Nucleic acid combination products and gastric cancer diagnostic kits

Abstract

The present application relates to a nucleic acid combination product and a gastric cancer diagnostic kit, including: a primer pair and a detection probe for detecting mRNA of the EVX1 gene, as well as a primer pair and a detection probe for detecting mRNA of the DMBX1 gene; a primer pair and a detection probe for detecting mRNA of the EVX1 gene, a primer pair and a detection probe for detecting mRNA of the DMBX1 gene, as well as a primer pair and a detection probe for detecting mRNA of the MAL gene; a primer pair and a detection probe for detecting mRNA of the EVX1 gene, as well as a primer pair and a detection probe for detecting mRNA of the PIWIL1 gene; a primer pair and a detection probe for detecting mRNA of the PIWIL1 gene, as well as a primer pair and a detection probe for detecting mRNA of the MAL gene; or a primer pair for detecting mRNA of the DMBX1 gene. The nucleic acid combination product is used to detect cfRNA with high sensitivity, strong specificity, and good gastric cancer diagnostic effect.

Description

Technical Field

[0001] The present application relates to the field of medical diagnosis technology, and in particular to a nucleic acid combination product and a gastric cancer diagnosis kit. Background Art

[0002] Gastric cancer (GC) is a highly invasive, highly molecularly and phenotypic malignant tumor of the gastric mucosal epithelium with the highest incidence. It is the fifth most common cancer and the fourth most common cause of cancer-related death in the world, and is more common in men.

[0003] Gastric cancer can be divided into squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma and carcinoid tumor according to histology. Among them, the most common histological type is gastric adenocarcinoma (STAD), which accounts for about 90% of gastric cancer cases. Gastric cancer is a multifactorial disease, mainly related to infection with Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV). H. pylori was described as a Class I carcinogen for gastric cancer by the World Health Organization's International Agency for Research on Cancer (IARC) in 1994. More than half of the world's population is infected with H. pylori, but only 2% of infected people will develop gastric cancer. EBV has been shown to affect the progression of gastric cancer in some cases (10%). In addition, family history, diet, alcoholism, smoking and unhealthy lifestyle also have a significant impact on the increased risk of gastric cancer.

[0004] Gastric cancer develops through a transitional process from a precancerous disease or lesion to cancer. Early detection and treatment are key to improving prognosis and reducing mortality. Early gastric cancer (EGC) refers to cancerous tissue located within the mucosa and submucosa, regardless of lymph node metastasis. With the increasing availability of gastroscopy, more and more patients with early-stage gastric cancer can now be diagnosed and treated surgically. In recent years, the overall incidence of gastric cancer has shown a slow downward trend worldwide. Because early-stage gastric cancer presents with nonspecific symptoms similar to those of chronic gastric diseases such as chronic gastritis and gastric ulcers, it is often missed. Most patients are already at an advanced stage by the time they are discovered, missing the optimal treatment window and resulting in a poor prognosis and low survival rate. Even for patients who undergo surgery, the 5-year survival rate is still <30%, while for patients with early-stage disease, the 5-year survival rate can reach >90% with timely treatment. Therefore, early diagnosis is key to improving prognosis and reducing mortality in gastric cancer patients. Currently, biomarker-based detection methods are a research hotspot for screening high-risk individuals for gastric cancer.

[0005] The recent surge in research on extracellular RNA (cfRNA) has opened the door to expanding the field of serological biomarkers for gastric cancer. Compared with other biomarkers, cfRNA has been shown to offer the following advantages: sensitivity and functionality, tissue specificity, low cost, clinical relevance to cancer, specificity for tumor type and subtype, and a relatively stable presence in plasma. It can distinguish cancer subtypes and trace their site of origin, reflecting phenotypic changes in local cancer sites and systemic host responses. It can be used as a biomarker for cancer diagnosis and tumor type identification, and is a promising tool for early cancer detection, prognosis, and monitoring. Numerous studies are exploring the potential of RNA species, such as microRNAs (microRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), Piwi-interacting RNAs (piRNAs), and mRNAs, as blood-based biomarkers for gastric cancer. cfRNA research has primarily focused on noncoding RNAs, particularly microRNAs. First discovered in 1993 by Lee et al. in the nematode G. elegans, miRNAs are small, non-coding, single-stranded RNAs (20-25 bp). They directly bind to the 3'-UTR of target mRNAs, degrading or inhibiting their translation. They regulate gene expression at the post-transcriptional level and are involved in the development and progression of GC and other tumors. With superior sensitivity and specificity compared to traditional proteomic markers and their high stability, miRNAs can serve as diagnostic and prognostic biomarkers for GC liquid biopsies.

[0006] In recent years, many studies have demonstrated changes in miRNA expression associated with gastric cancer and have shown that circulating miRNAs are more stable and abundant in the blood, making them easier to detect. Li et al. showed that miR-10b, miR-21, miR-126, miR-30a-5p, miR-338, let-7a, and miR-223 are independent predictors of overall survival and recurrence-free survival. 77 In addition, numerous research teams have discovered many miRNAs that function as biomarkers in GC. For example, high expression of miRNA-150, miRNA-20b, miRNA-142-5p, miRNA-214, and miRNA-375 and low expression of miRNA-433, miRNA-451, let-7g, and miRNA-125-5p are associated with short survival. For example, low levels of miRNA-126, miRNA-148, miRNA-146a, miRNA-218, miRNA-429, and miRNA-335, and high levels of miRNA-27a and miRNA-650 indicate lymph node metastasis. Distant metastasis often leads to advanced cancer stages and shorter survival. Circulating miR-21 and miR-376c levels have been found to be upregulated in the serum of patients with early-stage gastric cancer and have a positive predictive value of up to 90%. Circulating miR-196a and miR-196b, alone or in combination, have higher sensitivity and specificity than CEA or CA19-9, distinguishing GC patients from healthy controls. A panel of five miRNAs (miR-16, miR-25, miR-92a, miR-451, and miR-486-5p) has been found to be able to distinguish patients with early-stage non-cardia adenocarcinoma from those without cancer. A panel of 12 miRNAs was able to distinguish GC patients from healthy controls with an area under the curve (AUC) of 0.848 (sensitivity of 87% and specificity of 68.4%), compared to other biomarker tests showing an AUC of 0.647 for the ABC method and 0.576 for the Pg index and CEA. Furthermore, miRNA alterations appear to occur early in GC, helping to identify patients at high risk for GC development and thus improving the prognosis of GC patients. Currently, the levels of single miRNAs or combinations of multiple miRNAs have been studied in various biological fluids of cancer patients, but the results associated with disease status, stage, aggressiveness, and treatment response are highly inconsistent, with poor reproducibility and low interpretability. Multiple studies have shown consistent results for a few miRNAs, while the results for most miRNAs are conflicting. This is because miRNA levels can be affected by pre-analytical processing conditions, quantification strategies, and batch effects, and lack validation from large-scale multicenter and prospective studies.

[0007] In recent years, interest in messenger RNA (mRNA) isolated from body fluids as a potential non-invasive biomarker for cancer detection has grown. cfRNA was first discovered in 1999 in the plasma of patients with nasopharyngeal carcinoma, and subsequently in the serum of patients with melanoma. Roskams-Hieter et al. found that mRNA transcripts composed of a small number of genes in plasma can be used to distinguish cancer types and precancerous conditions in solid tumors and hematological malignancies. cfRNA sequencing has made it possible to identify mRNA biomarkers with specific tissue origin in plasma. Numerous cfRNA profiling studies have confirmed that cfRNA levels are increased in oncology patients and can serve as a suitable cancer marker. These results suggest that cfRNA may play an important role in the diagnosis and monitoring of cancer. Although several potential mRNA biomarkers, such as hTERT mRNA, have been discovered, However, limited progress has been made in the field of early GC diagnosis using cf-mRNA. This is attributed to the difficulties associated with its greater diversity, fragmentation, low abundance, low stability, poor quality and integrity, and contamination with cellular RNA. In contrast, lncRNAs and miRNAs are more stable, leading to the majority of studies focusing on analyzing noncoding RNAs, particularly miRNAs, which are more stable and abundant and thus easier to detect. Poor quality is a major obstacle to cf-mRNA analysis. Isolated RNA is typically degraded or fragmented into small fragments, resulting in variations in the expression of different regions of the same mRNA. To overcome this issue, primer design is crucial, requiring primers that probe multiple regions of the desired mRNA. Furthermore, research should focus on developing new methods to maintain cf-mRNA stability and standardized experimental procedures, including extraction, detection, and multicenter validation.

[0008] Gastroscopy and biopsy are the gold standard for gastric cancer diagnosis. They provide information on lesion depth, extent, and histopathology, enabling timely detection of gastric cancer at its earliest stages and improving the detection rate of early-stage gastric cancer. A study by Hamashima et al. found that endoscopic screening reduced gastric cancer mortality by 30% compared with a control group. However, gastroscopy is invasive, complex, and can lead to overdiagnosis. It is relatively expensive, has the potential for complications such as intestinal obstruction, and is dependent on testing equipment and endoscopist resources. Furthermore, the early diagnosis rate of gastric cancer in my country using gastroscopy is only approximately 5% to 10%. It is reported that up to 10% of early cancer lesions are missed within the three years before diagnosis. Therefore, endoscopic examination is not a universally applicable method for early gastric cancer screening.

[0009] Recent studies have demonstrated that combining serum tumor markers (CEA, CA19-9, and CA72-4) can improve gastric cancer diagnosis. Studies have shown that the combined use of serum CEA, AFP, CA19-9, and CA12-5 levels for gastric cancer diagnosis has an AUC of 0.950, which is higher than that of individual CEA, AFP, CA19-9, or CA12-5 tests. Combined testing of CA19-9 and CEA achieves higher specificity than CEA alone, with sensitivity increasing to 87%. Combined testing of CEA with CA12-5 and CA19-9 has higher sensitivity than CEA alone. The combination of CEA, CA19-9, and CA72-4 with TK1 (thymidine kinase 1, a cell proliferation biomarker) has an AUC greater than 0.820, significantly improving both the sensitivity and specificity of gastric cancer detection compared to using each biomarker individually. A meta-analysis by Zagari et al. demonstrated that the combined use of PGI, PGII, G-17, and H. pylori serological antibodies in atrophic gastritis had a sensitivity of 74.7% and a specificity of 95.6%, but this was not used for GC detection. Another validated gastric cancer-specific biomarker panel, the "GastroPanel," consisting of PGI, PGII, G-17, and H. pylori serological antibodies, demonstrated a diagnostic sensitivity of 39.9% and a specificity of 93.4% in a high-risk population. Accurate early diagnosis of gastric cancer is crucial for treatment and prognosis. Conventional blood protein tumor biomarkers such as CEA, CA19-9, and CA72-4 have insufficient specificity and sensitivity, and their role in gastric cancer diagnosis and screening, whether alone or in combination, is limited. Furthermore, these markers are often elevated in the late stages of the disease and may play a primary role in treatment monitoring and prognosis, rather than in the early detection or screening of GC. Therefore, the development of highly sensitive and specific blood biomarkers for early GC is urgently needed to provide new strategies for early gastric cancer screening. Summary of the Invention

[0010] Based on this, one or more embodiments of the present application provide a nucleic acid combination product and a gastric cancer diagnostic kit. The technical solution includes:

[0011] One or more embodiments of the present application provide a nucleic acid combination product, the nucleic acid combination product comprising:

[0012] (1) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, and a detection probe for a primer pair for detecting mRNA of the DMBX1 gene;

[0013] (2) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, a primer pair and a detection probe for detecting mRNA of the DMBX1 gene, and a primer pair and a detection probe for detecting mRNA of the MAL gene; (3) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, and a primer pair and a detection probe for detecting mRNA of the PIWIL1 gene;

[0014] (4) a primer pair and a detection probe for detecting mRNA of the PIWIL1 gene, and a primer pair and a detection probe for detecting mRNA of the MAL gene; or,

[0015] (5) Primer pairs and detection probes for detecting mRNA of the DMBX1 gene.

[0016] In some embodiments of the present application, the primer pairs for detecting the mRNA of the MAL gene include one or more pairs of primer pairs 9 to 12 defined as follows:

[0017] Detection primer pair 9 includes primers whose sequences are shown in SEQ ID NO.14 and SEQ ID NO.15;

[0018] The detection primer pair 10 includes primers having sequences shown as SEQ ID NO.16 and SEQ ID NO.17;

[0019] The detection primer pair 11 includes primers whose sequences are shown in SEQ ID NO.18 and SEQ ID NO.19; and,

[0020] The detection primer pair 12 includes primers having sequences shown as SEQ ID NO. 18 and SEQ ID NO. 20.

[0021] In some embodiments of the present application, the detection probes for the mRNA of the MAL gene include one or more of the detection probes P8 to P11:

[0022] The sequence of the detection probe P8 is shown in SEQ ID NO. 35;

[0023] The sequence of the detection probe P9 is shown in SEQ ID NO. 36;

[0024] The sequence of the detection probe P10 is shown in SEQ ID NO.37; and,

[0025] The sequence of the detection probe P11 is shown in SEQ ID NO.38.

[0026] In some embodiments of the present application, the primer pairs for detecting mRNA of the EVX1 gene include one or more pairs of primer pairs 5 to 8 defined below:

[0027] Detection primer pair 5 includes primers whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10;

[0028] The detection primer pair 6 includes primers having sequences shown as SEQ ID NO.11 and SEQ ID NO.10;

[0029] The detection primer pair 7 includes primers having sequences as shown in SEQ ID NO.11 and SEQ ID NO.12; and

[0030] The detection primer pair 8 includes primers whose sequences are shown as SEQ ID NO.13 and SEQ ID NO.10.

[0031] In some embodiments of the present application, the detection probes for the mRNA of the EVX1 gene include one or more of the detection probes P4 to 7:

[0032] The sequence of the detection probe P4 is shown in SEQ ID NO. 31;

[0033] The sequence of the detection probe P5 is shown in SEQ ID NO. 32;

[0034] The sequence of the detection probe P6 is shown in SEQ ID NO.33; and,

[0035] The sequence of the detection probe P7 is shown in SEQ ID NO.34.

[0036] In some embodiments of the present application, the primer pairs for detecting the mRNA of the DMBX1 gene include one or more pairs of primer pairs 1 to 4 defined below:

[0037] Detection primer pair 1 includes primers with sequences shown as SEQ ID NO.1 and SEQ ID NO.2;

[0038] Detection primer pair 2 includes primers whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0039] The detection primer pair 3 includes primers having sequences as shown in SEQ ID NO.5 and SEQ ID NO.6; and

[0040] The detection primer pair 4 includes primers whose sequences are shown as SEQ ID NO.7 and SEQ ID NO.8.

[0041] In some embodiments of the present application, the detection probe for the mRNA of the DMBX1 gene includes one or more of the detection probes P1 to P3:

[0042] The sequence of the detection probe P1 is shown in SEQ ID NO. 28;

[0043] The sequence of the detection probe P2 is shown in SEQ ID NO. 29; and,

[0044] The sequence of the detection probe P3 is shown in SEQ ID NO.30

[0045] In some embodiments of the present application, the primer pairs for detecting the mRNA of the PIWIL1 gene include one or more pairs of primer pairs 13 to 15 defined below:

[0046] The detection primer pair 13 includes primers having sequences shown as SEQ ID NO.21 and SEQ ID NO.22;

[0047] The detection primer pair 14 includes primers having sequences as shown in SEQ ID NO. 23 and SEQ ID NO. 22; and

[0048] The detection primer pair 15 includes primers whose sequences are shown as SEQ ID NO. 24 and SEQ ID NO. 25.

[0049] In some embodiments of the present application, the detection probes for the mRNA of the PIWIL1 gene include one or more of the detection probes P12 to P14:

[0050] The sequence of the detection probe P12 is shown in SEQ ID NO. 39;

[0051] The sequence of the detection probe P13 is shown in SEQ ID NO.40; and,

[0052] The sequence of the detection probe P14 is shown in SEQ ID NO.41.

[0053] In some embodiments of the present application, the nucleic acid combination product further includes: a detection primer pair and a detection probe for the mRNA of an internal reference gene.

[0054] In some embodiments of the present application, the sequences of the primer pair for detecting the mRNA of the internal reference gene are shown as SEQ ID NO. 26 and SEQ ID NO. 27.

[0055] In some embodiments of the present application, the sequence of the detection probe P15 for the mRNA of the internal reference gene is shown as SEQ ID NO.42.

[0056] In some embodiments of the present application, the mRNA detected by the nucleic acid combination product is cfRNA in a blood sample.

[0057] One or more embodiments of the present application also provide a use of the nucleic acid combination product in preparing a gastric cancer diagnostic kit.

[0058] One or more embodiments of the present application further provide a gastric cancer diagnosis kit, which includes: the nucleic acid combination product.

[0059] In some embodiments of the present application, the gastric cancer diagnostic kit further includes one or more of an RNA extraction reagent and an RT-qPCR reaction reagent.

[0060] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. DETAILED DESCRIPTION

[0061] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0063] the term

[0064] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0065] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0066] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0067] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0068] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0069] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0070] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0071] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0072] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0073] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0074] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0075] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0076] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0077] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0078] In a first aspect of the present application, a nucleic acid combination product is provided, comprising:

[0079] (1) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, and a primer pair and a detection probe for detecting mRNA of the DMBX1 gene;

[0080] (2) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, a primer pair and a detection probe for detecting mRNA of the DMBX1 gene, and a primer pair and a detection probe for detecting mRNA of the MAL gene;

[0081] (3) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, and a primer pair and a detection probe for detecting mRNA of the PIWIL1 gene;

[0082] (4) a primer pair and a detection probe for detecting mRNA of the PIWIL1 gene, and a primer pair and a detection probe for detecting mRNA of the MAL gene; or,

[0083] (5) Primer pairs and detection probes for detecting mRNA of the DMBX1 gene.

[0084] In some embodiments, the primer pairs for detecting the mRNA of the MAL gene include one or more pairs of primer pairs 9 to 12 as defined below:

[0085] Detection primer pair 9 includes primers whose sequences are shown in SEQ ID NO.14 and SEQ ID NO.15;

[0086] The detection primer pair 10 includes primers having sequences shown as SEQ ID NO.16 and SEQ ID NO.17;

[0087] The detection primer pair 11 includes primers whose sequences are shown in SEQ ID NO.18 and SEQ ID NO.19; and,

[0088] The detection primer pair 12 includes primers having sequences shown as SEQ ID NO. 18 and SEQ ID NO. 20.

[0089] In some embodiments, the detection probes for the mRNA of the MAL gene include one or more of the detection probes P8 to P11:

[0090] The sequence of the detection probe P8 is shown in SEQ ID NO. 35;

[0091] The sequence of the detection probe P9 is shown in SEQ ID NO. 36;

[0092] The sequence of the detection probe P10 is shown in SEQ ID NO.37; and,

[0093] The sequence of the detection probe P11 is shown in SEQ ID NO.38.

[0094] Optionally, the detection primers and detection probes for the mRNA of the MAL gene include: the detection primer pair 9 and the detection probe P8. Optionally, the detection primers and detection probes for the mRNA of the MAL gene include: the detection primer pair 10 and the detection probe P9. Optionally, the detection primers and detection probes for the mRNA of the MAL gene include: the detection primer pair 11 and the detection probe P10. Optionally, the detection primers and detection probes for the mRNA of the MAL gene include: the detection primer pair 12 and the detection probe P11.

[0095] In some embodiments, the primer pairs for detecting mRNA of the EVX1 gene include one or more pairs of primer pairs 5 to 8 defined below:

[0096] Detection primer pair 5 includes primers whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10;

[0097] The detection primer pair 6 includes primers having sequences shown as SEQ ID NO.11 and SEQ ID NO.10;

[0098] The detection primer pair 7 includes primers having sequences as shown in SEQ ID NO.11 and SEQ ID NO.12; and

[0099] The detection primer pair 8 includes primers whose sequences are shown as SEQ ID NO.13 and SEQ ID NO.10.

[0100] In some embodiments, the detection probes for the mRNA of the EVX1 gene include one or more of the detection probes P4 to 7:

[0101] The sequence of the detection probe P4 is shown in SEQ ID NO. 31;

[0102] The sequence of the detection probe P5 is shown in SEQ ID NO. 32;

[0103] The sequence of the detection probe P6 is shown in SEQ ID NO.33; and,

[0104] The sequence of the detection probe P7 is shown in SEQ ID NO.34.

[0105] Optionally, the primer pair and detection probe for detecting mRNA of the EVX1 gene include: detection primer pair 5 and detection probe P4. Optionally, the primer pair and detection probe for detecting mRNA of the EVX1 gene include: detection primer pair 6 and detection probe P5. Optionally, the primer pair and detection probe for detecting mRNA of the EVX1 gene include: detection primer pair 7 and detection probe P6. Optionally, the primer pair and detection probe for detecting mRNA of the EVX1 gene include: detection primer pair 9 and detection probe P7.

[0106] In some embodiments, the primer pairs for detecting the mRNA of the DMBX1 gene include one or more pairs of primer pairs 1 to 4 defined below:

[0107] Detection primer pair 1 includes primers with sequences shown as SEQ ID NO.1 and SEQ ID NO.2;

[0108] Detection primer pair 2 includes primers whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0109] The detection primer pair 3 includes primers having sequences as shown in SEQ ID NO.5 and SEQ ID NO.6; and

[0110] The detection primer pair 4 includes primers whose sequences are shown as SEQ ID NO.7 and SEQ ID NO.8.

[0111] In some embodiments, the detection probes for the mRNA of the DMBX1 gene include one or more of the detection probes P1 to P3:

[0112] The sequence of the detection probe P1 is shown in SEQ ID NO. 28;

[0113] The sequence of the detection probe P2 is shown in SEQ ID NO. 29; and,

[0114] The sequence of the detection probe P3 is shown in SEQ ID NO.30.

[0115] Optionally, the primer pair and detection probe for detecting DMBX1 gene mRNA include: detection primer pair 1 and detection probe P1. Optionally, the primer pair and detection probe for detecting DMBX1 gene mRNA include: detection primer pair 2 and detection probe P2. Optionally, the primer pair and detection probe for detecting DMBX1 gene mRNA include: detection primer pair 3 and detection probe P3. Optionally, the primer pair and detection probe for detecting DMBX1 gene mRNA include: detection primer pair 4 and detection probe P1.

[0116] Further optionally, the primer pair and detection probe for detecting the mRNA of the EVX1 gene include: detection primer pair 9 and detection probe P7, and the primer pair and detection probe for detecting the mRNA of the DMBX1 gene include: detection primer pair 2 and detection probe P2.

[0117] In some embodiments, the primer pairs for detecting mRNA of the PIWIL1 gene include one or more pairs of primer pairs 13 to 15 defined below:

[0118] The detection primer pair 13 includes primers having sequences shown as SEQ ID NO.21 and SEQ ID NO.22;

[0119] The detection primer pair 14 includes primers having sequences as shown in SEQ ID NO. 23 and SEQ ID NO. 22; and

[0120] The detection primer pair 15 includes primers whose sequences are shown as SEQ ID NO. 24 and SEQ ID NO. 25.

[0121] In some embodiments, the detection probes for the mRNA of the PIWIL1 gene include one or more of the detection probes P12 to P14:

[0122] The sequence of the detection probe P12 is shown in SEQ ID NO. 39;

[0123] The sequence of the detection probe P13 is shown in SEQ ID NO.40; and,

[0124] The sequence of the detection probe P14 is shown in SEQ ID NO.41.

[0125] Optionally, the primer pair and detection probe for detecting the mRNA of the PIWIL1 gene include: detection primer pair 13 and detection probe P12. Optionally, the primer pair and detection probe for detecting the mRNA of the PIWIL1 gene include: detection primer pair 14 and detection probe P13. Optionally, the primer pair and detection probe for detecting the mRNA of the PIWIL1 gene include: detection primer pair 15 and detection probe P14.

[0126] Further optionally, the detection primer pair and detection probe for EVX1 gene mRNA include: detection primer pair 9 and detection probe P7, and the detection primer pair and detection probe for PIWIL1 gene mRNA include: detection primer pair 14 and detection probe P13. In some embodiments, the nucleic acid combination product further includes: a detection primer pair and detection probe for the mRNA of an internal reference gene.

[0127] In some embodiments, the sequences of the primer pair for detecting the mRNA of the internal reference gene are shown as SEQ ID NO.26 and SEQ ID NO.27.

[0128] In some embodiments, the sequence of the detection probe P15 for the mRNA of the internal reference gene is shown as SEQ ID NO.42.

[0129] In some embodiments, the mRNA detected by the nucleic acid combination product is cfRNA in a blood sample.

[0130] In a second aspect of the embodiments of the present application, there is also provided a use of the nucleic acid combination product in preparing a gastric cancer diagnostic kit.

[0131] In a third aspect of the embodiments of the present application, a gastric cancer diagnosis kit is provided, wherein the gastric cancer diagnosis kit comprises: the nucleic acid combination product described above.

[0132] In some embodiments of the present application, the gastric cancer diagnostic kit further includes one or more of an RNA extraction reagent and an RT-qPCR reaction reagent.

[0133] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0134] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0135] Table 1

[0136] English abbreviation Full English name Full Chinese name AGC Advanced Gastric Cancer Advanced gastric cancer AUC Area Under Curve Area under the curve CA19-9 Carbohydrate Antigen 19-9 Carbohydrate antigen 19-9 CA72-4 Carbohydrate Antigen 72-4 Carbohydrate antigen 72-4 cfNA Circulating Nucleic Acid Circulating free nucleic acids cfDNA Circulating Cell-free DNA circulating free DNA cfRNA Circulating Cell-free RNA circulating free RNA CI Confidence Interval Confidence interval circRNA circular RNA circular RNA ctDNA Circulating Tumor DNA circulating tumor DNA EGC Early Gastric Cancer Early gastric cancer GC Gastric Cancer Gastric cancer GO Gene Ontology Gene Ontology lncRNA long non-coding RNA long noncoding RNA miRNA microRNA microRNA mRNA messenger RNA messenger RNA PCR Polymerase Chain Reaction polymerase chain reaction piRNA piwi-interacting RNA piwi-interacting RNA RT-PCR Reverse Transcription Polymerase Chain Reverse transcription-polymerase chain reaction STAD Stomach adenocarcinoma Gastric adenocarcinoma Support Vector Machine Support Vector Machine Support Vector Machine Sen Sensitivity Sensitivity Spe Specificity Specificity

[0137] 1. Construction of an early diagnosis model for GC based on cfRNA

[0138] 1.1 Differential expression analysis to screen cfRNA

[0139] The 100 genes obtained from differential expression analysis were input into the SVM classification algorithm model for training. The top 56 models trained by SVM with model performance approaching 100% including accuracy, sensitivity, specificity and Youden index were selected. Then, 23 RNAs with higher weights were screened from these models, including DEFA5, ESM1, HOTAIR, PGA5, ADIPOQ, EVX1, LINCO1234, PIWIL1, ASB5, HOXC11, ATP4A, CHGA, CIDEA, HOXC8, CMTM5, DMBX1, HOXC9, HOXC-AS3, KRT24, MAL, PGA3, RDH8, and SH3GL2.

[0140] The screening ideas for the cfRNA test set are as follows:

[0141] To validate the RNA of the 23 genes mentioned above by RT-qPCR, we first designed and validated primer probes. To avoid genomic contamination, the best approach is to use two primers targeting two exons. Using genomic DNA as a template will contain at least one intron, making the target sequence particularly large. Such large fragments cannot be detected by qPCR amplification.

[0142] (1) Theoretical value verification

[0143] Using the MAL gene as an example, we explain how to screen for a gene. First, use GeneBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) to query the CDS sequence of MAL. Import the CDS sequence into PrimerPremier 5 software or directly enter the gene's NM number into the Primer design tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast) to design primers based on the principles of primer design. Use Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast) to verify the theoretical values. Filter out genes and their primer pairs with less than 1000bp of contamination in the genome and transcriptome. Once the primer pair for the gene is determined, design the probe in Primer Premier 5 software according to the principles of probe design.

[0144] Using NM_002371.4, a total of 10 MAL primer pairs were designed. BLAST analysis was performed to eliminate primer pairs that did not meet the design guidelines, such as those with excessively high or low GC content or those prone to primer-dimer formation. Ultimately, four primer pairs, MAL-2, 4, 5, and 9-F, were obtained. After theoretical value verification, these four sequences were validated by PCR, and the final primer pairs were determined by observing the target product bands.

[0145] (2) RT-qPCR verification

[0146] First, whole blood was used to validate the different primer pairs for MAL. The CT values for GAPDH were around 20, and for all four MAL primer pairs, around 30. MAL-4 had the lowest CT value, indicating the highest expression level. Next, gel electrophoresis was performed on the PCR products of the four MAL primer pairs. The actual target products for all four primer pairs were consistent with the theoretical values. However, the lanes for MAL-2, 5, and 9-F all showed bands, indicating genomic contamination. In contrast, MAL-4-F showed clear bands without any other bands, and its CT value was the lowest, thus meeting the criteria of "high expression level, consistent target product, and no contamination," and was therefore selected.

[0147] After MAL-4-F was identified, it was further validated using serum. Similar to the other gene screening processes, this process filtered out genes with genomic contamination and CT values greater than 39, ultimately resulting in a validation set of six genes: DMBX1, ESM1, EVX1, MAL, PGA5, and PIWIL1.

[0148] 1.2 Target mechanism research

[0149] Blood cfRNA testing offers advantages such as being noninvasive, highly sensitive, overcoming tumor heterogeneity, and enabling real-time monitoring of disease progression. Research on blood cfRNA (mRNA) is increasingly demonstrating its potential value in the early diagnosis of GC. Related studies have shown that circulating mRNAs such as MACC1, PD-L1, GXYLT2, HTRA2, SHIP2, and GDF-15 have been identified as promising biomarkers for GC diagnosis. However, the application of cfRNA in GC diagnosis requires more than just biomarker development; efficient, sensitive, and cost-effective cfRNA detection methods are equally important. Commonly used cfRNA detection techniques include reverse transcription quantitative PCR (RT-qPCR), RNA sequencing (RNA-seq), and microarrays. RT-qPCR offers high sensitivity, specificity, and reproducibility, making it suitable for detecting the expression of individual cfRNAs. RNA-seq is a high-throughput sequencing technology that can be used to identify and quantify all mRNA molecules in plasma, including those expressed at very low levels, facilitating the discovery of new biomarkers and disease-associated genes. Compared with RT-qPCR, RNA-seq has the advantages of high throughput, high accuracy and comprehensiveness. Chip technology is a high-throughput cfRNA detection method that can simultaneously detect the expression levels of thousands of cfRNAs. Chip technology has the characteristics of high throughput, high specificity and high sensitivity, and is suitable for rapid screening of large amounts of cfRNA. These technologies each have their own advantages and limitations. The choice of which technology depends on factors such as the purpose of the research, the type of sample, the type and quantity of mRNA to be detected, and the experimental conditions. At present, RT-qPCR has become the detection method used by most laboratories due to its high sensitivity, high specificity, high repeatability and economical applicability. The embodiment of the present application also uses RT-qPCR to detect cfRNA in blood samples, and finally verifies that 4 cfRNA (mRNA) targets are obtained, namely DMBX1, EVX1, MAL and PIWIL1. Based on these 4 targets, the embodiment of the present application further constructed a diagnostic model for GC and explored its value in the diagnosis of GC. The primer pairs designed to detect these 4 targets are shown in the following table:

[0150] Table 2

[0151]

[0152]

[0153] DMBX1 (Diencephalon Mesencephalon Homeobox 1), diencephalon / midbrain homeobox 1, is a protein-coding gene that belongs to the homeobox gene family and contains a conserved DNA sequence called a homeobox. GO annotations related to DMBX1 include DNA-binding transcription factor activity and sequence-specific DNA binding. DMBX1 was originally isolated in chicks and mice and was called Atx. Later, because of its high sequence similarity with the orthodenticle homeobox (OTX) gene family, it was named "OTX3". There is a single DMBX1 gene in the genome of terrestrial vertebrates, while the teleost genome has at least two paralogs, such as zebrafish containing DMBX1a and DMBX1b genes. The expression of DMBX1 was found to be crucial in the central nervous system, especially in the midbrain and forebrain. For example, Ohtoshi et al. found that the expression pattern of DMBX1 in the mouse embryonic brain was mainly concentrated in specific developmental stages and regions. At 7.5 and 8.5 days In embryos of 14 days old, DMBX1 is mainly expressed in a subregion of the anterior head fold, while in embryos of 9.5 days old, its expression extends to the caudal diencephalon and midbrain and is limited to the neuroepithelium. It is worth noting that DMBX1 is species conserved, which suggests that it may play a similarly important role in human brain development and function, especially in early embryonic development. In addition, studies have shown that DMBX1 plays an important role in the neurogenesis of the midbrain and retina in zebrafish. For example, Wong et al. proposed that DMBX1 plays a role in the proliferation of retinal progenitor cells (RPCs) in zebrafish embryos. It is crucial for the transition between reproductive and postmitotic states, and the Fgf-vsx2-dmbx1-ccnd1 signaling axis was proposed. As the research deepened, DMBX1 was found to play a role in the normal regulation of energy homeostasis and behavior. In summary, DMBX1 plays an important role in postnatal embryonic survival, growth, limb development, central nervous system development, retinal development, cell cycle regulation, maintenance of feeding behavior and energy homeostasis. In recent years, researchers have found that DMBX1 is associated with the occurrence and development of various cancers. For example, Luo et al. explored the role of DMBX1 in The study examined the role of DMBX1 in the proliferation and cell cycle progression of lung adenocarcinoma cells. High levels of DMBX1 were found to be associated with enhanced proliferation in lung adenocarcinoma cells. DMBX1 promotes tumor cell proliferation and cell cycle progression by inhibiting the transcription of p21 (a cell cycle inhibitor) mediated by OTX2 (a homeobox transcription factor associated with eye development). This may cause tumor cells to evade normal cell cycle regulation, thereby promoting continued tumor growth and division. By influencing the interaction between OTX2 and p21, DMBX1 may provide new insights into the diagnosis and treatment of lung adenocarcinoma.In addition, the researchers found that DMBX1, as a target gene regulated by miR-455-5p / -3p and miR-23c, was associated with poor prognosis in patients with prostate cancer and endometrial cancer. Based on the above information, it is upregulated in prostate cancer, endometrial cancer and lung adenocarcinoma, and is associated with poor prognosis in tumor patients. This suggests that DMBX1 may promote tumor development by regulating cell cycle-related proteins. However, the results of the preliminary experiments of this application showed that DMBX1 expression was downregulated in MGC-803 and AGS cell lines, which may indicate that the effects of DMBX1 in different tumor cell lines are different, or are affected by specific experimental conditions. In order to further explore this phenomenon, more detailed molecular mechanism studies may be needed in the future, including the expression regulatory network of DMBX1 in different GC cell lines, and the specific action pathway of DMBX1 in GC development.

[0154] The EVX1 (Even-Skipped Homeobox 1) gene is a protein-coding gene. GO annotations associated with this gene include DNA-binding transcription factor activity and sequence-specific DNA binding. EVX1 is a member of the even-skipped homeobox family of proteins, characterized by a homeodomain closely related to the even-skipped (Eve) segmentation gene in Drosophila. Studies have shown that Eve is an important homeobox gene that controls segment patterning in Drosophila embryos. The Eve homeobox encodes a highly conserved DNA-binding domain that binds to two DNA consensus sequences: an AT-rich sequence and a GC-rich sequence. Studies have revealed that the mechanism by which homeobox proteins regulate specificity is through tandem binding of two homeodomains to a 10-bp DNA sequence. Residue Gln50 has also been shown to form direct and water-mediated hydrogen bonds with DNA bases. As a transcription factor, EVX1 is involved in various developmental processes. For example, Kalisz et al. revealed that the BMP4 / activin-EVX1-GSC pathway mediates cell fate selection during stripe-like development in mammalian embryos and embryonic stem cells. Similarly, Bell et al. identified EVX1 as a key downstream effector of BMP4 and WNT3A in posterior mesoderm patterning. Other researchers, analyzing the molecular mechanisms, have found that four different Eve mutations—Fushi Tarazu (FTZ), Enrailed (En), and Wingless (WG)—disrupt the normal Eve expression pattern. However, this disruption of Eve patterning is not due to promoter changes but rather involves abnormal Eve protein. Furthermore, EVX1 has been found to play a key role in the development and identity of spinal interneurons. In summary, EVX1 has been linked to embryonic stem cell development, spinal interneuron development, hand, foot, mouth, and genital syndrome, and preaxial defects. As research deepens, EVX1 has been found to be associated with disease. The formation of two fusion genes, ETV6::EVX2 and MSI2::EVX1 / HOXA13, activates the abnormal upregulation of the HOXD and HOXA gene clusters through enhancer hijacking, thereby affecting the normal development and function of T cells and ultimately leading to the development of pediatric immature T-cell acute lymphoblastic leukemia (T-ALL). Preliminary experimental results of this application showed that EVX1 expression was downregulated in MGC-803 and AGS cell lines, which may indicate that the role of EVX1 varies in different cell lines or is affected by specific experimental conditions.To further explore this phenomenon, more detailed molecular mechanism studies may be needed in the future, including the expression regulatory network of EVX1 in different GC cell lines and the specific action pathway of EVX1 in GC development.

[0155] MAL (Myelin and Lymphocyte protein, T Cell Differentiation Protein) is a T cell differentiation protein expressed in the middle and late stages of T cell differentiation. Genetic annotations associated with this gene include lipid binding and peptidase activator activities involved in apoptosis. The MAL family, first discovered and characterized in 2002, consists of seven members: MAL, MAL2, PLLP, MALL, MYADM, MYADML2, and CMTM8, located on different chromosomes. MAL is a highly hydrophobic integral membrane protein primarily localized to the apical zone of polarized epithelial cells and the dense myelin sheath of nervous system cells. It assists in transport vesicles to regulate protein sorting and is involved in myelin biogenesis and function. MAL is widely expressed in respiratory, nervous, genitourinary, gastrointestinal, and endocrine / exocrine epithelial tissues. MAL has a binary role in carcinogenesis: either an oncogene or a tumor suppressor. For example, MAL can act as an oncogenic factor in ovarian cancer and certain types of lymphoma, promoting tumor progression. In contrast, hypermethylation of the MAL promoter leads to its downregulation of expression, which enhances cellular functions that are conducive to cancer progression, such as proliferation and migration, or reduces cellular functions that prevent cancer progression, such as DNA repair, apoptosis, or elimination of malignant cells by the immune system. Abnormal expression of MAL has been reported in a variety of malignant tumors, such as lung cancer, breast cancer, bladder cancer, salivary gland cancer, etc. Overexpression of MAL has been shown to induce apoptosis of tumor cells, while its inhibition can lead to epithelial-mesenchymal transition (EMT), indicating its potential as a tumor cell proliferation inhibitor and a candidate for tumor immunotherapy. The experimental results of the previous study of this application showed that the expression of MAL was downregulated in the cell lines MGC-803 and AGS, and in serum, which is consistent with the above research results and the expression of tissues in the database.

[0156] PIWIL1 (Piwi Like RNA-mediated Gene Silencing 1) belongs to the human PIWI subfamily, and its GO annotations include nucleic acid binding and mRNA binding. The PIWI subfamily interacts and binds to piRNAs, silencing transposons to help maintain genomic integrity in germ cells. Loss of these proteins leads to severe fertility defects in species ranging from nematodes to mammals. With increasing research, PIWI proteins have been found to be expressed not only in the testis, contributing to male fertility, but also in various types of cancer. Inhibition of PIWI expression can prevent sperm production and cancer progression. PIWI proteins are involved in regulating a wide range of biological processes, including cancer epigenetics, transposon silencing, and translational repression. PIWIL1, a member of the PIWI subfamily, contains a PIWI domain and a C-terminal E3 domain. PIWIL1's E3 activity enables it to regulate the stability and activity of interacting proteins through ubiquitination, contributing to its involvement in gene silencing and epigenetic regulation. PIWIL1 is an oncogene first reported to be overexpressed in seminoma. For example, Wang et al. found significant correlations between the expression of human PIWI subfamily proteins and T stage, lymph node metastasis, and TNM stage. Furthermore, they found that PIWIL1 expression status was closely associated with tumor differentiation, tumor size, and TNM stage. PIWIL1 has been shown to potentially play a role in cancer in a piRNA-independent manner. For example, Li et al. found that in the absence of piRNAs, PIWIL1 promotes the degradation of the cell adhesion protein Pinin by acting as an activator of the APC / C E3 complex, thereby contributing to pancreatic cancer metastasis. Furthermore, studies have demonstrated that the formation of functional piRNA-silencing complexes involving PIWIL1 and piRNAs was not detected in colorectal cancer (CRC) cell lines. Notably, PIWIL1 has distinct functional modes in somatic cells and male germ cells. PIWI proteins are specifically expressed in the testis under physiological conditions, but their expression is aberrantly induced in human cancer cells. Furthermore, PIWIL1 has been shown to be absent from normal somatic tissues. This suggests that it may be a potential therapeutic target, as most non-cancerous cells are not susceptible to cytotoxic effects. Preliminary experimental results from this application showed that PIWIL1 expression was downregulated in MGC-803 and AGS cell lines, which may indicate that the role of PIWIL1 varies across cell lines or is affected by specific experimental conditions. To further explore this phenomenon, more detailed molecular mechanisms may be needed in the future, including the expression regulatory network of PIWIL1 in different GC cell lines and the specific pathways of action of PIWIL1 in GC development.

[0157] 2. Verification

[0158] 2.1 Whole blood collection and processing

[0159] Sample situation: 155 samples were selected (45 gastric cancer samples, 35 interference diseases, and 75 healthy people).

[0160] Samples were collected at Renji Hospital affiliated to Shanghai Jiao Tong University from September 2022 to May 2023. This study was approved by the Medical Ethics Committee of Renji Hospital affiliated to Shanghai Jiao Tong University, with ethics approval number: KY2023-062-B. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki. Written informed consent signed by all research subjects or their families was obtained for the collection and use of clinical data.

[0161] Sample inclusion criteria:

[0162] Gastric cancer enrollment criteria are as follows: The World Health Organization (WHO) classification of gastric cancer is recommended for histological classification. The staging system for gastric cancer is the joint staging system developed by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC). Histopathological examination serves as the gold standard, while the final diagnosis is based on the expertise of experienced clinicians.

[0163] Interference sample inclusion: patients diagnosed with other types of gastric diseases (such as gastric ulcers and gastritis) and with negative pathological results were excluded from the gastric cancer healthy group. Three conditions were selected: (1) according to the questionnaire, subjects with common chronic diseases (hypertension, diabetes, coronary heart disease), a history of tumor treatment, and a history of major surgery were excluded; (2) self-reported no obvious clinical symptoms; (3) physical examination results were within the normal range and no obvious abnormalities were found; and physical examination results were outside the normal range or abnormal but judged by the doctor to be clinically insignificant. Among them, the physical examination items included: LDCT, abdominal color Doppler ultrasound, four tumor markers, blood pressure, etc.

[0164] Use a vacuum tube with a red cap to collect 3 mL-5 mL of whole blood. Within 2 hours after collection, first centrifuge the blood sample (centrifugation conditions: 1200g, 4°C, 10 min) to obtain serum, transfer the supernatant after centrifugation to a 1.5 mL centrifuge tube, and centrifuge again (13000 rpm, 4°C, 10 min). Transfer the supernatant to a 1.5 mL centrifuge tube and store at -80°C until RNA extraction.

[0165] 2.2 Serum cfRNA extraction and detection

[0166] Total cfRNA was extracted from 1.5 mL of serum using the QIAamp Recycling Nucleic Acid Extraction Kit according to the manufacturer's instructions. RNA was eluted with 80 μL of enzyme-free water and stored at 4°C until RT-qPCR. The integrity of the extracted RNA was assessed using a Nanodrop and an Agilent RNA6000 Pico chip. RT-qPCR primers and probes were selected from Table 2. Specifically, for the DMBX1 gene, DMBX1-3, DMBX1-4, and detection probe P2 were used; for EVX1, EVX1-7, EVX1-8, and detection probe P7 were used; for the MAL gene, MAL-3, MAL-4, and detection probe P9 were used; for the PIWIL1 gene, PIWIL1-3, PIWIL1-4, and detection probe P13 were used; and for the GAPDH gene, GAPDH-1, GAPDH-2, and detection probe P15 were used.

[0167] Table 3. RT-qPCR system

[0168] 20μL system Reagent (μL) RT-qPCR mix 10.2 Primers (20 μM / each primer) 0.6 Probe (20 μM / each probe) 0.4 template 8.8

[0169] Table 4. RT-qPCR reaction procedure

[0170]

[0171] 2.3 Comparison of test results from different panels

[0172] The above 155 samples were tested for the targets shown in the following table. The results are shown in the following table:

[0173] Table 5. Comparison results of linear models (linear SVM model)

[0174] Target combination AUC Sen(%) Spe (%) DMBX1 0.70 80.0 74.3 EVX1 0.80 90.0 77.1 MAL 0.75 80.0 82.9 PIWIL1 0.70 60.0 85.7 DMBX1+EVX1 0.81 90.0 80.0 DMBX1+MAL 0.53 90.0 34.3 DMBX1+PIWIL1 0.70 60 82.8 EVX1+PIWIL1 0.81 90.0 80.0 MAL+PIWIL1 0.75 80.0 80.0 EVX1+MAL 0.80 80.0 85.7 DMBX1+EVX1+PIWIL1 0.78 90.0 71.4 DMBX1+MAL+PIWIL1 0.68 90.0 51.4 DMBX1+EVX1+MAL 0.75 90.0 68.6 EVX1+MAL+PIWIL1 0.79 90.0 74.3

[0175] The data in the above table takes the detection targets of DMBX1 and EVX1 as an example. Taking into account the detection of internal reference genes, two duplex RT-qPCRs are performed, namely DMBX1 and internal reference genes, and EVX1 and internal reference genes. Of course, in the process of detecting DMBX1 and EVX1, triple PCR of DMBX1, EVX1 and internal reference genes can also be performed. The same is true for other detection schemes.

[0176] Table 6. Comparison of the accuracy of nonlinear model detection (KNN-fine KNN (%) → SVM-fine Gaussian SVM (%) → ensemble-boosted tree (%) → ensemble-subspace KNN (%) → SVM-quadratic SVM (%))

[0177]

[0178] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0179] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. Application of nucleic acid combination products in the preparation of gastric cancer diagnostic kits; The nucleic acid combination product includes: (1) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, and a primer pair and a detection probe for detecting mRNA of the DMBX1 gene; (2) a primer pair and a detection probe for detecting mRNA of the EVX1 gene, a primer pair and a detection probe for detecting mRNA of the DMBX1 gene, and a primer pair and a detection probe for detecting mRNA of the MAL gene; or, (3) a primer pair and a detection probe for mRNA of the DMBX1 gene; The detection primer pair for the mRNA of the DMBX1 gene includes primers whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, and the sequence of the detection probe is shown in SEQ ID NO.29; The EVX1 gene mRNA detection primer pair includes primers whose sequences are shown in SEQ ID NO.13 and SEQ ID NO.10, and the detection probe sequence is shown in SEQ ID NO.34; The primer pair for detecting the mRNA of the MAL gene includes primers whose sequences are shown in SEQ ID NO.16 and SEQ ID NO.17, and the detection probe has a sequence shown in SEQ ID NO.

36.

2. The use according to claim 1, characterized in that The nucleic acid combination product further includes: a detection primer pair and a detection probe for the mRNA of the internal reference gene.

3. The use according to claim 2, characterized in that The nucleic acid combination product satisfies one or more of the following conditions (i) and (ii): (i) the sequences of the primer pair for detecting the mRNA of the internal reference gene are shown in SEQ ID NO. 26 and SEQ ID NO. 27; and, (ii) The sequence of the detection probe P15 for the mRNA of the internal reference gene is shown in SEQ ID NO.

42.

4. The use according to claim 1, characterized in that The mRNA detected by the nucleic acid combination product is cfRNA in the blood sample.

5. The use according to any one of claims 1 to 4, characterized in that The gastric cancer diagnostic kit further includes one or more of an RNA extraction reagent and an RT-qPCR reaction reagent.

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