CN113774123B - MicroRNA molecular marker combination for predicting immune response level of HIV infected person and application thereof - Google Patents
MicroRNA molecular marker combination for predicting immune response level of HIV infected person and application thereof Download PDFInfo
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Abstract
The present invention provides a set of microRNA molecular marker combinations for predicting the level of immune response in HIV-infected individuals following antiviral treatment, comprising the following 5 microRNA nucleic acid molecules: miR-580, miR-627, miR-138-5p, miR-16-5p and miR-323-3p. The content of the 5 microRNA molecular marker combinations in the plasma of patients with immune non-response after the antiviral treatment of HIV infected patients is significantly different from the content in the plasma of patients with immune response, so that the immune response of HIV infected patients after the antiviral treatment can be predicted and judged. The microRNA molecular marker combination diagnosis curative effect has the characteristics of simple operation, safety, no wound, high specificity, high sensitivity and easiness in large-scale screening.
Description
Technical Field
The invention relates to the field of HIV infection prognosis, in particular to a group of HIV infection disease progress molecular marker combinations: miR-580, miR-627, miR-138-5p, miR-16-5p and miR-323-3p can be used for efficacy evaluation after HIV infection antiviral treatment so as to effectively follow up and treat intervention measures for patients, prolong survival time and improve survival quality.
Background
microRNA (miRNA) is a recent research hotspot, which is a single-stranded small molecule RNA widely existing in eukaryotes, has no coding function, but is capable of binding to flanking regions of a gene sequence to repress or inhibit translation of target mRNA. miRNA has high conservation, time sequence and tissue specificity, and plays an important role in the fields of clinical diagnosis, treatment, prognosis evaluation and the like of various diseases such as tumor, hematopathy, virus infection and the like at present.
AIDS is a worldwide serious infectious disease, seriously threatens the health of people and affects the development of economy and society. At present, antiviral treatment can effectively control viruses, and immune functions represented by CD4+ T cells are effectively recovered (called as 'immune response' (person) ") after antiviral treatment on most of infected persons, so that life is prolonged. However, 15-30% of HIV-infected individuals are effectively inhibited by the virus, but CD4+ T cells are not effectively recovered, which is called an "immune non-responder" (hereinafter referred to as "non-responder"). If the immune non-responders cannot be treated as early as possible, the morbidity and mortality will continue to rise, reducing the life expectancy and quality of life. At present, antiviral treatment in China is increasingly popular, if immune response is effectively predicted after treatment before antiviral treatment, immune responders and non-responders are distinguished, follow-up visit of patients and treatment such as enhancing immunity are reasonably arranged in an individuation mode, the survival time and the survival quality of HIV infected persons can be improved, and the method has important significance for national AIDS prevention and treatment work.
At present, the modes of evaluating the curative effect of antiviral treatment of HIV infection diseases mainly comprise the following steps: cd4+ T cell numbers, immune activation, and other indicators. The CD4+ T cells are target cells infected by HIV, the quantity of the CD4+ T cells can effectively reflect the immune state of the organism, but the detection needs a flow cytometer, the instrument and corresponding reagents are high in price, the technical requirements are high, the method is not suitable for general development, and the quantity difference of the CD4+ T cells is not obvious enough before antiviral treatment of immune responders and non-responders, so that the future curative effect is not easy to be effectively predicted; although the indexes such as immune activation and the like can be reported to predict disease prognosis, the requirements on equipment are high, professional analysis is needed, fresh specimens are preferably selected as specimens, the storage and transportation are inconvenient, and the popularization is difficult.
Recent studies have shown that HIV infection is closely related to mirnas, which can affect disease progression by acting on the virus itself or on the immune system, and thus diagnosis of the immune response of an antiviral treatment of HIV infection may also have a corresponding effect. The miRNAs related to HIV infection have a plurality of different functions, and the research shows that the miRNAs are generally down-regulated after HIV infection, and the miR-17/92 gene cluster can inhibit HIV infection and the like. Although some studies have been conducted in this field, judging the variation in miRNA expression of immune response after HIV infection antiviral treatment prior to HIV infection antiviral treatment has not been studied so far, and in clinic and research, there is a need to find miRNA molecular markers or combinations thereof that can effectively predict the prognosis of HIV infection.
Disclosure of Invention
The present invention provides a set of miRNA molecular marker combinations for predicting the level of immune response in HIV-infected individuals following antiviral treatment, which is Tenofovir (TDF) +lamivudine (3 TC) +efavirenz (EFV).
Alternative antiviral treatment regimens may also be zidovudine (AZT) +lamivudine (3 TC) +efavirenz (EFV), zidovudine (AZT) +lamivudine (3 TC) +nevirapine (NVP), stavudine (D4T) +lamivudine (3 TC) +efavirenz (EFV).
The first aspect of the invention provides a group of miRNA molecular marker combinations for predicting the immune response level of HIV infected persons after antiviral treatment, which mainly comprise miR-580, miR-627, miR-138-5p, miR-16-5p and miR-323-3p, and the specific sequences are as follows:
TABLE 1
| miRNA name | miRNA sequences | Sequence number |
| miR-580 | 5’-UUGAGAAUGAUGAAUCAUUAGG-3’ | SEQ ID No.:1 |
| miR-627 | 5’-GUGAGUCUCUAAGAAAAGAGGA-3’ | SEQ ID No.:2 |
| miR-138-5p | 5’-AGCUGGUGUUGUGAAUCAGGCCG-3’ | SEQ ID No.:3 |
| miR-16-5p | 5’-UAGCAGCACGUAAAUAUUGGCG-3’ | SEQ ID No.:4 |
| miR-323-3p | 5’-CACAUUACACGGUCGACCUCU-3’ | SEQ ID No.:5 |
In a preferred embodiment, the miRNA molecular marker combination of the invention consists of miR-580, miR-627, miR-138-5p, miR-16-5p and miR-323-3p.
In a preferred embodiment, each miRNA is differentially expressed in at least one target plasma/serum and in at least one control plasma/serum. The target plasma/serum is from a patient who is immune responsive after antiviral treatment, and the control plasma/serum is from a patient who is immune non-responsive. The inventor discovers that the content of the 5 miRNA markers in the plasma/serum of the immune non-responsive patient is obviously improved compared with the expression level of the immune responsive patient, and can effectively distinguish whether the HIV infected patient has immune response after antiviral treatment.
Based on the findings, the invention provides basis for preparing the diagnosis kit for immune response after antiviral treatment.
Accordingly, in a second aspect the present invention provides the use of a set of miRNA molecular marker combinations as described above for the preparation of a diagnostic kit for the prognosis of an HIV infected person with antiviral therapy. Preferably, the diagnostic kit distinguishes patients who have an immune response following antiviral treatment from HIV-infected patients.
In one embodiment, the above diagnostic kit determines the level of immune response in an HIV-infected subject after antiviral treatment by detecting the level of the miRNA molecular marker combination of the present invention in the plasma of the HIV-infected subject.
Specifically, the diagnosis kit is used for judging whether the immunity is responsive or non-responsive by detecting the content of the 5 miRNA molecular marker combinations in the serum/plasma of the HIV infected person and utilizing a binary regression formula. The binary regression model formula is as follows:
Logit(p)＝37.204–0.190×Ct1+0.833×Ct2+1.210×Ct3–1.577×Ct4–1.712×Ct5
y＝1/(1+e -logit(p) )
y is more than or equal to 0.51, and is judged as immune non-response after antiviral treatment, and y <0.51 is judged as immune response.
Wherein Ct1, ct2 … … Ct5 represent Ct values of mir-580, mir-627, mir-138-5p, mir-16-5p, and mir-323-3p, respectively (Ct values are cycle numbers of PCR amplification curves exceeding a threshold).
In a specific embodiment, quantitative PCR methods are used to detect the level of the miRNA molecular marker combination of the invention in the serum/plasma of HIV-infected subjects.
In a third aspect the invention provides a kit for predicting the level of immune response in an HIV infected person following antiviral treatment, the kit comprising specific forward primers for a combination of 5 miRNA molecule markers of the invention.
In one embodiment, the specific forward primer of the above kit comprises one or more primers selected from the group consisting of: SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, the sequences of which are as follows.
TABLE 2
| miRNA name | Forward primer sequences | Sequence number |
| miR-580 | 5’-GGGTTGAGAATGATGAAT-3’ | SEQ ID No.:6 |
| miR-627 | 5’-GGTGAGTCTCTAAGAAAAG-3’ | SEQ ID No.:7 |
| miR-138-5p | 5’-TGGTGTTGTGAATCAGG-3’ | SEQ ID No.:8 |
| miR-16-5p | 5’-GCACGTAAATATTGGCG-3’ | SEQ ID No.:9 |
| miR-323-3p | 5’-GGCACATTACACGGTCG-3’ | SEQ ID No.:10 |
In a preferred embodiment, the kit of the present invention further comprises:
(1) The total RNA extraction reagent of the blood plasma/serum,
(2) The RNA is added with a polyA reagent,
(3) The reagent for RT-PCR,
(4) The PCR reagent is quantified and the PCR reagent is used for the quantitative PCR,
wherein the quantitative PCR reagent comprises a specific forward primer of the miRNA molecular marker combination.
In a more preferred embodiment, the total plasma/serum RNA extraction reagent in the kit of the present invention comprises an External Control sequence-1 (External Control-1) having the sequence 5'-CAACCTCCTAGAAAGA-3' (SEQ ID NO: 11), wherein the RNA plus polyA reagent comprises an External Control sequence-3 (External Control-3) having the sequence 5'-TGAGCAACGCGAACAA-3' (SEQ ID NO: 12), and wherein the RT-PCR reagent comprises a reverse transcription Primer (RT-Primer) having the sequence 5'-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO: 13).
In another more preferred embodiment, the quantitative PCR reagents in the kits of the invention described above comprise the universal reverse primers UPM-short and UPM-long having the sequences 5'-CTCACACGACTCACGACAC-3' (SEQ ID NO. 14) and 5'-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3' (SEQ ID NO. 15), respectively.
In a fourth aspect the invention provides the use of a kit of the invention as described above for predicting the level of immune response in an HIV-infected person after antiviral treatment.
The invention has higher diagnosis performance, can timely give immune intervention measures to patients with rapid disease progression by predicting the disease progression as early as possible, improves immunity, reduces morbidity and mortality, improves the prognosis of patients, and provides support for AIDS prevention and treatment.
Drawings
FIG. 1 is a graph ROC (receiver operating characteristic) plotted using Spss mapping software with true positive rate (sensitivity) on the ordinate and false positive rate (1-specificity) on the abscissa in patients with immune response and no response after antiviral treatment. The result of combining 5 miRNAs shows that the AUC (area under the curve) of the miRNA molecular marker combination reaches 0.932, and the miRNA molecular marker combination can effectively predict whether the immune response exists after antiviral treatment.
Detailed Description
The technical scheme of the present invention is further described by the detailed description and the accompanying drawings, but it is understood by those skilled in the art that: the following detailed description and examples are intended to illustrate the invention and are not to be construed as limiting the invention in any way. It will be apparent to those skilled in the art that many modifications can be made to the present invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention.
Those skilled in the art will appreciate that there are a number of antiviral regimens for the clinical treatment of HIV, and basically these regimens can be used to predict the level of immune response in HIV-infected individuals following antiviral treatment using the molecular marker combinations of the present invention, for example, but not limited to, alternative antiviral treatment regimens may also be zidovudine (AZT) +lamivudine (3 TC) +efavirenz (EFV), zidovudine (AZT) +lamivudine (3 TC) +nevirapine (NVP), stavudine (D4T) +lamivudine (3 TC) +efavirenz (EFV).
The following experimental methods are all conventional experimental methods in the art unless otherwise specified, and all experimental materials used, unless otherwise specified, are all readily available from commercial companies.
The microRNA molecular marker combination provided by the invention is 5 microRNA molecular marker combinations found from 444 candidate microRNAs through screening and verification of discovery (discovery), screening (translation), validation (verification), mixed detection and blind detection stages (blinded) by utilizing a high-throughput miRNA screening platform to compare and analyze immune responses of HIV infected persons with immune non-responders.
Example 1 plasma sample collection and preparation
During the period 1 in 2014 to 12 in 2016, 50 plasma samples satisfying immune non-response after antiviral treatment and 50 plasma samples satisfying immune response after antiviral treatment were collected in advance from the first affiliated hospital of the university of medical science.
The immune non-response inclusion group criteria were: after 12 months of antiviral treatment, cd4+ T lymphocyte cell counts increased less than 30% from baseline in HIV-infected persons. The immune response group criteria were: after 12 months of antiviral treatment, the cd4+ T lymphocyte count increased by 30% or more from baseline in HIV-infected persons. Wherein baseline is the pre-treatment cd4+ T lymphocyte count for HIV-infected persons.
Peripheral venous blood 10mL was collected, EDTA anticoagulated, and the plasma collection procedure was as follows: the whole blood sample was centrifuged at 3000rpm for 10min at 4℃in a centrifuge. 1mL of the upper plasma was aspirated with a pipette and carefully transferred to a 1.5mL sterile centrifuge tube without rnase. Each sample is marked. The plasma samples must be kept in an ultra-low temperature (-80 ℃) freezer within 4 hours.
EXAMPLE 2 extraction of Total RNA in plasma
Total miRNA in plasma was extracted using an RNA extraction kit (Bohr technology Co., ltd.) and 1. Mu.L (20 nM) of External Control sequence-1 (External Control-1) (Shanghai Biotechnology Co., ltd.) having a sequence of 5'-CAACCTCCTAGAAAGA-3' (SEQ ID No.: 11) was added to 250. Mu.L of plasma to monitor the quality of RNA extraction in plasma. The total RNA extracted was measured for concentration using Thermo NanoDrop 2000 c.
Example 3 quantitative detection of miRNA in plasma by three-step method
(1) Adding polyA tail:
i. a reaction solution containing polyA tail was prepared in a RNase-free PCR tube (Axygen Co., ltd., 200. Mu.L) to give a reaction solution of 20. Mu.L. mu.L (20 nM) of External Control sequence-3 (External Control-3) (synthesized by Shanghai Biotechnology Co., ltd.) having a sequence of 5'-TGAGCAACGCGAACAA-3' (SEQ ID No.: 12) was added to each 20. Mu.L of the system to monitor the tailing and reverse transcription quality of the miRNA.
TABLE 3 Table 3
( And (3) injection: the volume of RNA added was determined by the concentration of RNA, x=500 ng/RNA, and the enzymes used in this experiment were all products from Beijing Kuang Bo Biotechnology Co. )
The PCR tube with the prepared reaction solution was placed in a PCR instrument (Thermo) and incubated at 37℃for 1 hour.
(2) RT-PCR to obtain cDNA single strand:
i. to the reaction solution obtained in (1), 0.5. Mu.L (0.5 ng/. Mu.L) of a reverse transcription Primer RT-Primer (synthesized by Shanghai Biotechnology Co., ltd.) having a sequence of 5'-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID No.: 13) was added, and incubated at 70℃for 5 minutes, and immediately placed on ice for at least 2 minutes.
Preparing a reverse transcription reaction solution:
TABLE 4 Table 4
( And (3) injection: the products are all products of Beijing Kuang Bo biotechnology Co. )
Mixing the solutions obtained in i and ii, incubating at 50 ℃ for 50min, then preserving heat at 70 ℃ for 15min, and cooling on ice to obtain cDNA.
Diluting the product obtained in iii to a reverse transcription product containing 1ng total RNA in a 1 mu L system, subpackaging, and preserving at-20 ℃.
(3) quantitative qPCR detection:
i. the reaction solution was prepared in a 2mL EP tube (Axygen Co.):
TABLE 5
(Note: UPM-long, UPM-short were all synthesized by Invitrogen, and the remaining reagents were all from Takara CoGreen PCR Master Mix。)
Wherein the sequences of the universal reverse primers UPM-short and UPM-long are 5'-CTCACACGACTCACGACAC-3' (SEQ ID No.: 14) and 5'-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3' (SEQ ID No.: 15), respectively.
The prepared reaction solution was thoroughly mixed upside down and then dispensed into a 96 Kong Jiande PCR plate (Axygen Co.) at 18. Mu.L per well.
Specific forward primers (synthesized by Invitrogen) of the corresponding 5 miRNA molecular marker combinations were added using a row gun (Eppendoff, inc., 1-10. Mu.L) at 2. Mu.L (10. Mu.M) per well. The 5 specific forward marker sequences are shown in Table 6 below:
TABLE 6
| miRNA name | Forward primer sequences | Sequence number |
| miR-580 | 5’-GGGTTGAGAATGATGAAT-3’ | SEQ ID No.:6 |
| miR-627 | 5’-GGTGAGTCTCTAAGAAAAG-3’ | SEQ ID No.:7 |
| miR-138-5p | 5’-TGGTGTTGTGAATCAGG-3’ | SEQ ID No.:8 |
| miR-16-5p | 5’-GCACGTAAATATTGGCG-3’ | SEQ ID No.:9 |
| miR-323-3p | 5’-GGCACATTACACGGTCG-3’ | SEQ ID No.:10 |
Adding 10 mu L paraffin oil, sealing with special film (ABI company).
v. put into ABI-7900PCR quantitative detector, the procedure is set as follows:
TABLE 7
Drawing a melting curve, checking the specificity of the primer, and setting the procedure as follows: 95℃15s,60℃15s,95℃15s.
Example 4 significant Difference analysis Using Omicsoffice software
qPCR assays were performed according to the methods of examples 2 and 3 on 50 plasma samples satisfying immune non-response after antiviral treatment and 50 plasma samples satisfying immune response after antiviral treatment in example 1.
The results of measuring the expression level of 5 miRNA markers in 100 plasma samples are shown in the following table 8.
TABLE 8
| Detection markers | P value | MTP-MRP Ct difference |
| miR-580 | 3.95E-05 | 1.09 |
| miR-627 | 9.48E-06 | 1.21 |
| miR-138-5p | 9.98E-05 | 1.04 |
| miR-16-5p | 2.52E-14 | 2.13 |
| miR-323-3p | 3.72E-15 | 2.26 |
( And (3) injection: MTP is immune response after antiviral treatment, MRP is immune non-response, ct represents the cycle number of PCR amplification curve exceeding threshold. )
The results show that the content of 5 miRNA markers in the plasma of the immune non-responders is obviously increased relative to the content of the miRNA markers in the plasma of the immune responders.
Example 5 binary regression model equation Using SPSS
The 5 miRNA marker binary regression model constructed for each set of 50 samples in example 1 using SPSS software was formulated as follows:
Logit(p)＝37.204–0.190×Ct1+0.833×Ct2+1.210×Ct3–1.577×Ct4–1.712×Ct5
y＝1/(1+e -logit(p) )
wherein Ct1, ct2 … … Ct5 represent Ct values of mir-580, mir-627, mir-138-5p, mir-16-5p, and mir-323-3p, respectively.
The binary regression model plots the ROC curve as shown in fig. 1.
This formula was then applied to the blind test of the sample, where y.gtoreq.0.51 was judged as immune non-responsive after antiviral treatment, and y <0.51 was judged as immune responsive.
EXAMPLE 6 Blind testing of both immune-responsive and immune-non-responsive samples
According to the sample collection and preparation method of example 1, the extraction and qPCR detection of the steps of examples 2 and 3 were performed on 39 plasma samples (39 plasma samples were collected from HIV-infected patients via Tenofovir (TDF) +lamivudine (3 TC) +efavirenz (EFV) after the antiviral treatment course was completed) collected in advance from the first affiliated hospital of the university of chinese medical science, and Ct values of 5 miRNA marker detection were substituted into the binary regression model constructed in example 5 for verification, and the results are shown in table 9 below:
TABLE 9
| Grouping | Number of examples | Predicting correct number of cases | Accuracy rate of |
| Immune non-response | 20 | 19 | 95％ |
| Immune response | 19 | 18 | 94.74％ |
The 5 miRNA molecular marker combinations disclosed by the invention can be used for effectively predicting whether HIV infected patients have immune response after antiviral treatment, so that immune intervention measures can be timely given to patients with rapid disease progression, and morbidity and mortality are reduced.
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<110> affiliated first Hospital of university of medical science in China
Beijing Kuangbo Star Biotech Co., ltd
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Claims (7)
- Use of a specific forward primer of a microRNA molecular marker combination in the preparation of a diagnostic kit for predicting the immune response level of an HIV infected person after antiviral treatment, characterized in that the microRNA molecular marker combination consists of miR-580, miR-627, miR-138-5p, miR-16-5p and miR-323-3p, the sequences of which are shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively.
- 2. The use according to claim 1, wherein the diagnostic kit determines the level of immune response of the HIV-infected person after antiviral treatment by detecting the level of microRNA molecular marker combination in the blood plasma of the HIV-infected person.
- 3. Use according to claim 1 or 2, wherein the amount of microRNA molecular marker combination in the HIV infected patient's plasma/serum is detected using quantitative PCR methods.
- 4. The use according to claim 1 or 2, characterized in that the specific forward primer comprises more than one primer selected from the following sequences: SEQ ID No.:6, SEQ ID No.:7, SEQ ID No.:8, SEQ ID No.:9 and SEQ ID No.:10.
- 5. The use according to claim 4, wherein the diagnostic kit further comprises:(1) The total RNA extraction reagent of the blood plasma/serum,(2) The RNA is added with a polyA reagent,(3) The reagent for RT-PCR,(4) The PCR reagent is quantified and the PCR reagent is used for the quantitative PCR,wherein the quantitative PCR reagent comprises a specific forward primer of the microRNA molecular marker combination.
- 6. The use according to claim 5, wherein the total plasma/serum RNA extraction reagent comprises an external control sequence-1 of SEQ ID No.:11, wherein the RNA plus polyA reagent comprises an external control sequence-3 of SEQ ID No.:12, and wherein the RT-PCR reagent comprises a reverse transcription primer of SEQ ID No.: 13.
- 7. The use according to claim 5, wherein the quantitative PCR reagents comprise universal reverse primers UPM-short and UPM-long having the sequences SEQ ID NO. 14 and SEQ ID NO. 15, respectively.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101641440A (en) * | 2006-11-23 | 2010-02-03 | 米尔克斯治疗有限责任公司 | Oligonucleotides for modulating target RNA activity |
| CN102839172A (en) * | 2012-08-24 | 2012-12-26 | 中国医科大学附属第一医院 | HIV (Human immunodeficiency virus) infection disease progression molecule marker miR-503 |
| CN102839175A (en) * | 2012-08-24 | 2012-12-26 | 中国医科大学附属第一医院 | Molecular marker miR-526a for progress of human immunodeficiency virus (HIV) infectious disease |
| CN105567838A (en) * | 2016-02-02 | 2016-05-11 | 首都医科大学附属北京佑安医院 | Application of miR-331-5p as acute HIV infection detection marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101641440A (en) * | 2006-11-23 | 2010-02-03 | 米尔克斯治疗有限责任公司 | Oligonucleotides for modulating target RNA activity |
| CN102839172A (en) * | 2012-08-24 | 2012-12-26 | 中国医科大学附属第一医院 | HIV (Human immunodeficiency virus) infection disease progression molecule marker miR-503 |
| CN102839175A (en) * | 2012-08-24 | 2012-12-26 | 中国医科大学附属第一医院 | Molecular marker miR-526a for progress of human immunodeficiency virus (HIV) infectious disease |
| CN105567838A (en) * | 2016-02-02 | 2016-05-11 | 首都医科大学附属北京佑安医院 | Application of miR-331-5p as acute HIV infection detection marker |
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