Kit for detecting multiple microRNA levels and method for screening disease markers by using kit

Abstract

The invention discloses a kit for detecting multiple microRNA levels and a method for screening disease markers by using the kit, and belongs to the technical field of molecular biology. According to the kit, at least 96 microRNAs in a sample can be detected at a time, 376 microRNAs can be detected at a time by utilizing a 384 pore plate, all potential markers are covered, the microRNAs of all targets can be completed only by extracting a single sample once, the detection time is short, and the sample demand is low. The kit has no limitation on sample collection, after the standard substance is introduced, all the samples do not need to be detected at the same time, the problem that the screened markers are not excellent enough due to insufficient sample quantity is effectively prevented, and the phenomenon that RNA is degraded due to waiting for the samples is also avoided. By utilizing the method provided by the invention, different microRNA levels of disease patients and healthy subjects can be analyzed, and reliable markers can be obtained more quickly and more effectively.

Description

Technical Field

[0001] The present application relates to the field of molecular biology technology, and in particular to a kit for detecting levels of multiple microRNAs and a method for screening disease markers using the kit. Background Art

[0002] MicroRNAs are a class of single-stranded, non-coding RNA molecules approximately 19-25 nucleotides in length. These microRNAs typically target one or more mRNAs and regulate gene expression by inhibiting the translation of target genes. Existing studies have shown that common cancers are associated with altered microRNA expression, and that microRNAs are often located in genomic regions associated with cancer. Therefore, it is believed that microRNAs may play a dual role as tumor suppressor genes and oncogenes. Mature microRNAs form miRNA-protein complexes with other proteins, making them very stable in vitro and offering greater advantages over mRNA as tumor biomarkers. The confirmed abnormal expression of microRNAs in cancer further highlights their potential application as diagnostic and prognostic biomarkers.

[0003] There are thousands of confirmed human microRNAs, and identifying effective markers for specific indications from these thousands of microRNAs is a lengthy and tedious process. Currently, potential markers are typically screened using existing technologies. Once candidate microRNAs are identified, samples are collected from both case and control groups. After collection, samples are extracted and then individually analyzed for effective markers.

[0004] In the existing marker screening process, the collected samples need to be extracted and then tested and analyzed for each microRNA. As the number of microRNAs to be tested increases, the screening cycle will be longer, so the number of microRNAs to be screened will be very small, which will increase the possibility of missing truly effective markers. Secondly, the screening cycle is too long, which means that the nucleic acid after sample extraction needs to be stored for a long time and tested multiple times by freeze-thaw cycles. However, the extracted microRNA is not protected by proteins and has a short sequence, which is very unstable. In the subsequent testing process, experimental bias will be caused by sample degradation. Moreover, because each sample must undergo multiple tests, the required sample volume is also relatively large, so the sample restriction requirements are also relatively high. Before the screening begins, all samples of the case group and the control group need to be collected. Because it is difficult to add new samples during the screening process, there will be a long sample collection period before the screening begins, or because the number of samples collected in the early stage is insufficient, the screened markers are not excellent enough. Finally, the content of different microRNAs in the human body varies greatly. During the screening process, many low-concentration targets may also be missed because they cannot be detected. Summary of the Invention

[0005] In order to solve at least one of the above technical problems, the technical solution adopted in this application is as follows.

[0006] In a first aspect, the present application provides a kit for detecting the levels of multiple microRNAs, wherein the multiple microRNAs include 2*n groups, where n≥1, each group includes 47 microRNAs, and there is no overlap between any two groups of microRNAs. The kit includes the following reagents: A standard and its specific forward and reverse primers, wherein the standard is an RNA sequence different from the microRNA sequence in the human body; Specific forward and reverse primers for each microRNA; 2n pre-amplification master mixes, each containing a set of microRNA-specific forward and reverse primers and a set of standard-specific forward and reverse primers; n reverse transcription premixes, each containing two sets of microRNA-specific reverse transcription primers and a standard-specific reverse transcription primer, Therefore, one reverse transcription master mix corresponds to two pre-amplification master mixes.

[0007] In some embodiments of the present application, the microRNA-specific reverse transcription primer comprises a 5'-terminal stem-loop structure and a 3'-terminal miRNA-specific sequence. The 5'-terminal stem-loop structure is 12 to 24 nucleotides and can be designed by those skilled in the art using conventional design software such as MFOLD; the 3'-terminal 6-8 nucleotides are complementary to the corresponding microRNA.

[0008] In some embodiments of the present application, in the microRNA-specific forward and reverse primers: the forward primer targets the 3' end of the microRNA reverse transcription product; the reverse primer targets the reverse transcription product and / or the 5' end of the microRNA reverse transcription product.

[0009] In some embodiments of the present application, the reverse primer targets a portion of the reverse transcription product and a portion of the complementary portion of the stem-loop structure. In other embodiments of the present application, the reverse primer targets only the complementary portion of the stem-loop structure, i.e., directly selecting a portion from the stem-loop structure. In some embodiments of the present application, the kit further comprises a reverse transcriptase and a DNA polymerase.

[0010] In some embodiments of the present application, the kit includes a primer plate, which includes at least 2*n third reaction well groups, each third reaction well group corresponds to one group of microRNA, and includes at least 48 wells, and the specific forward and reverse primers corresponding to 47 microRNAs and the specific forward and reverse primers of the standard are respectively placed in different third reaction wells.

[0011] In some embodiments of the present application, n=4, i.e., including 376 microRNAs, the primer plate is a 384-well plate, which can be divided into 48 wells as a group, and 8 groups can be obtained, corresponding to 8 pre-amplification premixes and 4 reverse transcription premixes.

[0012] The second aspect of the present application provides a method for screening disease markers using any one of the kits described in the first aspect of the present application, comprising the following steps: S1, obtain blood RNA samples from multiple disease patients and multiple healthy control subjects; S2, for each blood nucleic acid sample, detecting the levels of the multiple microRNAs: S21, mixing the blood RNA sample with the n reverse transcription premix solutions in n first reaction wells, respectively, wherein the reverse transcription premix solution in each first reaction well is different, and performing reverse transcription; S22, for any first reaction well, taking a portion of the reaction product and mixing it with a pre-amplification pre-mix solution corresponding to the reverse transcription pre-mix solution added to the first reaction well in a second reaction well, and taking another portion of the reaction product and mixing it with another pre-amplification pre-mix solution corresponding to the reverse transcription pre-mix solution added to the first reaction well in another second reaction well to perform PCR pre-amplification; S23, for any second reaction well, take 48 reaction products and mix them with 47 corresponding microRNA-specific forward and reverse primers and standard-specific forward and reverse primers in 48 third reaction wells, respectively, with each third reaction well containing a different specific forward and reverse primer. Perform qPCR detection and obtain the CT value of each third reaction well using the melting curve method; S3. Analyze the differences in CT values ​​of different microRNAs in disease patients and healthy control subjects. Those with significant differences are candidate microRNA markers for the disease.

[0013] In some embodiments of the present application, in step S21, the reverse transcription program is: 25˚C 10 min; 30˚C 10 min; 35˚C 10 min; 40˚C 10 min; 95˚C 5 min; and storage at 25˚C.

[0014] In some embodiments of the present application, in step S22, the PCR pre-amplification program is: 25°C 10 s; 95°C 10 min; 40°C 5 min; 95°C 10 s, 60°C 30 s, 9-13 cycles; storage at 4°C. In some specific embodiments of the present application, in step S22, the PCR pre-amplification program is: 25°C 10 s; 95°C 10 min; 40°C 5 min; 95°C 10 s, 60°C 30 s, 11 cycles; storage at 4°C.

[0015] In some embodiments of the present application, in step S23, the qPCR detection program is: 95°C 10 min; 40°C 5 min; 95°C 10 s, 60°C 40 s, 40 cycles; 95°C 15 s; 60°C 1 min; 95°C 15 s.

[0016] In some embodiments of the present application, each sample is tested 3 to 10 times, the CT values ​​of each Spike-in test are counted, and the average CT value is calculated, and the CT value of each microRNA is corrected: CT adjusted =CT original +(CT SP-average -CT SP-now ) Among them, CT SP-average Indicates the average CT value of Spike-in, CT SP-now Indicates the CT value of Spike-in in this test, CT original Indicates the CT value of microRNA before correction in this test, CT adjusted It represents the corrected CT value of microRNA in this test.

[0017] In some embodiments of the present application, the disease is cancer, such as gastric cancer, kidney cancer, bladder cancer, colon cancer, pancreatic cancer, etc.

[0018] A third aspect of the present application provides use of a detection reagent of a microRNA combination in the preparation of a kit for diagnosing gastric cancer, wherein the microRNA combination includes at least one of miRNA-21, miRNA-93 and miRNA-183.

[0019] Compared with the prior art, this application has the following beneficial effects: The kit of the present application can detect at least 96 types of microRNAs in a sample at one time. Using a 384-well plate, 376 types of microRNAs can be detected at one time, covering all potential markers. A single sample only needs to be extracted once to complete the detection of all target microRNAs, with short detection time and low sample demand.

[0020] The kit of the present application has no restrictions on sample collection. After the introduction of standard substances, some experimental operation errors can be calibrated to obtain more accurate results. There is no need to test all samples at the same time, which effectively prevents the problem of insufficient quality of the screened markers due to insufficient sample quantity, and also avoids the phenomenon of RNA degradation due to waiting for samples.

[0021] In addition, the method of the present application includes a pre-amplification step, which can detect low concentrations of targets in samples, effectively reducing the possibility of missed screening due to low target concentrations.

[0022] By using the method of the present application to analyze the levels of different microRNAs in disease patients and healthy subjects, reliable markers can be obtained more quickly and efficiently.

[0023] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other objects, features and advantages of the exemplary embodiments of the present application will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present application are shown in an illustrative and non-limiting manner, in which: Figure 1 Schematic diagram of the layout of the primer plate including specific forward and reverse primers for microRNAs numbered G1-G376 in Example 1 of the present application is shown; Figure 2 A schematic diagram of the process for detecting multiple microRNAs in Example 2 of the present application is shown; Figure 3 The figure shows the comparison of SD values ​​before and after correction of microRNAs numbered G1-G47 in Example 2 of the present application; Figure 4 The figure shows the CT value distribution of the microRNA numbered G58 in Example 3 of the present application in healthy controls (Control) and gastric cancer patients (Gastric Cancer); Figure 5 The ROC curve for the microRNA numbered G58 in Example 3 of the present application for distinguishing healthy controls from gastric cancer patients is shown; Figure 6 Schematic diagram of the layout of the primer plate including different grouping situations in Example 4 of the present application is shown; Figure 7 The CT values ​​of the detections in Example 4 of the present application including different grouping situations are shown, and “Ud” means not detected.

[0025] Figure 8 A curve diagram of the detected CT values ​​including different grouping situations in Example 4 of the present application is shown.

[0026] Figure 9 Schematic diagram showing the layout of the primer plate for different pre-amplification cycle numbers in Example 5 of the present application; Figure 10 The CT values ​​for different pre-amplification cycle numbers in Example 5 of the present application are shown. DETAILED DESCRIPTION

[0027] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, particularly for definitions of relevant terms in the art disclosed therein. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0028] The numerical ranges in this application are approximate, so unless otherwise stated, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For ranges containing values ​​less than 1 or containing fractions greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For ranges containing single-digit numbers less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0029] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination thereof.

[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer and more understandable, the present application is further described in detail below in conjunction with the embodiments.

[0031] The following examples are provided herein to illustrate preferred embodiments of the present application. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present application and, therefore, can be considered preferred embodiments of the present application. However, those skilled in the art will appreciate, based on this specification, that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present application.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the disclosures and materials cited therein are hereby incorporated by reference.

[0033] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0035] Example 1: Preparation of microRNA marker screening kit 1. Screening and grouping of markers There are a total of 376 candidate microRNA targets: hsa-let-7i-5p, hsa-miR-10a-5p, hsa-miR-122-5p, hsa-miR-1246, hsa-miR-129-5p, hsa-miR-132-3p, hsa-miR-141-3p, hsa-miR-146a-5p, hsa-miR-147b-3p, hsa-miR-149-5p, hsa-miR-151a-3p, hsa-miR-152-3p, hsa-miR-15b-5p, hsa-miR-181d-5p, hsa-miR-190b-5p, hsa-miR-193a-3p, hsa-miR-208b-3p, hsa-miR-20a-5p, hsa-miR-215-5p, hsa-miR-221-3p, hsa-miR-!222-3p, hsa-miR-22-3p, hsa-miR-25-3p, hsa-miR-324-5p, hsa-miR-32-5p, hsa-miR-339-5p, hsa-miR-34c-5p, hsa-miR-365b-5p, hsa-miR-374b-5p, hsa-miR-376c-3p, hsa-miR-377-3p, hsa-miR-410-3p, hsa-miR-4306, hsa-miR-494-3p, hsa-miR-500b-5p, hsa-miR-502-3p, hsa-miR-517-5p, hsa-miR-522-3p, hsa-miR-541-5p, hsa-miR-559, hsa-miR-582-5p, hsa-miR-621, hsa-miR-648, hsa-miR-657, hsa-miR-658, hsa-miR-664a-3p, hsa-let-7f-5p, hsa-miR-107, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-128-3p, hsa-miR-1285-3p, hsa-miR-1290, hsa-miR-130a-3p, hsa-miR-137-3p, hsa-miR-138-1-3p, hsa-miR-139-5p, hsa-miR-!21, hsa-miR-188-5p, hsa-miR-191-5p, hsa-miR-1!97-3p, hsa-miR-198, hsa-miR-199b-5p, hsa-miR-19b-3p, hsa-miR-200b-3p, hsa-miR-208a-3p It should be noted that there seems to be a "!" in front of some "miR" in the original text. It's not clear if this is a correct symbol or an error. If it's an error, it should be removed during translation. The above translation is based on the text you provided as is.hsa-miR-214-3p, hsa-miR-148a-3p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-302d-3p, hsa-miR-34b-5p, hsa-miR-361-5p, hsa-miR-376a-3p hsa-miR-378c, hsa-miR-383-5p, hsa-miR-421, hsa-miR-425-5p, hsa-miR-431-3p, hsa-miR-454-3p, hsa-miR-513a-3p, hsa-miR-516b-5p, hsa-miR -518b、hsa-miR-520a-3p、hsa-miR-520e-3p、hsa-miR-523-5p、hsa-miR-548l、hsa-miR-576-5p、hsa-miR-616-5p、hsa-miR-622、hsa-miR-625-5p、 hsa-miR-628-5p, hsa-miR-767-3p, hsa-miR-770-5p, hsa-miR-874-3p, hsa-let-7a-5p, hsa-miR-10b-5p, hsa-miR-125a-5p, hsa-miR-127-3p, hsa-miR-1299, hsa-miR-1323, hsa-miR-133a-3p, hsa-miR-135a-5p, hsa-miR-148b-3p, hsa-miR-16-5p, hsa-miR-552-3p, hsa-miR-186-5p, hsa-miR-1 87-3p、hsa-miR-196a-5p、hsa-miR-203a-3p、hsa-miR-206、hsa-miR-20b- 5p、hsa-miR-216a-5p、hsa-miR-27b-3p、hsa-miR-301a-3p、hsa-miR-30c- 5p、hsa-miR-3150b-3p、hsa-miR-320d、hsa-miR-329-3p、hsa-miR-362-5p、hsa-miR-369-3p、hsa-miR-424-5p、hsa-miR-433-3p、hsa-miR-449b-5p 、hsa-miR-450a-5p、hsa-miR-452-5p、hsa-miR-490-3p、hsa-miR-497-5p、hsa-miR-508-3p、hsa-miR-518c-3p、hsa-miR-520c-3p、hsa-miR-525-5p、hsa-miR-532-5p、hsa-miR-551a、hsa-miR-574-3p、hsa-miR-602、hsa-miR-629-5p、hsa-miR-642a-5p、hsa-miR-760、hsa-miR-888-5p、hsa-miR-92b-3p、hsa-miR-9-5p、hsa-let-7d-5p、hsa-let-7g-5p、hsa-miR-1181、hsa-miR-1202、hsa-miR-142-5p、hsa-miR-181b-5p、hsa-miR-190a-5p、hsa-miR-193b-3p、hsa-miR-194-5p、hsa-miR-202-5p、hsa-miR-205-5p、hsa-miR-210-3p、hsa-miR-211-5p、hsa-miR-23a-3p、hsa-miR-29b-3p、hsa-miR-3065-5p、hsa-miR-30d-5p、hsa-miR-3130-5p、hsa-miR-320e、hsa-miR-330-3p、hsa-miR-338-5p、hsa-miR-34a-5p、hsa-miR-411-5p、hsa-miR-432-5p、hsa-miR-483-5p、hsa-miR-484、hsa-miR-495-3p、hsa-miR-499a-5p、hsa-miR-501-3p、hsa-miR-510-5p、hsa-miR-513b-5p、hsa-miR-515-5p、hsa-miR-520f-3p、hsa-miR-544a、hsa-miR-545-3p、hsa-miR-548b-5p、hsa-miR-550a-3p、hsa-miR-93、hsa-miR-573、hsa-miR-579-3p、hsa-miR-609、hsa-miR-618、hsa-miR-650、hsa-miR-654-3p、hsa-miR-659-3p、hsa-miR-671-5p、hsa-miR-744-5p、hsa-miR-890、hsa-let-7e-5p、hsa-miR-101-3p、hsa-miR-103b、hsa-miR-105-3p、hsa-miR-1224-5p、hsa-miR-1228-3p、hsa-miR-1271-5p、hsa-miR-1291、hsa-miR-1296-5p、hsa-miR-140-3p、hsa-miR-146b-5p、hsa-miR-147a、hsa-miR-150-5p、hsa-miR-155-5p、hs a-miR-15a-5p、hsa-miR-181c-5p、hsa-miR-185-5p、hsa-miR-18b-5p、hsa -miR-196b-5p、hsa-miR-19a-3p、hsa-miR-204-5p、hsa-miR-183、hsa-miR-24-3p、hsa-miR-302a-5p、hsa-miR-31-5p、hsa-miR-320b、hsa-miR-323b -3p、hsa-miR-326、hsa-miR-337-3p、hsa-miR-33a-5p、hsa-miR-370-3p、 hsa-miR-372-3p、hsa-miR-374a-5p、hsa-miR-449c-5p、hsa-miR-451a、hs a-miR-486-5p、hsa-miR-493-5p、hsa-miR-500a-3p、hsa-miR-503-5p、hsa-miR-506-3p、hsa-miR-519a-3p、hsa-miR-519e-5p、hsa-miR-590-5p、hsa -miR-627-5p、hsa-miR-642b-3p、hsa-miR-652-3p、hsa-miR-885-3p、hsa-miR-100-5p、hsa-miR-106b-5p、hsa-miR-1207-5p、hsa-miR-1226-3p、hs a-miR-1284, hsa-miR-1306-5p, hsa-miR-144-5p, hsa-miR-154-5p, hsa-miR-17-3p, hsa-miR-195-5p, hsa-miR-1973, hsa-miR-200a-3p, hsa-miR-2 00c-3p、hsa-miR-218-5p、hsa-miR-219a-2-3p、hsa-miR-224-5p、hsa-miR-2355-3p、hsa-miR-29a-3p、hsa-miR-30e-5p、hsa-miR-328-3p、hsa-miR -342-3p、hsa-miR-363-3p、hsa-miR-375-3p、hsa-miR-4270、hsa-miR-455-3p、hsa-miR-487b-3p、hsa-miR-505-3p、hsa-miR-507、hsa-miR-509-3p、hsa-miR-518e-3p、hsa-miR-519c-3p、hsa-miR-524-5p、hsa-miR-548a-3p、hsa-miR-548d-5p、hsa-miR-567、hsa-miR-596、hsa-miR-597-5p、hsa-miR-598-3p、hsa-miR-599、hsa-miR-612、hsa-miR-637、hsa-miR-641、hsa-miR-663b、hsa-miR-668-3p、hsa-miR-765、hsa-miR-922、hsa-miR-95-3p、hsa-miR-106a-5p、hsa-miR-1260a、hsa-miR-1267、hsa-miR-1303、hsa-miR-135b-5p、hsa-miR-136-3p、hsa-miR-1-3p、hsa-miR-143-3p、hsa-miR-181a-5p、hsa-miR-182-5p、hsa-miR-212-3p、hsa-miR-23c、hsa-miR-26a-5p、hsa-miR-28-5p、hsa-miR-301b-3p、hsa-miR-30a-5p、hsa-miR-320a-3p、hsa-miR-335-5p、hsa-miR-376b-3p、hsa-miR-378b、hsa-miR-379-5p、hsa-miR-382-5p、hsa-miR-449a、hsa-miR-485-5p、hsa-miR-491-5p、hsa-miR-513c-5p、hsa-miR-517c-3p、hsa-miR-520h、hsa-miR-526a-5p、hsa-miR-539-5p、hsa-miR-542-3p、hsa-miR-575、hsa-miR-592、hsa-miR-610、hsa-miR-613、hsa-miR-645、hsa-miR-651-5p、hsa-miR-660-5p、hsa-miR-708-5p、hsa-miR-802、hsa-miR-23b-3p、hsa-miR-940、hsa-miR-96-5p、hsa-miR-98-5p、hsa-miR-99a-5p、hsa-let-7c-5p、hsa-miR-103a-3p、hsa-miR-124-3p、hsa-miR-1257、hsa-miR-1304-5p、hsa-miR-130b-3p、hsa-miR-134-5p、hsa-miR-145-5p, hsa-miR-1471, hsa-miR-153-3p, hsa-miR-1825, hsa-miR-184, hsa-m iR-18a-5p, hsa-miR-1915-5p, hsa-miR-192-5p, hsa-miR-199a-3p, hsa-miR-216b-5p, h sa-miR-223-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-299-5p, hsa-miR-29c-3p, hs a-miR-30b-5p, hsa-miR-320c, hsa-miR-340-5p, hsa-miR-345-5p, hsa-miR-365a-3p, hs a-miR-367-3p, hsa-miR-378a-3p, hsa-miR-381-3p, hsa-miR-409-3p, hsa-miR-423-5p , hsa-miR-429, hsa-miR-488-3p, hsa-miR-517a-3p, hsa-miR-518f-3p, hsa-miR-526b-3 p、hsa-miR-551b-3p、hsa-miR-561-3p、hsa-miR-570-3p、hsa-miR-572、hsa-miR-584-5p、hsa-miR-761、hsa-miR-769-5p、hsa-miR-873-5p、hsa-miR-92a-3p、hsa-miR-99b-5p. They are numbered G1, G2, ..., G375 and G376 in sequence.

[0036] The 376 microRNA targets were divided into four major groups (A / B / C / D), each containing 94 microRNA targets. Each major group was divided into two subgroups (A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2), each containing 47 microRNA targets. Homologous microRNA targets need to be divided into different subgroups. As shown in Table 1: Table 1: Target grouping

[0037] The standard is an exogenously synthesized small nucleic acid sequence Spike-in, whose sequence is not the microRNA target sequence in the human body. The Spike-in sequence selected in this example is: 5'-AAUUUACGUAACGUAUAUCGAGGC-3'.

[0038] 2. Reverse transcriptase Concentration: 160U / μL, brand: Promega.

[0039] 3. Reverse transcription master mix There are four groups, A / B / C / D, each containing 32.5% reverse transcription premix (5X), 1.1mM dNTPs, 5.5mM MgCl2, 40nM target-specific reverse transcription primers, and nuclease-free water. Group A contains specific reverse transcription primers for targets G1-G47, G189-G235, and Spike-in; Group B contains specific reverse transcription primers for targets G48-G94, G236-G282, and Spike-in; Group C contains specific reverse transcription primers for targets G95-G141, G283-G329, and Spike-in; and Group D contains specific reverse transcription primers for targets G142-G188, G330-G376, and Spike-in.

[0040] The target-specific reverse transcription primers were designed with reference to the primer design method disclosed in the Chinese invention patent publication CN103210092A.

[0041] The reverse transcription primer sequence of Spike-in is: 5'-ACCCAGTGATGGGTGCCTCGATAT-3'.

[0042] 4. PCR enzyme DNA polymerase, concentration 5 U / μL, brand Klear Taq.

[0043] 5. Pre-amplification master mix Contains 8 groups: A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2, all containing 40% pre-amplification master mix (10X), MgCl2 concentration ≥ 20mM, target-specific forward and reverse primers at a concentration of 150nM, and the rest is nuclease-free water; Group A1 contains specific forward and reverse primers for targets G1-G47 and Spike-in; Group B1 contains specific forward and reverse primers for targets G48-G94 and Spike-in; Group C1 contains specific forward and reverse primers for targets G95-G141 and Spike-in; Group D1 contains specific forward and reverse primers for targets Specific forward and reverse primers for targets G142-G188 and Spike-in; Group A2 contains specific forward and reverse primers for targets G189-G235 and Spike-in; Group B2 contains specific forward and reverse primers for targets G236-G282 and Spike-in; Group C2 contains specific forward and reverse primers for targets G283-G329 and Spike-in; Group D2 contains specific forward and reverse primers for targets G330-G376 and Spike-in.

[0044] The target-specific forward and reverse primers were designed with reference to the primer design method disclosed in Chinese invention patent publication CN103210092A.

[0045] The forward primer sequence of Spike-in is: 5'-ACCCAGCTTCCAATTTACGTAACG-3' The reverse primer sequence of Spike-in is: 5'-CCAGTGATGGGTGCCTCGATATA-3' 6. PCR Master Mix Contains 40% PCR premix (10X), 10mM MgCl2, and the rest is nuclease-free water.

[0046] 7. Standards Contains 0.1 nM spike-in standard.

[0047] 8. Primer Plate Prepare the forward and reverse primers of target G1-G376 and Spike-in into equal concentration solutions. After preparation, mix them in equal proportions according to different microRNAs and dilute them to a concentration of 0.75 μM. After mixing, use the pipetting workstation to press Figure 1 Sequencing: Add primers to a 384-well plate, 4 μL per well, move into an oven, and dry at 55°C for 80 min. After drying, seal the plate with a sealing film and seal it in an aluminum foil bag.

[0048] Example 2: Use of microRNA marker screening kit The same serum sample was tested 6 times using the kit of Example 1, and the difference between the uncorrected data of G1-G47 and the data corrected with Spike-in in Group A1 was analyzed. Figure 2 And the following steps: 1. Sample Preparation Dilute the standard Spike-in 30-fold and add it to the lysis buffer in the extraction kit, ensuring that 10 μL is added to each sample. Extract the samples according to the kit's instructions. The extraction kit used is from Miyin Biotech, model number: TQ-XQ-R-032.

[0049] 2. Reverse Transcription Take an eight-tube strip and add the extracted RNA from the sample to the eight-tube strip, 5 μL per well, add 4 wells, and mark them as A / B / C / D; prepare the reaction solution of group A, take 9.25 μL of the reverse transcription premix of group A, add 0.75 μL of reverse transcriptase, mix well, and add 10 μL of the mixture to well A of the eight-tube strip; repeat the operation for the reaction solutions of groups B / C / D; mix well and perform reverse transcription.

[0050] Reverse transcription program settings: 25˚C 10 min; 30˚C 10 min; 35˚C 10 min; 40˚C 10 min; 95˚C 5 min; store at 25˚C.

[0051] 3. Pre-amplification Prepare an eight-tube strip, labeled A1 / B1 / C1 / D1 / A2 / B2 / C2 / D2. Add the reverse-transcribed samples to the strip. Add sample A to wells A1 / A2, 4 μL per well. Repeat for samples B / C / D. Prepare the eight pre-amplification reaction solutions according to the ratios in Table 2 and add them to the corresponding wells. Mix thoroughly and proceed with pre-amplification.

[0052] Table 2: Pre-amplification reaction mix ratio

[0053] The pre-amplification program was set as follows: 25°C for 10 s; 95°C for 10 min; 40°C for 5 min; 95°C for 10 s, 60°C for 30 s, 11 cycles; and stored at 4°C.

[0054] 4. qPCR Detection 1950 μL of PCR premix was added to a 15 mL centrifuge tube. 5798 μL of nuclease-free water and 52 μL of PCR enzyme were added, mixed, and transferred to a deep-well plate, adding 864 μL to each well (one column (8 wells)). After centrifugation, the pre-amplified samples were transferred to the deep-well plate in order from top to bottom (A1 / B1 / C1 / D1 / A2 / B2 / C2 / D2), 36 μL per well. After mixing, the mixture was transferred to a primer plate using a dispenser, adding 15 μL of each mix to each well in two columns. After centrifugation, the reaction was performed in a reaction machine using the following protocol: 95°C for 10 min; 40°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 40 s; 95°C for 15 s; 60°C for 1 min; and 95°C for 15 s.

[0055] Follow steps 1-4 to complete 6 tests on the same serum sample, export the 6 test data for group A1, and analyze the calibration effect as follows: (1) Calculate the CT average value of the 6-time Spike-in detection data: CT SP-average =(CT SP1+CT SP 2+CT SP 3+CT SP 4+CT SP 5+CT SP 6) / 6 Example: CT SP-average =(15.9+16.3+16.0+16.2+16.4+15.8) / 6=16.1 According to the CT value of the Spike-in group in each experiment and the CT SP-average The difference between the two values ​​was used to calibrate the CT value of each microRNA: CT adjusted =CT original +(CT SP-average -CT SP-now ) Among them, CT SP-average Indicates the average CT value of Spike-in, CT SP-now Indicates the CT value of Spike-in in this test, CT original Indicates the CT value of microRNA before correction in this test, CT adjusted It represents the corrected CT value of microRNA in this test.

[0056] Calculate the SD of the six test data before and after calibration for each microRNA. The SD results of the six test data before and after calibration for microRNAs numbered G1 to G47 are as follows: Figure 3 shown.

[0057] Depend on Figure 3 It can be seen that after the detection data is calibrated by Spike-in, the data SD is significantly reduced, which means that the data detection stability has been significantly improved. The existence of Spike-in can calibrate some experimental operation errors, so that the experiment can obtain more accurate results.

[0058] Example 3: Application of microRNA marker screening kit in screening of small nucleic acid markers for gastric cancer A total of 272 serum clinical samples (136 gastric cancer patients and 136 controls) from Zhejiang Cancer Hospital were tested, and the calibrated data were analyzed to identify small nucleic acid markers that were differentially expressed in gastric cancer.

[0059] Using the kit produced in Example 1, 272 serum clinical samples were tested according to the detection method of Example 2; After the detection is completed, the detection data is calibrated according to the calibration method in Example 2 for each group of detected microRNAs.

[0060] Based on the CT values ​​of each microRNA detected in gastric cancer and control samples, receiver operating characteristic (ROC) curves were plotted and the area under the curve (AUC) for each microRNA was calculated. This yielded the AUCs for 376 microRNA targets, which were then evaluated according to the AUC criteria in Table 3 to identify microRNAs associated with gastric cancer.

[0061] Table 3: AUC judgment criteria

[0062] The results showed that among the 376 microRNAs, three had an AUC greater than 0.75: G58, G180, and G212. Based on the criteria in Table 3, these three microRNA targets can effectively distinguish between gastric cancer patients and healthy subjects, have high diagnostic value, and can be used as markers for gastric cancer diagnosis. The information of the three microRNAs is shown in Table 4: Table 4: Information on potential microRNA markers for gastric cancer

[0063] Among them, the microRNA with target number G58 (i.e., miRNA-21) has the following CT values ​​in gastric cancer and control samples: Figure 4 As shown, the ROC curve is as follows Figure 5 As shown, the AUC for distinguishing gastric cancer from controls was 0.809, indicating that this microRNA is a good marker for diagnosing or predicting gastric cancer.

[0064] Example 4: Determination of microRNA number groups in the microRNA marker screening kit Because microRNA fragments are short and have many similar sequences, mixing too many microRNA targets during the pre-amplification phase can lead to nonspecific amplification. Therefore, it's important to select an appropriate number of microRNA targets for grouping to optimize detection. In this example, different numbers of microRNA targets were mixed, with 24, 48, 72, and 96 microRNA targets selected for pre-amplification in each group. Serum samples and a control sample (HPLC water) underwent extraction, reverse transcription, pre-amplification, and PCR. Each sample was tested twice to determine the grouping scheme.

[0065] 1. Reagent Preparation Reverse transcription premix: Contains 32.5% reverse transcription premix (5X), 1.1mM dNTP concentration, 5.5mM MgCl2, 40nM G1-G96 target-specific reverse transcription primer concentration, and the rest is nuclease-free water.

[0066] Pre-amplification master mix: Contains 4 groups: A1 / B2 / C1 / D1, each group contains 40% pre-amplification master mix (10X), 20mM MgCl2 concentration, 150nM target-specific forward and reverse primer concentration, and the rest is nuclease-free water; among them, group A1 contains specific reverse transcription primers for targets G1-G24; group B1 contains specific reverse transcription primers for targets G1-G48; group C1 contains specific reverse transcription primers for targets G1-G72; group D1 contains specific reverse transcription primers for targets G1-G96.

[0067] PCR premix: Contains 40% PCR premix (10X), 10 mM MgCl2, and the rest is nuclease-free water.

[0068] Reverse transcriptase: concentration 160 U / μL, brand Promega.

[0069] PCR enzyme: DNA polymerase, concentration 5 U / μL, brand Klear Taq.

[0070] Primer plate: Prepare the forward and reverse primers of target G1-G96 into equal concentration solutions. After preparation, mix them in equal proportions according to different microRNAs and dilute them to a concentration of 0.75 μM. After mixing, use the pipetting workstation to press Figure 6 Sequencing: Add primers to a 384-well plate, 4 μL per well, move into an oven, and dry at 55°C for 80 min. After drying, seal the plate with a sealing film and seal it in an aluminum foil bag.

[0071] 2. Sample Preparation The samples were extracted according to the instructions of the extraction kit. The extraction kit used was brand: Miyin Bio; model: TQ-XQ-R-03.

[0072] 3. Reverse Transcription Take an eight-tube strip and add the extracted RNA to each well, adding 5 μL to two wells. Prepare the reverse transcription reaction solution by taking 27.75 μL of reverse transcription premix and adding 2.25 μL of reverse transcriptase. Mix thoroughly, then add 10 μL of the mixture to two wells of the eight-tube strip. Mix thoroughly and proceed with reverse transcription. The reverse transcription program is as follows: 25°C for 10 minutes; 30°C for 10 minutes; 35°C for 10 minutes; 40°C for 10 minutes; 95°C for 5 minutes; and store at 25°C.

[0073] 4. Pre-amplification Take an eight-tube strip, labeled A1 / B1 / C1 / C1 / D1 / D1, and mix the reverse-transcribed sample thoroughly before adding it to each of the eight tube strips. Prepare the four pre-amplification reaction solutions according to the ratios in Table 5 and add them to the corresponding wells. Mix thoroughly before pre-amplification.

[0074] Table 5: Pre-amplification reaction mix ratio

[0075] The pre-amplification program was set as follows: 25°C for 10 s; 95°C for 10 min; 40°C for 5 min; 95°C for 10 s, 60°C for 30 s, 11 cycles; and stored at 4°C.

[0076] 5. qPCR Detection Add 1950 μL of PCR premix to a 15 mL centrifuge tube, add 5798 μL of nuclease-free water, and 52 μL of PCR enzyme. Mix thoroughly, then transfer to a deep-well plate, 864 μL per well, for one column (6 wells). Centrifuge the pre-amplified samples and transfer them to the deep-well plate in order from top to bottom (A1 / B1 / C1 / C1 / D1 / D1), 36 μL per well. After mixing thoroughly, use a dispenser to transfer the mixture to the primer plate, 15 μL per well. Centrifuge and react in a reaction chamber using the following protocol: 95°C for 10 min; 40°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 40 s; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

[0077] Two serum samples and two control samples (HPLC water) were tested according to the above-mentioned detection method in steps 2-5. The results showed that when 24 or 48 microRNA primers were mixed for pre-amplification, the control samples showed no detection; when 72 or 96 microRNAs were mixed, some microRNAs in the control samples had CT values ​​(such as Figure 7 shown).

[0078] The amplification curves of serum samples showed that the plateau phase of some wells mixed with 96 microRNAs was low and the CT value was large. The amplification curves of the premixed solution groups of 24, 48, and 72 microRNAs were relatively consistent, and there was no significant difference in CT (e.g. Figure 8 and Table 6).

[0079] Table 6: CT values ​​of different groups

[0080] In summary, pre-amplification can be performed with either 24 or 48 microRNAs grouped together. However, mixing primers for 24 microRNAs for pre-amplification can be relatively complex. Therefore, pre-amplification with 48 microRNAs grouped together is relatively convenient.

[0081] Example 5: Determination of the number of pre-amplification cycles in the microRNA marker screening kit Because some microRNA targets are present at extremely low levels in the human body, they require pre-amplification before detection in the subsequent PCR process. Selecting the optimal number of pre-amplification cycles is crucial for accurately detecting the microRNA target content in a sample. In this example, different pre-amplification cycle numbers (5, 7, 9, 13, and 15) were used to test the same serum sample and a control sample (HPLC water) twice each to determine the optimal cycle number.

[0082] 1. Reagent Preparation Reverse transcription master mix: Contains 32.5% reverse transcription master mix (5X), 1.1mM dNTP concentration, 5.5mM MgCl2 concentration, 40nM G1-G48 target-specific reverse transcription primer concentration, and the rest is nuclease-free water.

[0083] Pre-amplification master mix: Contains 40% pre-amplification master mix (10X), 20mM MgCl2, 150nM G1-G48 target-specific forward and reverse primers, and the rest is nuclease-free water.

[0084] PCR premix: Contains 40% PCR premix (10X), 10 mM MgCl2, and the rest is nuclease-free water.

[0085] Reverse transcriptase: concentration 160 U / μL, brand Promega.

[0086] PCR enzyme: DNA polymerase, concentration 5 U / μL, brand Klear Taq.

[0087] Primer plate: Prepare the forward and reverse primers of target G1-G48 into equal concentration solutions. After preparation, mix them in equal proportions according to different microRNAs and dilute them to a concentration of 0.75 μM. After mixing, use the pipetting workstation to press Figure 9 Sequencing: Add primers to a 384-well plate, 4 μL per well, move to an oven, dry at 55°C for 80 min, and seal the plate with a sealing film.

[0088] 2. Sample Preparation The samples were extracted according to the instructions of the extraction kit. The extraction kit used was brand: Miyin Bio; model: TQ-XQ-R-032.

[0089] 3. Reverse Transcription Take an eight-tube strip and add the extracted RNA from four samples (two serum samples and two control samples) to each well, 5 μL per well. Prepare reverse transcription reaction solution A by taking 55.5 μL of reverse transcription premix and adding 4.5 μL of reverse transcriptase. After mixing, add 10 μL of the mixture to each well of the eight-tube strip. After mixing, reverse transcription is performed. The reverse transcription program is as follows: 25˚C for 10 min; 30˚C for 10 min; 35˚C for 10 min; 40˚C for 10 min; 95˚C for 5 min; and storage at 25˚C.

[0090] 4. Pre-amplification Take an eight-tube strip and add the reverse-transcribed samples to each of the four wells. Prepare the pre-amplification reaction solution according to the ratio in Table 7 and add it to the corresponding wells. Mix thoroughly and perform pre-amplification.

[0091] Table 7: Pre-amplification reaction mix ratio

[0092] The pre-amplification program was set as follows: 25°C for 10 s; 95°C for 10 min; 40°C for 5 min; 95°C for 10 s, 60°C for 30 s, 5 cycles; and storage at 4°C.

[0093] 5. qPCR Detection Add 1950 μL of PCR premix to a 15 mL centrifuge tube, add 5798 μL of nuclease-free water, and 52 μL of PCR enzyme. Mix thoroughly, then transfer to a deep-well plate, 864 μL per well, for one column (4 wells). Centrifuge the pre-amplified samples and transfer them sequentially from top to bottom of the deep-well plate, 36 μL per well. After mixing thoroughly, use a dispenser to transfer the mixture to a primer plate, 15 μL per well. Centrifuge and react in a reaction chamber using the following protocol: 95°C for 10 min; 40°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 40 s; 95°C for 15 s; 60°C for 1 min; and 95°C for 15 s.

[0094] Modify the pre-amplification reaction conditions in step 4 to 7, 9, 11, 13, and 15 cycles, and then test the serum samples and the control sample (HPLC water) according to the detection method in steps 3-5 above. The results are as follows Figure 10As shown, when the preamplification cycle number was set to 5, all control samples had no CT values, and most serum samples had no CT values. When the preamplification cycle number was set to 7, serum samples had CT values, but some were abnormal. When the preamplification cycle number was set to 15, both control and serum samples had CT values, and the CT values ​​of serum samples were slightly lower than those of serum samples with 9 to 13 cycles. At preamplification cycle numbers of 9, 11, and 13, serum samples showed normal results with no significant differences in CT values, and no CT values ​​were detected in control samples.

[0095] The number of pre-amplification cycles was selected to be 9 to 13 cycles to ensure the correct detection of the content of microRNA target in the sample.

[0096] In addition, it should be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A kit for detecting multiple microRNA levels, characterized in that: The plurality of microRNAs include 2*n groups, where n≥1, each group includes 47 microRNAs, and there is no overlap between any two groups of microRNAs. The kit includes the following reagents: A standard and its specific forward and reverse primers, wherein the standard is an RNA sequence different from the microRNA sequence in the human body; Specific forward and reverse primers for each microRNA; 2n pre-amplification master mixes, each containing a set of microRNA-specific forward and reverse primers and a set of standard-specific forward and reverse primers; n reverse transcription premixes, each containing two sets of microRNA-specific reverse transcription primers and a standard-specific reverse transcription primer, Therefore, one reverse transcription master mix corresponds to two pre-amplification master mixes.

2. The kit according to claim 1, wherein Also included are reverse transcriptase and DNA polymerase.

3. The kit according to claim 1 or 2, characterized in that The kit includes a primer plate, which includes at least 2*n third reaction well groups, each third reaction well group corresponds to one group of microRNA, and includes at least 48 wells. Specific forward and reverse primers corresponding to 47 microRNAs and specific forward and reverse primers of the standard are respectively placed in different third reaction wells.

4. A method for screening disease markers using the kit according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, obtain blood RNA samples from multiple disease patients and multiple healthy control subjects; S2, for each blood nucleic acid sample, detecting the levels of the multiple microRNAs: S21, mixing the blood RNA sample with the n reverse transcription premix solutions in n first reaction wells, respectively, wherein the reverse transcription premix solution in each first reaction well is different, and performing reverse transcription; S22, for any first reaction well, taking a portion of the reaction product and mixing it with a pre-amplification pre-mix solution corresponding to the reverse transcription pre-mix solution added to the first reaction well in a second reaction well, and taking another portion of the reaction product and mixing it with another pre-amplification pre-mix solution corresponding to the reverse transcription pre-mix solution added to the first reaction well in another second reaction well to perform PCR pre-amplification; S23, for any second reaction well, take 48 reaction products and mix them with 47 corresponding microRNA-specific forward and reverse primers and standard-specific forward and reverse primers in 48 third reaction wells, respectively, with each third reaction well containing different specific forward and reverse primers. Perform qPCR detection and obtain the CT value of each third reaction well using the melting curve method; S3. Analyze the differences in CT values ​​of different microRNAs in disease patients and healthy control subjects. Those with significant differences are candidate microRNA markers for the disease.

5. The method according to claim 4, wherein In step S21, the reverse transcription program is as follows: 25˚C for 10 min; 30˚C for 10 min; 35˚C for 10 min; 40˚C for 10 min; 95˚C for 5 min; and storage at 25˚C.

6. The method according to claim 4, wherein In step S22, the PCR pre-amplification program is: 25°C for 10 seconds; 95°C for 10 minutes; 40°C for 5 minutes; 95°C for 10 seconds, 60°C for 30 seconds, 9 to 13 cycles; and storage at 4°C.

7. The method according to claim 6, wherein In step S22, the PCR pre-amplification program is: 25°C for 10 seconds; 95°C for 10 minutes; 40°C for 5 minutes; 95°C for 10 seconds, 60°C for 30 seconds, 11 cycles; and storage at 4°C.

8. The method according to claim 4, wherein In step S23, the qPCR detection program is: 95°C for 10 min; 40°C for 5 min; 95°C for 10 s, 60°C for 40 s, 40 cycles; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

9. The method according to any one of claims 4 to 8, characterized in that Each sample was tested 3 to 10 times, the CT values ​​of each Spike-in test were counted, and the average CT value was calculated. The CT value of each microRNA was corrected: CT adjusted =CT original +(CT SP-average -CT SP-now ) Among them, CT SP-average Indicates the average CT value of Spike-in, CT SP-now Indicates the CT value of Spike-in in this test, CT original Indicates the CT value of microRNA before correction in this test, CT adjusted It represents the corrected CT value of microRNA in this test.

10. Use of a detection reagent comprising a microRNA combination in the preparation of a kit for diagnosing gastric cancer, characterized in that: The microRNA combination includes at least one of miRNA-21, miRNA-93 and miRNA-183.

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