Application of diagnostic marker hsacirc0001021 in peripheral blood of spinal tuberculosis

Abstract

The invention discloses application of a diagnostic marker hsacirc0001021 in peripheral blood of spinal tuberculosis and application of circular RNA hsacirc0001021 in diagnosis and treatment of spinal tuberculosis in the peripheral blood of spinal tuberculosis. The circular RNA hsacirc0001021 comprises a kit, test paper and a chip. The expression of the hsacirc0001021 disclosed by the invention in the peripheral blood of a spinal tuberculosis patient is obviously up-regulated compared with that of a normal person; as the current clinical auxiliary diagnosis technology for spinal tuberculosis is not perfect, hsacirc0001021 has the potential of serving as a biological marker related to spinal tuberculosis and can be used for early diagnosis of spinal tuberculosis; according to the kit disclosed by the invention, the risk of spinal tuberculosis is predicted through the relative expression quantity of the hsacirc0001021, the spinal tuberculosis is screened by detecting the expression level of the hsacirc0001021 in peripheral blood of a patient, and a detection result shows that the expression of the hsacirc0001021 is remarkably up-regulated compared with that of a healthy person, so that the condition that the patient suffers from the spinal tuberculosis is indicated; generally, the relative expression quantity of circRNA in peripheral blood of a spinal tuberculosis patient is also related to diagnosis, prognosis or treatment of the spinal tuberculosis patient.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and specifically relates to the application of the diagnostic marker hsa_circ_0001021 in peripheral blood of spinal tuberculosis. Background Art

[0004] Research has shown that there are differential expressions of circRNAs in the peripheral blood of tuberculosis patients, which are closely related to the occurrence and development of tuberculosis and can be used as new biomarkers for early diagnosis and prognosis evaluation of tuberculosis. However, for the screening and detection of spinal tuberculosis, there are still few relevant studies, and it is necessary to further explore new effective markers and evaluate their specificity and accuracy. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies in the existing screening and detection of spinal tuberculosis, and provide an application of circular RNA hsa_circ_0001021 in the peripheral blood of spinal tuberculosis for the diagnosis and treatment of spinal tuberculosis, so as to solve the problems mentioned in the background art.

[0006] To solve the above problems, the present invention provides a technical solution:

[0007] The application of the diagnostic marker hsa_circ_0001021 in the peripheral blood of spinal tuberculosis, and the application of circular RNA hsa_circ_0001021 in the peripheral blood of spinal tuberculosis for the diagnosis and treatment of spinal tuberculosis, including: kits, test strips, and chips.

[0008] Preferably, the hsa_circ_0001021 as a marker contains PCR primers for detecting the content of hsa_circ_0001021 in the product for screening spinal tuberculosis-related infections.

[0009] Preferably, the sequences of the primers are shown in SEQ NO:2 and SEQ NO:3.

[0010] Preferably, the kit further includes primers for amplifying an internal reference gene.

[0011] Preferably, the sequences of the primers for amplifying the internal reference gene are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0012] Preferably, the internal reference gene is glyceraldehyde-3-phosphate dehydrogenase GAPDH.

[0013] The kit disclosed by the present invention predicts the risk of spinal tuberculosis through the relative expression level of hsa_circ_0001021, screens spinal tuberculosis by detecting the expression level of hsa_circ_0001021 in the peripheral blood of patients. The detection result shows that the expression of hsa_circ_0001021 is significantly up-regulated compared with that of healthy people, indicating that the patient has spinal tuberculosis.

[0014] The present invention relates to the application of circular RNA hsa_circ_0001021 in the diagnosis and treatment of spinal tuberculosis in peripheral blood. Generally, the relative expression level of circRNA in the peripheral blood of spinal tuberculosis patients is also related to the diagnosis, prognosis or treatment of spinal tuberculosis patients. Products for the diagnosis, prognosis or treatment of spinal tuberculosis include: products for detecting the expression level of hsa_circ_0001021 by fluorescence quantitative PCR, in situ hybridization or high-throughput sequencing.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The hsa_circ_0001021 disclosed by the present invention is significantly up-regulated in the peripheral blood of spinal tuberculosis patients compared with that of normal people. In view of the fact that the current clinical auxiliary diagnosis technology for spinal tuberculosis is not yet perfect, therefore, this hsa_circ_0001021 has the potential to be used as a biomarker related to spinal tuberculosis and can be used for the early diagnosis of spinal tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For the sake of easy explanation, the present invention will be described in detail by the following specific embodiments and drawings.

[0018] Figure 1 It is a schematic diagram of the functional annotation of hsa_circ_0001021 in the parental gene;

[0019] Figure 2 It is a schematic diagram of the amplification curve of hsa_circ_0001021;

[0020] Figure 3 It is a schematic diagram of the melting curve of hsa_circ_0001021;

[0021] Figure 4 It is a schematic diagram of the relative expression levels of hsa_circ_0001021 in the peripheral blood of spinal tuberculosis patients and healthy people;

[0022] Figure 5 It is a schematic diagram of the ROC curve for diagnosis. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following combines the attached Figures 1-5A further description of the present invention is provided. The following examples are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention. For the experimental methods without specific conditions noted in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the manufacturer; for the reagents, materials and instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase:

[0024] Application of the diagnostic marker hsa_circ_0001021 in peripheral blood of spinal tuberculosis. The present invention provides the application of circular RNA hsa_circ_0001021 in peripheral blood of spinal tuberculosis for the diagnosis and treatment of spinal tuberculosis. Its position in the genome is chr2:64524299 - 64593005, and its structure in the parental gene AFTPH is as Figure 1 shown;

[0025] Figure 1 In it, AFTPH: human tail - associated protein; chr2:64524299 - 64593005: chromosome 2, starting position 64524299, ending position 6459300; full - length gene sequence: 1967bp; RefSeq of mRNA: NM_017657; exon: exon; back - splicing junction: reverse splicing site.

[0026] Figure 2 In it, AmplifieationPlot: amplification curve; Cycle: cycle.

[0027] Figure 3 In it, MeltCurve: melting curve; Temperature: temperature; Derivativereporter: strong fluorescence intensity.

[0028] Figure 4 In it, Control: control group; Tuberculosis: spinal tuberculosis group; expression: expression level; ***: P < 0.001.

[0029] Figure 5 In it, AUC: area under the ROC curve.

[0030] Example:

[0031] In the early stage, the gene research group of the present invention selected 3 patients diagnosed with spinal tuberculosis who visited the Department of Orthopedics of Ningxia Medical University General Hospital, collected their peripheral blood and compared it with that of 3 healthy people for high - throughput sequencing. High - expression and well - differentiated circRNA molecules were screened out for screening and verification analysis. hsa_circ_0001021 was selected and, through subsequent verification and combined with statistical analysis, it was used for the early diagnosis of spinal tuberculosis.

[0032] Research objects and samples:

[0033] Thirty patients with spinal tuberculosis admitted to the General Hospital of Ningxia Medical University from January 2019 to December 2020 were selected as the experimental group. All patients were diagnosed by clinical manifestations, imaging, laboratory tests, histopathology, tissue GeneXpert MTB / RIF testing, etc. Thirty healthy patients of similar gender and age were collected during the same period as the control group. 5 ml of fasting venous blood was collected in the early morning with an EDTA anticoagulant tube, left standing in a 4°C refrigerator for 20 minutes, and then centrifuged at 2000 revolutions per minute for 20 minutes. Carefully divide the upper plasma and the lower blood cells into 1.5 ml enzyme-free EP tubes labeled with patient name, medical record number, serial number, specimen type, collection date, etc. After inverting and mixing them evenly, quickly transfer them to an -80°C refrigerator for storage or proceed to the next experiment.

[0034] RNA extraction:

[0035] Take the peripheral blood out of the -80°C refrigerator and let it thaw naturally at room temperature. After pipetting and mixing evenly, aspirate 500 μl into a 4 ml enzyme-free EP tube, add 1500 μl of Trizol to it, and oscillate on a vortex oscillator for 30 seconds. Let it stand at room temperature for 10 minutes to facilitate sufficient lysis, and centrifuge at 12000×g at 4°C for 10 minutes; after centrifugation, slowly open the EP tube and aspirate the supernatant into a new 1.5 ml enzyme-free EP tube, add 300 μl of chloroform, which becomes a pink emulsion, and let it stand at room temperature for 10 min. Centrifuge at 12000×g at 4°C for 15 min, and the sample will be divided into three layers: a red organic phase, a middle layer, and an upper colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the aqueous phase to a new tube. Add 750 μl of isopropanol, mix well and let it stand at room temperature for 10 minutes, and centrifuge at 12000×g at 4°C for 10 minutes. After carefully discarding the upper supernatant, a small white precipitate can be seen at the bottom of the EP tube, which is the total RNA; add 1 ml of 75% ethanol, invert and shake for 30 seconds, centrifuge at 12000×g at 4°C for 10 minutes, repeat the above steps three times, then carefully discard the supernatant, place the EP tube with the opening downward in a clean bench, and after the ethanol has fully evaporated, add 30 μl of enzyme-free water preheated at 55°C for 10 minutes and pipette and mix well. The sample is stored at -80°C.

[0036] Total RNA quality control:

[0037] Use a NanoDrop ND-2000 spectrophotometer to measure the purity and concentration of RNA. The RNA concentration and the optical density (OD) values at 260 nm - 280 nm can be automatically generated. An OD260 / 280 between 1.80 and 2.00 indicates high RNA purity, and record the RNA concentration.

[0038] cDNA synthesis:

[0039] Prepare the reverse transcription reaction mixture (Takara) on ice according to the components in Table 1. After mixing, perform reverse transcription at 37°C for 15 min, 85°C for 5 s, and hold at 4°C. The reverse-transcribed cDNA is placed on ice for the next reaction or stored at -20°C.

[0040] Table 1 Reverse transcription reaction system

[0041]

[0042] Fluorescent quantitative PCR:

[0043] Prepare the fluorescent quantitative PCR reaction mixture on ice according to the components in Table 2. After mixing, place the PCR plate on the StepOnePlus Real-Time PCR System instrument for PCR reaction. Perform the reaction according to the following program, set the reaction conditions as: pre-denaturation at 95°C for 30 s, PCR reaction at 95°C for 5 s → 60°C for 30 s → repeat the above cycle 40 times, and perform melting curve analysis according to the program of 95°C for 15 s → 60°C for 60 s → 95°C for 15 s to obtain the corresponding Ct value. When verifying by qRT-PCR, its melting curve shows a single peak (see Figure 2 、 Figure 3 ), the primers have good specificity. For the rest of the PCR instruments (Applied Biosystems 7300 / 7500 Fast Real-Time PCR System and StepOnePlus Real-Time PCR System), set according to the corresponding programs in the instructions. GAPDH is used as an internal reference, and the relative expression level is calculated using 2-ΔΔCt.

[0044] Table 2 Fluorescent quantitative PCR reaction system

[0045]

[0046] Specific PCR primers for hsa_circ_0001021:

[0047] Forward primer: 5’-CCCAAAACGCACAGTGTACC-3’

[0048] Reverse primer: 5’-TGGTGGTGGGGATGAAGAGT-3’

[0049] Specific PCR primers for the internal reference gene GAPDH:

[0050] Forward primer: 5’-CAGGAGGCATTGCTGATGAT-3’

[0051] Reverse primer: 5’-GAAGGCTGGGGCTCATTT-3’

[0052] In this invention, SPSS 20.0 and GraphPad Prism 8 were used for statistical analysis. The expression level of hsa_circ_0001021 in the peripheral blood of spinal tuberculosis patients was higher than that in the control group, and the difference was statistically significant (P<0.001), as shown in Figure 4 ; the analysis results of the Receiver Operating Characteristic (ROC) curve showed that the expression level of hsa_circ_0001021 in peripheral blood could be used as a diagnostic indicator for spinal tuberculosis. The area under the ROC curve (AUC) was 0.888 (95% CI: 0.776 - 0.956). When the cut-off value was 0.604, the sensitivity was 70.37% and the specificity was 90.00%, as shown in Figure 5 , and the results showed that hsa_circ_0001021 could be used as a diagnostic biomarker in the peripheral blood of spinal tuberculosis.

[0053] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the equivalents of the appended claims.

Claims

1. Application of the diagnostic marker hsa_circ_0001021 in peripheral blood of spinal tuberculosis, characterized in that: Application of circular RNA hsa_circ_0001021 detection reagent in the preparation of potential therapeutic drugs for spinal tuberculosis; Includes: test kits, test strips, and chips.

2. The use of the diagnostic marker hsa_circ_0001021 in peripheral blood for spinal tuberculosis according to claim 1, characterized in that: The detection reagent is a PCR primer for detecting the content of hsa_circ_0001021.

3. The use of the diagnostic marker hsa_circ_0001021 in peripheral blood for spinal tuberculosis according to claim 1, characterized in that: The sequences of the PCR primers are shown in SEQ ID NO:2 and SEQ ID NO:

3.

4. The use of the diagnostic marker hsa_circ_0001021 in peripheral blood for spinal tuberculosis according to claim 1, characterized in that: The kit also includes primers for amplifying an internal reference gene.

5. The use of the diagnostic marker hsa_circ_0001021 in peripheral blood for spinal tuberculosis according to claim 4, characterized in that: The sequences of the primers for amplifying the internal reference gene are shown in SEQ ID NO:4 and SEQ ID NO:

5.

6. The use of the diagnostic marker hsa_circ_0001021 in peripheral blood for spinal tuberculosis according to claim 4, characterized in that: The internal reference gene is glyceraldehyde-3-phosphate dehydrogenase GAPDH.

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