CN113862350A - Diagnosis marker hsa _ circ _0043898 in peripheral blood of spinal tuberculosis and application thereof - Google Patents

Diagnosis marker hsa _ circ _0043898 in peripheral blood of spinal tuberculosis and application thereof Download PDF

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CN113862350A
CN113862350A CN202111409144.9A CN202111409144A CN113862350A CN 113862350 A CN113862350 A CN 113862350A CN 202111409144 A CN202111409144 A CN 202111409144A CN 113862350 A CN113862350 A CN 113862350A
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tuberculosis
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王立楠
牛宁奎
师志云
郭乐
屈昱良
王辉
张含菲
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General Hospital of Ningxia Medical University
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Abstract

The diagnosis marker hsa _ circ _0043898 in peripheral blood of spinal tuberculosis and the application thereof comprise the following contents: the application of hsa _ circ _0043898 as a marker in preparing a peripheral blood diagnostic marker product for spinal tuberculosis; products for spinal tuberculosis-related infection screening include reagents or drugs that detect the level of hsa _ circ _0043898 expression in peripheral blood cells of a patient; the hsa _ circ _0043898 is used as a marker and contains PCR primers for detecting the content of hsa _ circ _0043898 in the preparation of products for spinal tuberculosis related infection screening; according to the application, the sequence of the primer is shown as SEQ ID NO.2 and SEQ ID NO. 3; a kit for spinal tuberculosis-related infection screening; the application of the compound as a marker in preparing a medicine for diagnosing or prognosis of spinal tuberculosis related infection. The hsa _ circ _0043898 disclosed by the invention is obviously up-regulated in peripheral blood of tuberculosis of spine patients compared with normal expression, and aiming at the defect that the current diagnosis technology for tuberculosis of spine is incomplete, the hsa _ circ _0043898 has the potential of being used as a biomarker related to tuberculosis of spine and can be used for early diagnosis of tuberculosis of spine.

Description

Diagnosis marker hsa _ circ _0043898 in peripheral blood of spinal tuberculosis and application thereof
Technical Field
The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to application of hsa _ circ _0043898 as a marker in preparation of products or medicines for spinal tuberculosis related infection screening.
Background
Tuberculosis is still a chronic infectious disease which seriously harms human health worldwide at present, and is one of infectious diseases which are key prevention and control in China and are generated by the world health organization. Osteoarticular tuberculosis is a common secondary extrapulmonary tuberculosis, while spinal tuberculosis accounts for the first site of osteoarticular tuberculosis of the whole body, accounting for about 5% of all tuberculosis and 50% of bone and joint tuberculosis. If the tuberculosis of the spine cannot be diagnosed and treated in time, serious complications such as spinal cord and nerve compression, spinal instability, kyphosis, paralysis and the like can occur. In recent years, clinical and basic research on tuberculosis of spine has progressed to a certain extent, but the present situations of difficult early diagnosis, increased intractable cases, poor drug resistance, poor compliance, high recurrence rate and the like are still faced. Is limited by the undefined pathogenesis of tuberculosis of spine, and the prevention and early diagnosis of tuberculosis of spine are still difficult, which becomes a difficult point for preventing and treating the disease. Therefore, the research of the specific early diagnosis marker in the peripheral blood of the tuberculosis patient has important significance.
With the rapid development of molecular biology technology and the continuous and deep research on the pathogenesis of tuberculosis of spine, more and more related biomarkers are continuously discovered and reported. From the research progress of epigenetics, some non-coding RNAs have application prospects as disease biomarkers. Circular RNA (circular RNA) is a non-coding RNA which is widely present in organisms, and unlike traditional linear RNA, circular RNA molecules are in a closed circular structure, are not influenced by RNA exonuclease, and are stable and not easy to degrade.
Research shows that the circRNA in peripheral blood and lesion tissues of tuberculosis patients has differential expression, is closely related to the occurrence and development of tuberculosis, can be used as a novel biological diagnostic marker and prognosis evaluation of tuberculosis in early stage, is still less aiming at spinal tuberculosis screening detection, and needs to further explore a novel effective marker and evaluate the specificity and accuracy of the marker.
Disclosure of Invention
The invention aims to provide application of hsa _ circ _0043898 as a marker in preparation of drugs for diagnosis, prognosis or treatment of spinal tuberculosis aiming at the defects of the existing spinal tuberculosis screening and detection.
In order to achieve the purpose, the invention provides the following technical scheme:
the diagnosis marker hsa _ circ _0043898 in peripheral blood of spinal tuberculosis and the application thereof comprise the following aspects:
1. the application of hsa _ circ _0043898 as a marker in preparing a diagnostic marker product for the peripheral blood of spinal tuberculosis, wherein the sequence of the diagnostic marker product is shown as SEQ ID NO. 1.
2. Products for spinal tuberculosis-related infection screening include reagents or drugs that detect the expression level of hsa _ circ _0043898 in peripheral blood cells of a patient, and the detection results show that hsa _ circ _0043898 expression is significantly upregulated compared to healthy persons.
3. The hsa _ circ _0043898 is used as a marker and contains PCR primers for detecting the content of hsa _ circ _0043898 in the preparation of products for spinal tuberculosis related infection screening; the Primer sequence is Forward Primer sequence (5-3'): AGCTTCGTGTCCAACCTGTT (SEQ NO: 2); reverse Primer sequence (5 '-3'): GAATGGCAGGACACAACAGC (SEQ ID NO. 3).
4. The primer has the sequence shown in SEQ ID NO.2 and SEQ ID NO.3 according to the application described in the previous 3.
5. A kit for screening spinal tuberculosis related infection, the PCR primer of the 3 or 4 and a primer for amplifying an internal reference gene; further, the reference gene is GAPDH. The Primer sequence for amplifying GAPDH is Forward Primer sequence (5 '-3'): CAGGAGGCATTGCTGATGAT, (SEQ ID NO. 4); reverse Primer sequence (5 '-3'): GAAGGCTGGGGCTCATTT, (SEQ ID NO. 5).
6. The kit according to the above 5, wherein the reference gene is Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
7. The kit as described in the preceding 6, characterized in that the primer sequences for amplifying GAPDH are shown as SEQ ID NO.4 and SEQ ID NO. 5.
8. Application of hsa _ circ _0043898 as a marker in preparation of drugs for diagnosis, prognosis or treatment of spinal tuberculosis-related infection.
The kit disclosed by the invention predicts the risk of spinal tuberculosis through the relative expression of hsa _ circ _ 0043898. Spinal tuberculosis is screened by detecting the expression level of hsa _ circ _0043898 in peripheral blood of a patient, and the detection result shows that the expression of hsa _ circ _0043898 is obviously up-regulated compared with that of a healthy person, which indicates that the spinal tuberculosis is suffered.
The application of the hsa _ circ _0043898 as a marker in preparing the medicine for diagnosis, prognosis or treatment of spinal tuberculosis is disclosed. Usually, the relative expression of circRNA in peripheral blood of tuberculosis patients is also related to the diagnosis, prognosis or treatment of tuberculosis patients, and products for diagnosis, prognosis or treatment of tuberculosis include: products were tested for hsa _ circ _0043898 expression levels by fluorescent quantitative PCR, in situ hybridization, or high throughput sequencing.
Compared with the prior art, the invention has the beneficial effects that:
the hsa _ circ _0043898 disclosed by the invention is obviously up-regulated in peripheral blood of a tuberculosis patient compared with normal people, and is incomplete aiming at the current clinical auxiliary diagnosis technology for the tuberculosis of the spine, so that the hsa _ circ _0043898 has the potential of being used as a biomarker related to the tuberculosis of the spine, and can be used for early diagnosis of the tuberculosis of the spine.
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FIG. 1 is a schematic diagram of the functional annotation of hsa _ circ _0043898 in the parental gene;
FIG. 2 is a schematic representation of the hsa _ circ _0043898 amplification curve;
FIG. 3 is a schematic representation of the hsa _ circ _0043898 dissolution curve;
FIG. 4 is a graph showing the relative expression levels of hsa _ circ _0043898 in peripheral blood of tuberculosis patients and healthy persons;
FIG. 5 is a schematic diagram of a ROC curve for diagnosis.
In fig. 1, EZH 1: drosophila zeste gene enhancer human homolog 1;
chr17:40879652 and 40882936: chromosome 17, start position 40879652, end position 40882936; the full-length sequence of the gene is as follows: 348 bp; RefSeq of mRNA: NM _ 001991; exon: an exon; back-fusing junction: a reverse splice site.
In FIG. 2, amplification Plot: an amplification curve; cycle: and (6) circulating.
In FIG. 3, Melt cut: a dissolution profile; temperature: (ii) temperature; derivative reporter: strong light fluorescence.
In fig. 4, Control: a control group; tuberculosis: tuberculosis of spine group; expression: the expression amount; ***: p < 0.001.
In fig. 5, AUC: area under ROC curve.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby. The experimental procedures, for which specific conditions are not noted in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturer; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.
The invention provides application of hsa _ circ _0043898 as a diagnostic marker in preparing a product for spinal tuberculosis diagnosis, wherein the position in the genome is chr17: 40879652-.
Examples
In the early stage of the gene subject group, 3 patients who are diagnosed in Ningxia medical university Hospital department and are diagnosed as spinal tuberculosis patients are selected, peripheral blood of the patients is collected to perform high-throughput sequencing with 3 healthy people, and circRNA molecules with high expression and good difference are screened out to be screened, verified and analyzed. The invention selects hsa _ circ _0043898, and combines with statistical analysis through subsequent verification to be used for early diagnosis of spinal tuberculosis.
Study subjects and samples:
30 spinal tuberculosis patients collected in Ningxia medical university Hospital from 2019, 1 month to 2020, 12 months are selected as an experimental group, and the patients are confirmed by clinical manifestations, imaging, laboratory tests, histopathology, Gene Xpert MTB/RIF tests and the like. 30 healthy patients with similar sex and age are collected at the same time as a control group, 5ml of early morning fasting venous blood is collected by an EDTA anticoagulation tube, the control group is stood in a refrigerator at 4 ℃ for 20 minutes, then the control group is centrifuged at 2000 rpm for 20 minutes, upper layer plasma and lower layer blood cells are carefully packed in a 1.5ml enzyme-free EP tube written with information such as patient name, case number, specimen type, collection date and the like, and the control group is quickly transferred to a refrigerator at minus 80 ℃ for storage or next experiment after being inverted and mixed uniformly.
RNA extraction:
taking out peripheral blood from a refrigerator at minus 80 ℃ to naturally unfreeze at room temperature, blowing and uniformly mixing by a pipette, sucking 500 mu l to 4ml of an enzyme-free EP tube, adding 1500 mu l of Trizol, oscillating on a vortex oscillator for 30 seconds, standing at room temperature for 10 minutes to facilitate full lysis, and centrifuging at 12000 Xg at 4 ℃ for 10 minutes; after the centrifugation was completed, the EP tube was slowly opened and the supernatant was pipetted into a new 1.5ml enzyme-free EP tube, 300. mu.l of chloroform was added to turn into a pink emulsion, and the mixture was left at room temperature for 10 min. After centrifugation at 12000 Xg for 15min at 4 ℃ the sample will separate into three layers: the red organic phase, the middle and upper colorless aqueous phases, with RNA predominantly in the aqueous phase, was transferred to a new tube. Adding 750 mul of isopropanol, uniformly mixing, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 12000 Xg and 4 ℃, carefully discarding supernatant liquid, and then observing small white precipitates at the bottom of an EP tube, namely total RNA; adding 1ml of 75% ethanol, reversely shaking for 30 seconds, centrifuging for 10 minutes at 12000 Xg and 4 ℃, repeating the steps for three times, carefully discarding the supernatant, placing an EP tube into a super clean bench with an open end downwards, adding 30 mu l of enzyme-free water preheated at 55 ℃ for 10 minutes after the ethanol is fully volatilized, and blowing and uniformly mixing. Samples were stored to-80 ℃.
And (3) controlling the total RNA quality:
the RNA purity and concentration were measured by a NanoDrop ND-2000 spectrophotometer, the RNA concentration and Optical Density (OD) values at 260nm to 280nm were automatically generated, OD260/280 between 1.80 to 2.00 indicates high RNA purity, and the RNA concentration was recorded.
And (3) cDNA synthesis:
a reverse transcription reaction mixture (Takara) was prepared on ice according to the components in Table 1, and after mixing, reverse transcription was carried out at 37 ℃ for 15min, 85 ℃ for 5second, and 4 ℃ for hold. The reversed cDNA was placed on ice for further reaction or stored at-20 ℃.
TABLE 1 reverse transcription reaction System
Figure BDA0003373547930000061
Fluorescent quantitative PCR:
preparing fluorescent quantitative PCR reaction mixed liquor (Takara) on ice according to the components in the table 2, uniformly mixing, placing a PCR plate on a StepOnePelus Real-Time PCR System instrument for PCR reaction, and performing according to the following procedures under the set reaction conditions: the pre-denaturation step is carried out at 95 ℃ for 30 seconds, the PCR reaction step is carried out at 95 ℃ for 5 seconds → 60 ℃ for 30 seconds → the above cycle is carried out 40 times, and the program of 95 ℃ for 15 seconds → 60 ℃ for 60 seconds → 95 ℃ for 15 seconds is analyzed according to the melting curve to obtain the corresponding Ct value. The single peak of the dissolution curve (see figure 2 and figure 3) is verified by qRT-PCR, and the specificity of the primer is good. The remaining PCR instruments (Applied Biosystems 7300/7500Fast Real-Time PCRSystem and StepOneNus Real-Time PCRSystem) were programmed accordingly to the instructions. GAPDH as internal reference, 2-ΔΔCtAnd calculating the relative expression amount.
TABLE 2 fluorescent quantitative PCR reaction System
Figure BDA0003373547930000071
hsa _ circ _0043898 specific PCR primers:
a forward primer: 5'-AGCTTCGTGTCCAACCTGTT-3'
Reverse primer: 5'-GAATGGCAGGACACAACAGC-3' are provided.
The internal reference gene is GAPDH specific PCR primer:
a forward primer: 5'-CAGGAGGCATTGCTGATGAT-3'
Reverse primer: 5'-GAAGGCTGGGGCTCATTT-3' are provided.
The statistical analysis was performed using SPSS20.0 and GraphPad Prism 8. The expression level of hsa _ circ _0043898 in the periphery of tuberculosis patients was higher than that of the control group, and the difference was statistically significant (P <0.001), as shown in FIG. 4; the results of Receiver Operating Characteristic curve (ROC) analysis showed that the expression level of hsa _ circ _0043898 in peripheral blood was used as a diagnostic indicator for spinal tuberculosis, the area under the ROC curve (AUC) was 0.777 (95% CI: 0.642-0.880), and the cut-off was 0.5057, the sensitivity was 72.00% and the specificity was 78.57%, as shown in FIG. 5. The results show that hsa _ circ _0043898 can be used as a diagnostic marker in peripheral blood of spinal tuberculosis.
The above-described embodiments are intended to disclose the preferred embodiments of the present invention, but the disclosed embodiments are not limited to the above-described embodiments, and the above-described embodiments and descriptions are only illustrative of the principles of the present invention, and various changes and modifications can be made without departing from the spirit and scope of the invention, which fall within the scope of the claimed invention.
Sequence listing
<110> Ningxia medical university general Hospital
<120> diagnosis marker hsa _ circ _0043898 in peripheral blood of spinal tuberculosis and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 348
<212> DNA
<213> Homo sapiens
<400> 1
ctggacacct gttctgctgt tgtgtcctgc cattctcctg aagaacagag gcacactgta 60
aaacccaaca cttccccttg cattctataa gattacagca agatggaaat accaaatccc 120
cctacctcca aatgtatcac ttactggaaa agaaaagtga aatctgaata catgcgactt 180
cgacaactta aacggcttca ggcaaatatg ggtgcaaagg ctttgtatgt ggcaaatttt 240
gcaaaggttc aagaaaaaac ccagatcctc aatgaagaat ggaagaagct tcgtgtccaa 300
cctgttcagt caatgaagcc tgtgagtgga cacccttttc tcaaaaag 348
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcttcgtgt ccaacctgtt 20
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaatggcagg acacaacagc 20
<210> 4
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caggaggcat tgctgatgat 20
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaaggctggg gctcattt 18

Claims (8)

1. The diagnostic marker hsa _ circ _0043898 in the peripheral blood of spinal tuberculosis and the application thereof are characterized in that the sequence of the diagnostic marker is shown in SEQ ID NO.1 when the diagnostic marker is used as the marker in the preparation of the diagnostic marker product for the peripheral blood of spinal tuberculosis.
2. The use according to claim 1, wherein the product for screening tuberculosis of the spine comprises a reagent or drug for detecting the expression level of hsa _ circ _0043898 in peripheral blood cells of the patient.
3. The use according to claim 1, wherein hsa _ circ _0043898 contains PCR primers for detecting hsa _ circ _0043898 as markers in the preparation of a product for screening spinal tuberculosis-related infections.
4. The use of claim 3, wherein the primer has the sequence shown in SEQ ID No.2 and SEQ ID No. 3.
5. A kit for screening tuberculosis in the spine by using hsa _ circ _0043898 as a marker, which comprises the PCR primer according to claim 3 or 4 and a primer for amplifying an internal reference gene.
6. The kit of claim 5, wherein the reference gene is Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
7. The kit of claim 6, wherein the primer sequences for amplifying GAPDH are shown in SEQ ID No.4 and SEQ ID No. 5.
8. The diagnosis marker hsa _ circ _0043898 in peripheral blood of tuberculosis of spine and the application thereof are characterized in that hsa _ circ _0043898 is used as the marker in the preparation of drugs for diagnosis, prognosis or treatment of tuberculosis of spine related infection.
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