table 4 specifically: Table 4. Evaluation results of the detection performance of detecting actual clinical samples with SNORD65 as the detection target

[0078] Continued Table 4. Evaluation results of the detection performance of detecting actual clinical samples with SNORD65 as the detection target

[0079] For these 51 actual clinical samples, compare the identification results of oral squamous cell carcinoma obtained by using SNORD65 as the detection target with the clinical pathological detection results of the samples to be tested. By applying statistical methods, it is calculated that: the sensitivity of using SNORD65 as the detection target reaches 76.92%, the specificity reaches 92.00%, the positive predictive value reaches 90.91%, the negative predictive value reaches 79.31%, and the accuracy reaches 84.31%.

[0080] Example 6 Binomial logistic regression model for the identification of oral squamous cell carcinoma with SNORD52 and SNORD65 as detection targets, study on positive judgment value and evaluation of detection performance; According to the calculated dCt in Example 4 and Example 5 基因1 、dCt 基因2 , using SPSS software, binomial logistic regression analysis was performed to obtain the comprehensive evaluation value calculation formula Logit = 9.478 - 0.536 * dCt 基因1 - 0.673 * dCt 基因2 . The comprehensive evaluation value of each clinical sample was calculated according to the above formula, and the Receiver Operating Characteristic (ROC) curve ( Figure 3 ) was analyzed by SPSS software. The area under the ROC curve (Area Under Curve, AUC) was 0.9538. The closer the AUC value is to 1, the better the model performance. The critical value was determined according to the maximum Youden index. The critical value was 0.7375. If the comprehensive evaluation value ≥ the critical value, it was determined that the probability of the sample being oral squamous cell carcinoma increased; if the comprehensive evaluation value < the critical value, it was determined that the probability of the sample being oral squamous cell carcinoma decreased.

[0081] The detection results of SNORD52 and SNORD65 expression levels were compared with the clinicopathological results to evaluate the detection performance of identifying oral squamous cell carcinoma with SNORD52 and SNORD65 as detection targets. The specific results are shown in Table 5: Table 5. Evaluation results of the detection performance of detecting actual clinical samples with SNORD52 and SNORD65 as detection targets

[0082] Continued Table 5. Evaluation results of the detection performance of detecting actual clinical samples with SNORD52 and SNORD65 as detection targets

[0083] For these 51 actual clinical samples, the identification results of oral squamous cell carcinoma obtained using SNORD52 and SNORD65 as detection targets were compared with the clinicopathological detection results of the samples examined. By applying statistical methods, it was calculated that: the sensitivity of using SNORD52 and SNORD65 as detection targets reached 92.31%, the specificity reached 100.00%, the positive predictive value reached 92.31%, the negative predictive value reached 92.59%, and the accuracy reached 96.08%.

[0084] Finally, it should be noted that the above-exemplified embodiments are only the preferred embodiments of the present invention and do not limit the technical solutions of the present invention. Those of ordinary skill in the art can make non-unexpected changes to the form, content, and details of the present invention without departing from the principle of the present invention, and these changes do not deviate from the scope defined by the claims of the present invention.

Claims

1. A composition for differentiating oral squamous cell carcinoma based on saliva sample detection, characterized in that, The composition is used to identify oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in saliva samples; The composition includes: specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes, and universal reverse primers; When used to detect the expression of SNORD52 in saliva samples: The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.1; The nucleotide sequence of the specific detection forward primer is as shown in SEQ ID NO.4; The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.5; The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10; When used to detect the expression of SNORD65 in saliva samples: The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.2; The nucleotide sequence of the specific detection forward primer is as shown in SEQ ID NO.6; The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.7; The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.

10.

2. The composition according to claim 1, characterized in that, The composition further includes positive control products and negative control products.

3. The composition according to claim 2, wherein The positive control products are a mixture of artificially synthesized single-stranded SNORD52 mimics, artificially synthesized single-stranded SNORD65 mimics, and artificially synthesized RNA mimics of the internal reference gene U68; The nucleotide sequence of the artificially synthesized single-stranded SNORD52 mimics is as shown in SEQ ID NO.11; The nucleotide sequence of the artificially synthesized single-stranded SNORD65 mimics is as shown in SEQ ID NO.12; The nucleotide sequence of the artificially synthesized RNA mimics of the internal reference gene U68 is as shown in SEQ ID NO.

13.

4. The composition according to claim 2, wherein The negative control products are nuclease-free water.

5. The composition according to claim 3, characterized in that, When used to detect the internal reference gene U68: The nucleotide sequence of the corresponding specific reverse transcription stem-loop primer is as shown in SEQ ID NO.3; The nucleotide sequence of the corresponding specific detection forward primer is as shown in SEQ ID NO.8; The nucleotide sequence of the corresponding specific detection probe is as shown in SEQ ID NO.9; The nucleotide sequence of the corresponding universal reverse primer is as shown in SEQ ID NO.

10.

6. The composition according to claim 1, wherein The composition is used for the auxiliary judgment of oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in saliva samples and combining with a binary logistic regression model.

7. A kit for identifying oral squamous cell carcinoma, characterized in that, The kit includes the composition according to any one of claims 1-6.

8. The kit according to claim 7, characterized in that, The kit further includes: a snoRNA reverse transcription reaction solution, and the components of the snoRNA reverse transcription reaction solution include: RNA reverse transcriptase, 5× buffer, 0.1M DTT, dNTPs, and RNase inhibitor.

9. The kit according to claim 8, characterized in that, The kit further includes: a PCR reaction solution, and the components of the PCR reaction solution include DNA polymerase, KCl, MgCl2, Tris-HCl, and dNTPs.

10. The kit according to claim 9, wherein The kit further includes: 50× ROX Reference Dye and nuclease-free water.

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