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

Abstract

The present invention relates to a composition and use for identifying oral squamous cell carcinoma based on saliva sample detection, and belongs to the field of molecular biology technology. The composition of the present invention identifies 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. The composition of the present invention detects the expression of SNORD52 and / or SNORD65 in a saliva sample and, in combination with a binomial logistic regression model, assists in the diagnosis of oral squamous cell carcinoma, has high sensitivity and specificity, and can improve the accuracy of identifying oral squamous cell carcinoma. The present invention also provides a kit containing the composition, which detects saliva samples and has the characteristics of low sample consumption, non-invasive sampling, convenience and speed, and low risk.

Description

Technical Field

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

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, accounting for over 90% of oral cancers. Globally, the annual incidence is approximately 300,000 cases, primarily affecting middle-aged and elderly men, although the proportion of younger patients has increased in recent years. OSCC is closely associated with smoking, alcohol abuse, human papillomavirus (HPV) infection (particularly HPV types 16 / 18), and chronic oral irritation (such as poorly fitting dentures and sharp tooth stumps). OSCC survival is closely related to the stage at diagnosis. Early-stage (stages I-II) patients can achieve a 5-year survival rate of 60%-80% with combined surgery and radiotherapy. However, most patients have already progressed to advanced stages (stages III-IV) by the time of diagnosis, with a 5-year survival rate that plummets to less than 30%. Patients with lymph node metastasis, positive resection margins, or distant metastases have an extremely poor prognosis, with a recurrence rate as high as 50%-60%, often occurring within 2 years of treatment. Furthermore, the oral cavity is located at the beginning of the digestive and respiratory tracts, and is densely populated with vital organs located on the face. Therefore, in clinical practice, radical surgery based on the "tumor-free principle" is not feasible for patients with advanced or recurrent cancer. Therefore, seeking a molecular biomarker that is minimally invasive, easy to detect, and highly specific and sensitive for the auxiliary diagnosis of oral squamous cell carcinoma is of great clinical significance for both early diagnosis and follow-up monitoring.

[0003] Liquid biopsies are often viewed as an extension of in vitro molecular diagnostics, extending sampling from relevant tissues to fluids such as blood, saliva, and cerebrospinal fluid. They offer non-invasive, convenient, and low-risk sampling. Furthermore, liquid biopsies enable continuous testing, which is crucial for early cancer diagnosis, disease progression monitoring, and therapeutic efficacy assessment during cancer treatment.

[0004] Small nucleolar RNAs (snoRNAs) are short noncoding RNAs (ncRNAs) widely found in the nucleolus of eukaryotes. They range in size from 60 to 300 nucleotides and are involved in the modification and maturation of ribosomal RNA (rRNA). A growing number of studies have shown that snoRNAs are abnormally expressed in tumor tissues, cells, and body fluids, and that they are involved in regulating tumor biological behavior. Therefore, it would be of great significance to screen for snoRNAs with significant differential expression in the saliva of OSCC patients and develop reagents to detect these differentially expressed snoRNAs in saliva samples for the auxiliary diagnosis of oral squamous cell carcinoma. Summary of the Invention

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

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A composition for identifying oral squamous cell carcinoma based on saliva sample detection, wherein the composition identifies oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in the saliva sample;

[0008] The composition comprises: a specific reverse transcription stem-loop primer, a specific detection forward primer, a specific detection probe and a universal reverse primer;

[0009] When used to detect SNORD52 expression in saliva samples:

[0010] The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.1;

[0011] The nucleotide sequence of the specific detection forward primer is as SEQ ID NO.4;

[0012] The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.5;

[0013] The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10;

[0014] When used to detect SNORD65 expression in saliva samples:

[0015] The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.2;

[0016] The nucleotide sequence of the specific detection forward primer is as SEQ ID NO.6;

[0017] The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.7;

[0018] The nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.10.

[0019] In the above technical solution, the composition also includes a positive quality control product and a negative quality control product.

[0020] In the above technical solution, the positive quality 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;

[0021] The nucleotide sequence of the artificially synthesized single-chain SNORD52 mimics is shown in SEQ ID NO.11;

[0022] The nucleotide sequence of the artificially synthesized single-chain SNORD65 mimics is shown in SEQ ID NO.12;

[0023] The nucleotide sequence of the artificially synthesized RNA mimics of the internal reference gene U68 is shown in SEQ ID NO.13.

[0024] In the above technical solution, the negative quality control product is nuclease-free water.

[0025] In the above technical solution, when used to detect the artificially synthesized internal reference gene U68:

[0026] The nucleotide sequence of the corresponding specific reverse transcription stem-loop primer is shown in SEQ ID NO.3;

[0027] The nucleotide sequence of the corresponding specific detection forward primer is shown in SEQ ID NO.8;

[0028] The nucleotide sequence of the corresponding specific detection probe is shown in SEQ ID NO.9;

[0029] The nucleotide sequence of the corresponding universal reverse primer is shown in SEQ ID NO.10.

[0030] In the above technical solution, the composition assists in the diagnosis of oral squamous cell carcinoma by detecting the expression of SNORD52 and / or SNORD65 in saliva samples and combining the binomial logistic regression model.

[0031] A kit for identifying oral squamous cell carcinoma, comprising the above composition.

[0032] In the above technical solution, the kit further comprises: a snoRNA reverse transcription reaction solution, wherein the components of the snoRNA reverse transcription reaction solution include: RNA reverse transcriptase, 5× buffer, 0.1M DTT, dNTPs and RNase inhibitor.

[0033] In the above technical solution, the kit further comprises: a PCR reaction solution, wherein the components of the PCR reaction solution include DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs.

[0034] In the above technical solution, the kit further comprises: 50×ROX Reference Dye and nuclease-free water.

[0035] The beneficial effects of the present invention are:

[0036] (1) The composition of the present invention can detect changes in the expression of SNORD52 and / or SNORD65 in saliva samples, and such changes in expression are closely related to oral squamous cell carcinoma. Therefore, by detecting the expression of SNORD52 and / or SNORD65 in saliva samples and combining it with a binomial logistic regression model, an auxiliary judgment of oral squamous cell carcinoma is made, which has high sensitivity and specificity and can improve the accuracy of oral squamous cell carcinoma identification.

[0037] (2) The composition of the present invention is detected by real-time fluorescence quantitative PCR. The difference (dCt) between the detection cycles (Ct value) of 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 a comprehensive evaluation value is calculated. The oral squamous cell carcinoma is determined by comparing the comprehensive evaluation value with the critical value. This detection method does not require electrophoresis, hybridization, or other operational steps, reduces false positives, reduces contamination, and is simple to operate.

[0038] (3) The composition of the present invention is provided with an internal reference gene U68, which can effectively avoid missed detection and determine the quality of the sample; a positive quality control product is provided. If the positive quality control product is tested positive, it means that the detection system is working normally and there will be no false negative results or missed detection; a negative quality control product is provided. If the negative quality control product is tested negative, it means that the detection system is working normally. Such a design makes the detection method rigorous and effectively avoids the possibility of missed detection and wrong detection.

[0039] (4) The test kit containing the composition of the present invention is a saliva sample, which has the characteristics of small sample volume, non-invasive sampling, convenience and speed, and low risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Figure 1 The difference in expression of SNORD52 and SNORD65 in saliva between healthy subjects and patients with oral squamous cell carcinoma screened by high-throughput sequencing method;

[0042] Figure 2 This is the expression difference diagram of SNORD52 and SNORD65 detected by fluorescence quantitative PCR;

[0043] Figure 3 This is the ROC curve for the identification of oral squamous cell carcinoma using SNORD52 and SNORD65 expression as detection markers. DETAILED DESCRIPTION

[0044] The inventive concept of the present invention is as follows: snoRNA expression profile sequencing is performed on saliva samples from patients with oral squamous cell carcinoma and healthy controls to screen for differentially expressed snoRNAs. These differentially expressed snoRNAs are then validated using fluorescent quantitative PCR, confirming that snoRNAs SNORD52 and / or SNORD65 show significant expression differences between saliva samples from patients with oral squamous cell carcinoma and those from healthy controls. Saliva sample testing results indicate that a specific snoRNA combination, including SNORD52 and / or SNORD65, has excellent performance in the differential diagnosis of patients with oral squamous cell carcinoma. In summary, the specific snoRNA combination of the present invention, including SNORD52 and / or SNORD65, can be used in the preparation of reagents or kits for the identification of oral squamous cell carcinoma. The present invention also provides a method for detecting the expression of SNORD52 and / or SNORD65 in saliva samples, a composition capable of detecting the expression of SNORD52 and / or SNORD65, and a kit containing the composition, and constructs a specific reverse transcription reaction system and a fluorescent quantitative PCR reaction system. The kit and detection method of the present invention can quickly and accurately identify oral squamous cell carcinoma by detecting saliva samples, which can save medical resources for the country and has very broad clinical application prospects.

[0045] The present invention provides a composition for identifying oral squamous cell carcinoma based on saliva sample detection. The composition detects the expression of SNORD52 and / or SNORD65 in saliva samples and combines it with a binomial logistic regression model to assist in the diagnosis of oral squamous cell carcinoma, thereby effectively improving the sensitivity and specificity of auxiliary diagnosis of oral squamous cell carcinoma.

[0046] The composition comprises:

[0047] Specific reverse transcription stem-loop primer, specific detection forward primer, specific detection probe and universal reverse primer;

[0048] When used to detect SNORD52 expression in saliva samples:

[0049] The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.1;

[0050] The nucleotide sequence of the specific detection forward primer is as SEQ ID NO.4;

[0051] The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.5;

[0052] The nucleotide sequence of the universal reverse primer is as shown in SEQ ID NO.10;

[0053] When used to detect SNORD65 expression in saliva samples:

[0054] The nucleotide sequence of the specific reverse transcription stem-loop primer is as shown in SEQ ID NO.2;

[0055] The nucleotide sequence of the specific detection forward primer is as SEQ ID NO.6;

[0056] The nucleotide sequence of the specific detection probe is as shown in SEQ ID NO.7;

[0057] The nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.10.

[0058] Furthermore, the composition also includes a positive quality control product and a negative quality control product.

[0059] The positive quality control product is a mixture of artificially synthesized single-stranded SNORD52 mimics with a nucleotide sequence such as SEQ ID NO.11, artificially synthesized single-stranded SNORD65 mimics with a nucleotide sequence such as SEQ ID NO.12, and artificially synthesized RNA mimics of the internal reference gene U68 with a nucleotide sequence such as SEQ ID NO.13; the negative quality control product is nuclease-free water.

[0060] Furthermore, when used to detect the internal reference gene U68:

[0061] The nucleotide sequence of the corresponding specific reverse transcription stem-loop primer is shown in SEQ ID NO.3;

[0062] The nucleotide sequence of the corresponding specific detection forward primer is shown in SEQ ID NO.8;

[0063] The nucleotide sequence of the corresponding specific detection probe is shown in SEQ ID NO.9;

[0064] The nucleotide sequence of the corresponding universal reverse primer is shown in SEQ ID NO.10.

[0065] Furthermore, specific reverse transcription stem-loop primers of nucleotide sequences such as 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.

[0066] Furthermore, artificially synthesized single-stranded SNORD52 mimics with a nucleotide sequence such as SEQ ID NO.11, artificially synthesized single-stranded SNORD65 mimics with a nucleotide sequence such as SEQ ID NO.12, and artificially synthesized RNA mimics of the internal reference gene U68 with a nucleotide sequence such as SEQ ID NO.13 were mixed in equal proportions to form a positive quality control product.

[0067] The nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.13 are as follows:

[0068]

[0069] The present invention also provides a kit for identifying oral squamous cell carcinoma, which comprises the above composition.

[0070] Furthermore, the kit also includes a snoRNA reverse transcription reaction solution, the main components of which are RNA reverse transcriptase, 5× buffer, 0.1M DTT, dNTPs and RNase inhibitor.

[0071] Furthermore, the kit also includes a PCR reaction solution, the main components of which are DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs.

[0072] Furthermore, the kit also includes 50×ROX Reference Dye and nuclease-free water.

[0073] The kit for identifying oral squamous cell carcinoma of the present invention detects a saliva sample and the method of use comprises the following steps:

[0074] (1) Use an extraction kit to extract snoRNA (SNORD52 or SNORD6) from saliva samples. The extracted snoRNA should be used immediately. If not used temporarily, store it below -70°C.

[0075] (2) Using the snoRNA extracted in step (1) as a template, the positive quality control and the negative quality control as templates, and using the reverse transcription stem-loop primer pool as primers to reverse transcribe and prepare cDNA;

[0076] Reverse transcription was performed according to the following reaction system and amplification conditions:

[0077]

[0078] Amplification program settings:

[0079]

[0080] The obtained cDNA was used as a template for amplification reaction.

[0081] (3) Perform quantitative PCR on the prepared cDNA;

[0082] Using the prepared cDNA as a template, SNORD52 fluorescent quantitative PCR amplification is performed using 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; SNORD65 fluorescent quantitative PCR amplification is performed using 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; and an internal reference gene U68 fluorescent quantitative PCR amplification is performed using 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;

[0083] The SNORD52 quantitative PCR detection system was prepared according to the following reaction system:

[0084]

[0085] The SNORD65 quantitative PCR detection system was prepared according to the following reaction system:

[0086]

[0087] The internal reference gene U68 quantitative PCR detection system was prepared according to the following reaction system:

[0088]

[0089] Perform PCR amplification according to the following amplification conditions:

[0090]

[0091] (4) Determination of test results.

[0092] Specifically, the test result determination includes the following steps:

[0093] ① Determine whether the detection system is qualified: the positive control product SNORD52 detection Ct value, SNORD65 detection Ct value and internal reference gene U68 detection Ct value are all ≤30; the negative control products SNORD52, SNORD65 and internal reference gene U68 have no detection signal or the detection Ct value is all >43; the detection system is determined to be qualified;

[0094] ② Determine whether the sample meets the requirements: the sample is qualified if the Ct value of the sample internal reference gene U68 is ≤30;

[0095] ③ Calculate the difference between the detection cycle number (Ct value) of SNORD52, SNORD65 and the internal reference gene U68, dCt, to obtain the relative quantitative value of each oral squamous cell carcinoma-related marker. Marked as: dCt 基因1 、dCt 基因2 ; Then according to the formula Logit=9.478-0.536* dCt 基因1 -0.673* dCt 基因2 Calculate the comprehensive evaluation value;

[0096] ④ If the comprehensive evaluation value is ≥ the critical value, the probability that the sample may be oral squamous cell carcinoma increases; if the comprehensive evaluation value is < the critical value, the probability that the sample may be oral squamous cell carcinoma decreases.

[0097] Furthermore, a cutoff value was set based on the expression levels of SNORD52 and SNORD65 in saliva samples from patients with oral squamous cell carcinoma and healthy controls, as well as the expression level of the internal reference gene U68, in combination with a binomial logistic regression model to distinguish between oral squamous cell carcinoma and non-oral squamous cell carcinoma. The cutoff value was 0.7375.

[0098] In order to more thoroughly and comprehensively understand the contents of the present invention, the present invention will be described in detail 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 in the present invention. These descriptions are generally understood by ordinary technicians in the technical field of the present invention, and may make several unintended improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

[0099] Example 1

[0100] Sequencing of the full snoRNA expression profile of saliva samples to screen for differentially expressed snoRNAs;

[0101] The saliva samples of 16 patients with oral squamous cell carcinoma confirmed by clinical pathology and 9 healthy subjects (sample information is shown in Table 1) were sequenced for the whole snoRNA expression profile to screen for differentially expressed snoRNAs. Figure 1 Shown by: Figure 1 It can be seen that SNORD52 and SNORD65 were screened and showed significant expression differences between the two groups of saliva samples.

[0102] Table 1. Sample information of high-throughput sequencing of full snoRNA expression profiles

[0103]

[0104] Example 2

[0105] Fluorescence quantitative PCR method was used to verify the expression levels of differentially expressed snoRNAs screened by high-throughput sequencing;

[0106] Based on the differentially expressed snoRNAs in saliva samples of oral squamous cell carcinoma patients and healthy people screened by high-throughput sequencing, specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes and universal reverse primers were designed. The expression levels of the differentially expressed snoRNAs screened by high-throughput sequencing were verified by fluorescent quantitative PCR detection method.

[0107] The results of the study found that the expression of SNORD52 and SNORD65 was significantly different in the saliva samples of 26 patients with oral squamous cell carcinoma confirmed by clinical pathology and 25 healthy people (see Table 2 for sample information), as shown in Figure 2. Figure 2 shown.

[0108] Table 2. Information of differentially expressed snoRNA samples screened by fluorescence quantitative PCR

[0109]

[0110] Table 2. Fluorescence quantitative PCR screening of differentially expressed snoRNA samples

[0111]

[0112] Example 3

[0113] Designing the specific reverse transcription stem-loop primer, specific detection forward primer, specific detection probe, and universal reverse primer used in the present invention;

[0114] The sequences of SNORD52, SNORD65, and the internal reference gene U68 involved in this embodiment were obtained from the GeneBank database. Specific reverse transcription stem-loop primers, specific detection forward primers, specific detection probes, and universal reverse primers were designed based on the relevant sequences. The designed nucleotide sequences are detailed in the detailed description of the embodiment.

[0115] The specific reverse transcription stem-loop primer, specific detection forward primer, specific detection probe and universal reverse primer have been repeatedly verified and have the advantages of strong detection specificity, stable product structure and few dimer structures.

[0116] Specifically, the specific reverse transcription stem-loop primer of this embodiment has good reverse transcription specificity for the target snoRNA and high reverse transcription efficiency; the specific detection forward primer and specific detection probe show good specificity and stability during fluorescent quantitative PCR amplification, ensuring the specificity of PCR amplification while ensuring its amplification efficiency.

[0117] Example 4

[0118] Evaluation of the detection performance of SNORD52 alone for identifying oral squamous cell carcinoma;

[0119] Using SNORD52 as a single target, the target expression in clinical saliva samples was measured to identify oral squamous cell carcinoma. The performance of SNORD52 as a target for identifying oral squamous cell carcinoma was evaluated using clinical pathology results as the gold standard for diagnosis.

[0120] The specific steps are as follows:

[0121] Step 1: Sample collection:

[0122] Same as Example 2.

[0123] Step 2: Extract snoRNA (SNORD52) from saliva samples:

[0124] Tiangen miRcute Serum / Plasma miRNA isolation kit was used to extract snoRNA from saliva samples. The specific steps are as follows:

[0125] (1) Add 900 μL of lysis buffer MZA to every 200 μL of saliva, shake on an oscillator for 30 seconds until completely homogenized, and invert to mix;

[0126] (2) Place at room temperature for 5 minutes to allow the nucleic acid-protein complex to completely separate;

[0127] (3) Add 200 μL of chloroform, shake on an oscillator for 15 seconds to mix, and let it stand at room temperature for 5 minutes;

[0128] (4) Centrifuge at 12000 rpm for 15 minutes at 4°C, remove the supernatant, and transfer it to a new RNase-free centrifuge tube;

[0129] (5) Add 2 volumes of anhydrous ethanol, mix well, transfer the resulting solution and precipitate into the adsorption column miRelute, let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 30 seconds at room temperature, and discard the waste liquid;

[0130] (6) Add 700 μL of deproteinized MRD solution to the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0131] (7) Add 500 μL of rinse solution RW to the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0132] (8) Add 500 μL of rinse solution RW to the adsorption column miRelute, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0133] (9) Place the miRelute adsorption column into the collection tube and centrifuge at 12,000 rpm for 1 min to remove residual liquid;

[0134] (10) Place the miRelute adsorption column on a clean bench for a while to allow it to dry completely.

[0135] (11) Transfer the miRelute adsorption column 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 12000 rpm for 2 min;

[0136] (12) The concentration and purity of the snoRNA solution obtained by centrifugation were detected using Nanodrop;

[0137] (13) Store the qualified snoRNA solution in a -20℃ refrigerator for future use.

[0138] Step 3: Reverse transcription to prepare cDNA:

[0139] Using the snoRNA extracted in step 2 as a template, reverse transcription was performed using the reverse transcription stem-loop primer pool as a primer to prepare cDNA;

[0140] Perform reverse transcription according to the following reaction system and amplification conditions:

[0141]

[0142] Amplification program settings:

[0143]

[0144] The obtained cDNA was used as a template for amplification reaction.

[0145] Step 4: Perform quantitative PCR on the prepared cDNA:

[0146] The SNORD52 quantitative PCR detection system was prepared according to the following reaction system:

[0147]

[0148] The internal reference gene U68 quantitative PCR detection system was prepared according to the following reaction system:

[0149]

[0150] Perform PCR amplification according to the following amplification conditions:

[0151]

[0152] like Figure 2 As shown in the middle left figure: The relative quantitative value of the SNORD52 marker is obtained by calculating the difference in detection cycle (Ct value) dCt between SNORD52 and the internal reference gene U68. t The test and analysis results showed that there was a significant difference in the expression of SNORD52 in the saliva samples of patients with oral squamous cell carcinoma and healthy people. p <0.0001.

[0153] Step 5: Determination of test results

[0154] Specifically, the test result determination includes the following steps:

[0155] ① Calculate the difference 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 ;

[0156] ④ If dCt 基因1 ≤ critical value, the probability of judging that the sample may be oral squamous cell carcinoma increases; if dCt 基因1 > the critical value, the probability of judging that the sample may be oral squamous cell carcinoma is reduced;

[0157] The cutoff value was determined based on the expression levels of SNORD52 and the internal reference gene U68 in saliva samples from patients with oral squamous cell carcinoma and healthy controls, combined with the maximum value of the Youden index using a receiver operating characteristic (ROC) curve. The cutoff value was 5.715.

[0158] Step 6: Compare the SNORD52 expression level test results with the clinical pathological results to evaluate the performance of the test for identifying oral squamous cell carcinoma using SNORD52 as the sole detection target:

[0159] The results of oral squamous cell carcinoma identification obtained by using SNORD52 as the detection target alone were compared with the clinical pathological test results of the tested samples, as shown in Table 3:

[0160] Table 3. Detection performance evaluation results of actual clinical samples using SNORD52 as the detection target

[0161]

[0162] Table 3. Performance evaluation results of detecting actual clinical samples using SNORD52 as the detection target

[0163]

[0164] For these 51 actual clinical samples, the identification results of oral squamous cell carcinoma obtained using SNORD52 as the detection target were compared with the clinical pathological test results of the tested samples. The statistical method was used to calculate that the sensitivity of 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%.

[0165] Example 5

[0166] Evaluation of the performance of the assay using SNORD65 alone as a detection target for identifying oral squamous cell carcinoma:

[0167] Using SNORD65 as a single target, the target expression in clinical saliva samples was measured to identify oral squamous cell carcinoma. The performance of SNORD65 as a target for identifying oral squamous cell carcinoma was evaluated using clinical pathology results as the gold standard for diagnosis.

[0168] The specific steps are as follows:

[0169] Step 1: Sample collection:

[0170] Same as Example 2.

[0171] Step 2: Extract snoRNA (SNORD65) from saliva samples:

[0172] Same as Example 4.

[0173] 3. Reverse transcription to prepare cDNA:

[0174] Same as Example 4.

[0175] 4. Perform quantitative PCR on the prepared cDNA:

[0176] The SNORD65 quantitative PCR detection system was prepared according to the following reaction system:

[0177]

[0178] The internal reference gene U68 quantitative PCR detection system was prepared according to the following reaction system:

[0179]

[0180] Perform PCR amplification according to the following amplification conditions:

[0181]

[0182] like Figure 2 As shown in the middle right figure: The relative quantitative value of the SNORD65 marker is obtained by calculating the difference in detection cycle (Ct value) dCt between SNORD65 and the internal reference gene U68. t The test and analysis results showed that there was a significant difference in the expression of SNORD65 in the saliva samples of patients with oral squamous cell carcinoma and healthy people. p <0.0001.

[0183] Step 5: Determination of test results:

[0184] Specifically, the test result determination includes the following steps:

[0185] ① Calculate the difference 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 ;

[0186] ④ If dCt 基因2 ≤ critical value, the probability of judging that the sample may be oral squamous cell carcinoma increases; if dCt 基因2 > the critical value, the probability of judging that the sample may be oral squamous cell carcinoma is reduced;

[0187] The cutoff value was determined based on the expression levels of SNORD65 and the internal reference gene U68 in saliva samples from patients with oral squamous cell carcinoma and healthy controls, combined with the maximum value of the Youden index using a receiver operating characteristic (ROC) curve. The cutoff value was 8.125.

[0188] Step 6: Compare the SNORD65 expression level test results with the clinical pathological results to evaluate the performance of the test for identifying oral squamous cell carcinoma using SNORD65 as the sole detection target:

[0189] The results of oral squamous cell carcinoma identification obtained by using SNORD65 as the detection target alone were compared with the clinical pathological test results of the tested samples, as shown in Table 4:

[0190] Table 4. Detection performance evaluation results of actual clinical samples using SNORD65 as the detection target

[0191]

[0192] Table 4. Performance evaluation results of detecting actual clinical samples using SNORD65 as the detection target

[0193]

[0194] For these 51 actual clinical samples, the identification results of oral squamous cell carcinoma obtained using SNORD65 as the detection target were compared with the clinical pathological test results of the tested samples. The statistical method was used to calculate that the sensitivity of using SNORD65 as the detection target reached 76.92%, the specificity reached 92.00%, the positive predictive value reached 90.91%, the negative predictive value reached 79.31%, and the accuracy reached 84.31%.

[0195] Example 6

[0196] Binomial logistic regression model, positive judgment value study and detection performance evaluation for the identification of oral squamous cell carcinoma using SNORD52 and SNORD65 as detection targets;

[0197] According to the dCt calculated in Example 4 and Example 5 基因1 、dCt 基因2 Using SPSS software, binomial logistic regression analysis, the comprehensive evaluation value calculation formula was obtained: 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 curve (ROC) was analyzed using SPSS software. Figure 3 ) analysis, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.9538. The closer the AUC value is to 1, the better the model performance. The critical value was determined based on the maximum value of the Youden index. The critical value was 0.7375. If the comprehensive evaluation value ≥ the critical value, the probability of the sample being oral squamous cell carcinoma increases; if the comprehensive evaluation value is < the critical value, the probability of the sample being oral squamous cell carcinoma decreases.

[0198] The results of SNORD52 and SNORD65 expression level detection were compared with the clinical pathological results to evaluate the detection performance of SNORD52 and SNORD65 as detection targets for identifying oral squamous cell carcinoma. The details are shown in Table 5:

[0199] Table 5. Detection performance evaluation results of actual clinical samples using SNORD52 and SNORD65 as detection targets

[0200]

[0201] Table 5. Performance evaluation results of detecting actual clinical samples using SNORD52 and SNORD65 as detection targets

[0202]

[0203] 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 clinical pathological test results of the tested samples. The statistical method was used to calculate 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%.

[0204] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the technical solutions of the present invention. Persons skilled in the art may make unpredictable changes in the form, content, and details of the present invention without departing from the principles of the present invention, and such changes do not deviate from the scope of the present invention as defined by the claims.

Claims

1. Use of a reagent for specifically detecting the expression level of SNORD52 and / or SNORD65 in a saliva sample in the preparation of a kit for identifying oral squamous cell carcinoma.

2. The use according to claim 1, characterized in that The kit comprises a composition for identifying 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; When used to detect SNORD52 expression 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 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 SNORD65 expression 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 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 shown in SEQ ID NO.

10.

3. The use according to claim 2, characterized in that The composition also includes a positive quality control and a negative quality control.

4. The use according to claim 3, characterized in that The positive quality 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-chain SNORD52 mimics is shown in SEQ ID NO.11; The nucleotide sequence of the artificially synthesized single-chain SNORD65 mimics is shown in SEQ ID NO.12; The nucleotide sequence of the artificially synthesized RNA mimics of the internal reference gene U68 is shown in SEQ ID NO.

13.

5. The use according to claim 3, characterized in that The negative quality control product is nuclease-free water.

6. The use according to claim 4, 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 shown in SEQ ID NO.3; The nucleotide sequence of the corresponding specific detection forward primer is shown in SEQ ID NO.8; The nucleotide sequence of the corresponding specific detection probe is shown in SEQ ID NO.9; The nucleotide sequence of the corresponding universal reverse primer is shown in SEQ ID NO.

10.

7. The use according to claim 2, characterized in that The kit further comprises: a snoRNA reverse transcription reaction solution, wherein the components of the snoRNA reverse transcription reaction solution include: RNA reverse transcriptase, 5× buffer, 0.1M DTT, dNTPs and RNase inhibitor.

8. The use according to claim 7, characterized in that The kit further comprises: a PCR reaction solution, wherein the components of the PCR reaction solution include DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs.

9. The use according to claim 8, characterized in that The kit also includes: 50×ROX Reference Dye and nuclease-free water.

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