CN102839173A - Molecule marker miR-200c for progress of HIV (Human Immunodeficiency Virus) infected disease - Google Patents

Abstract

The invention provides a molecule marker miR-200c for a progress of a HIV (Human Immunodeficiency Virus) infected disease and an application thereof in the preparation of a diagnostic reagent for the progress of the HIV infected disease. The content of the miR-200c of a HIV infected rapid progressing patient in a peripheral blood mononuclear cell is lower than the content of the miR-200c of a typical progressing patient in the peripheral blood mononuclear cell, and the progressing degree of the disease after being infected for one year can be forecast. The invention also provides the diagnostic reagent for diagnosing the progress of the HIV infected disease. The application of the molecule marker miR-200c for the progress of the HIV infected disease provided by the invention for diagnosing the progress of the disease has the characteristics of simpleness in operation, safety, noninvasive property, high specificity, high sensitivity and easiness in mass screening.

Description

The HIV progress molecular marker miR-200c that catches

Technical field

The present invention relates to HIV and infect the Prognosis field; Relate in particular to a kind of HIV progress molecular marker miR-200c that catches; It can be applicable at HIV infection carrying out in early days prognosis evaluation; Intervening measures such as the patient follows up a case by regular visits to timely and effectively to give, treatment to prolong survival time, improve life quality.

Background technology

MicroRNA (miRNA) is a hot research in recent years; It is a kind of strand microRNA that extensively is present in the eukaryote; Do not have an encoding function; But the flank region that it can be incorporated into gene order checks or suppresses the translation of said target mrna, has conservative property, sequential property and the tissue specificity of height, has brought into play vital role in the fields such as clinical diagnosis, treatment and prognostic evaluation of multiple diseases such as tumour, hemopathy, virus infection at present.

AIDS is global serious infectious diseases, serious threat people's health and the development that influences economic society.After HIV infected, PD speed was different, generally got into the AIDS stadium through 8-10, was called " typical progress person ".But the infected of 10%, infect in 3 years even 1 year the CD4+T cell significantly drop to 350/below the microlitre, be called " rapid progress person ".As not reaching treatment as early as possible by regular follow-up after the rapid progress person diagnosis, then sickness rate and mortality ratio will continue to rise, and risk is propagated in increase.At present; China constantly increases through property approach HIV the infected, and rapid progress person ratio is higher in male sex's contact, if can effectively predict prognosis in early days in the HIV infection; Distinguish rapid progress person and typical progress person; Individuation is rationally arranged following up a case by regular visits to and treating of patient, not only can improve HIV the infected's survival time and life quality, and is also significant to the AIDS preventing and controlling work of country.

At present, catch progress and the mode of prognostic evaluation of HIV mainly contains following several kinds: other indexs such as CD4+T cell quantity, virus load, immune activation.The CD4+T cell quantity is the target cell that HIV infects, and can effectively reflect immune status, but its detection needs flow cytometer; Instrument and corresponding reagent cost an arm and a leg; Technical requirements is high, be not suitable for generally carrying out, and rapid progress person and typical progress person infects early stage at HIV; CD4+T cell quantity difference is remarkable inadequately, is difficult for the PD in future is effectively predicted; It is bigger that virus load infects early stage fluctuation at HIV, and not parallel fully with clinical immunological status; Though indexs such as immune activation have the measurable disease prognosis of report, and are high to equipment requirements, need professional's analysis, sample is preferably selected fresh specimens for use, stores transportation inconvenience, is difficult to promote.

Research in recent years shows, HIV infects closely related with miRNA, thereby miRNA can influence PD through acting on virus itself or acting on immunity system, so the diagnosis that makes progress that catches also has corresponding effect to HIV.Infect the more and effect of relevant miRNA kind with HIV and differ, existing research is illustrated in HIV and infects back miRNA and generally reduce, and the miR-17/92 gene cluster can suppress HIV infection etc.Study though carried out some in this field, the miRNA that HIV infects early stage disease rapid progress person expresses variation also not research at present, in clinical and research, need seek the miRNA mark that can effectively predict disease prognosis.

Summary of the invention

The invention provides a kind of new HIV and catch the relevant miRNA of progress as the molecular marker of PD, this miRNA is miR-200c, and its sequence is 5 '-UAAUACUGCCGGGUAAUGAUGGA-3 ' (SEQ ID NO:1).

The contriver finds; The content of miR-200c in early stage (120 days) the rapid progress person PMNC of HIV infection is compared typical progress person and is obviously reduced; Can predict the PD situation after a year that infects; This shows, the miR-200c of reduction and HIV catch the progress and prognosis closely related.

On the basis of above-mentioned discovery, the invention provides the purposes of miR-200c in preparation diagnosis HIV catches the diagnostic reagent that makes progress.

Particularly; Said diagnostic reagent is through detecting the content of miR-200c in testee's PMNC; And infect typical progress person's M.L. with HIV and compare and diagnose the HIV progress that catches; Wherein, miR-200c significantly descends with respect to typical case progress person at the content that HIV infects in the rapid progress person PMNC.In a specific embodiment, use the method for quantitative PCR to detect miR-200c content in testee's PMNC.

The diagnostic kit that the present invention also provides a kind of HIV of diagnosis to catch progress, it comprises:

(1) PMNC total RNA extraction reagent,

(2) RNA adds polyA reagent,

(3) RT-PCR reagent,

(4) quantitative PCR reagent.

Wherein, comprise the catch specificity forward primer of progress molecular marker miR-200c of HIV in the quantitative PCR reagent, preferably, its sequence is 5 '-GTAATAGTGCCCGGTAA-3 ' (SEQ ID NO:7).

In a specific embodiment, comprise in the said PMNC total RNA extraction reagent that sequence is the External Control-1 of 5 '-CAACCTCCTAGAAAGA-3 ' (SEQ ID NO:2); Said RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:3); Comprise in the said RT-PCR reagent that sequence is the RT-Primer (wherein, V is A or C or G, and N is A or T or C or G) of 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4); Comprise in the said quantitative PCR reagent that sequence is respectively general reverse primer UPM-short and the UPM-long of 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:5) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:6).

Use HIV of the present invention catch progress molecular marker miR-200c diagnosis HIV catch progress have simple to operate, safety no wound, high specific, high sensitivity and the characteristics that are easy to a large amount of examinations.

Embodiment

1. the extraction of total RNA in the PMNC

Extract 11 HIV infection rapid progress persons and infect 120 days 5ml blood; Extract 6 HIV simultaneously and catch typical progress person and 4 each 5ml of normal controls blood as contrast; Density gradient centrifugation is collected PMNC; Use the PBS1ml re-suspended cell, place the RNase/DNase-free centrifuge tube of 1.5ml.

Use RNA to extract test kit (Beijing Quanto Biotechnology Co., Ltd.) and in PMNC, extract total RNA, add the extraction quality that the External Control-1 that 1 μ l (20nM) sequence is 5 '-CAACCTCCTAGAAAGA-3 ' (SEQ ID NO:2) (it is synthetic that worker's biotechnology ltd is given birth in Shanghai) monitors RNA in the PMNC in per 250 μ l PMNCs.The total RNA that extracts uses Thermo NanoDrop2000c to measure concentration.

2. the miRNA in the three-step approach detection by quantitative PMNC

(1) add the polyA tail:

I. in the PCR pipe (Axygen company, 200 μ l) of no RNA enzyme, prepare the reaction solution that adds the polyA tail, system is 20 μ l.The External Control-2 (it is synthetic that worker's biotechnology ltd is given birth in Shanghai) that adds 1 μ l (20nM) sequence in per 20 μ l systems and be 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:3) monitors tailing and the reverse transcription quality of miRNA.

(annotate: the RNA volume of adding is confirmed by the concentration of RNA, x=500ng/RNA concentration, and this tests the product that employed enzyme is Beijing Quanto Biotechnology Co., Ltd..）

Ii., the PCR pipe that configures reaction solution will be housed to be put into 37 ℃ in PCR appearance (Thermo) and hatched 1 hour.

(2) RT-PCR obtains the cDNA strand:

I. in the reaction solution that (1) obtains, add the RT-Primer that 0.5 μ l (0.5ng/ μ l) sequence is 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4) (it is synthetic that worker's biotechnology ltd is given birth in Shanghai); Hatch 5min for 70 ℃, be put on ice ice bath 2min at least immediately.

Ii. prepare inverse transcription reaction liquid:

(annotate: used product is the product of Beijing Quanto Biotechnology Co., Ltd..）

Iii. the solution that i and ii is obtained mixes, and hatches 70 ℃ of insulation 15min behind the 50min for 50 ℃, puts cooled on ice, obtains cDNA.

Iv. the product that iii is obtained is diluted to the reverse transcription product that contains the total RNA of 1ng in the 1 μ l system ,-20 ℃ of preservations after the packing.

(3) qPCR detection by quantitative:

I. in 2ml EP pipe (Axygen company), prepare reaction solution:

(annotate: UPM-long, UPM-short are all synthetic in Invitrogen company, and all the other reagent are all from

Green PCR Master Mix of Beijing Quanto Biotechnology Co., Ltd..）

Wherein, the sequence of general reverse primer UPM-short and UPM-long be respectively 5 '-CTCACACGACTCACGACAC-3 ' (SEQIDNO:5) with 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:6).

Ii. after the reaction solution that configures is fully put upside down mixing, divide in the 96 holes point end PCR plate of packing into (Axygen company) every hole 18 μ l.

Iii. using the volley of rifle fire (Eppendoff company, 1-10 μ l) adding sequence is the miR-200c specificity forward primer (Invitrogen company is synthetic) of 5 '-GTAATAGTGCCCGGTAA-3 ' (SEQ ID NO:7), every hole 2 μ l (10 μ M).

Iv. seal with special-purpose pad pasting (ABI company) after adding 10 μ l Yellow Protopet 2A fluid-tights.

V. put into ABI7900PCR detection by quantitative appearance, program setting is:

Vi. draw melting curve, the specificity of inspection primer, program setting is: 95 ℃ of 15s, 60 ℃ of 15s, 95 ℃ of 15s.

3. adopt RealTime StatMiner software to carry out data analysis

Can record HIV with aforesaid method and infect the average Ct value that the rapid progress person infects miR-200c in 120 days, typical progress person and each sample PMNC of normal control and be respectively 7.45,1.74 and 12.24, the result shows that the relative content of miR-200c in rapid progress person PMNC significantly descends than typical progress person.Single factor Cox risk regression analysis and K-M survival analysis show that miR-200c can infect the PD after early prediction infects a year at HIV.

< 110>No. 1 Hospital Affiliated to Chinese Medical Univ Beijing Quanto Biotechnology Co., Ltd.

 

< 120>the HIV progress molecular marker miR-200c that catches

 

<160> 7

 

<170> PatentIn?version?3.3

 

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gtaatagtgc?ccggtaa 17

Claims (11)

1. the HIV progress molecular marker miR-200c that catches, its sequence is shown in SEQ ID NO:1.

2. the said HIV of the claim 1 progress molecular marker miR-200c that catches infects the purposes in the diagnostic reagent of prognosis at preparation HIV.

3. the said purposes of claim 2, wherein, said diagnostic reagent is through detecting the content of miR-200c in testee's PMNC, and infects typical progress person's M.L. with HIV and compare and judge the HIV progress that catches.

4. the said purposes of claim 3, wherein, miR-200c significantly descends with respect to typical case progress person at the content that HIV infects in the rapid progress person PMNC.

5. the said purposes of claim 3 wherein, uses the method for quantitative PCR to detect miR-200c content in testee's PMNC.

One kind diagnose HIV catch the progress diagnostic kit, it comprises:

(1) PMNC total RNA extraction reagent,

(2) RNA adds polyA reagent,

(3) RT-PCR reagent,

(4) quantitative PCR reagent;

Wherein, comprise the catch specificity forward primer of progress mark miR-200c of the said HIV of claim 1 in the quantitative PCR reagent.

7. the said diagnostic kit of claim 6 wherein, comprises in the said PMNC total RNA extraction reagent that sequence is the External Control-1 of SEQ ID NO:2.

8. the said diagnostic kit of claim 6, wherein, said RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of SEQ ID NO:3.

9. the said diagnostic kit of claim 6 wherein, comprises in the said RT-PCR reagent that sequence is the RT-Primer of SEQ ID NO:4.

10. the said diagnostic kit of claim 6 wherein, comprises in the said quantitative PCR reagent that sequence is respectively the general reverse primer UPM-short and the UPM-long of SEQ ID NO:5 and 6.

11. the said diagnostic kit of claim 6, wherein, said HIV catches the sequence of specificity forward primer of progress molecular marker miR-200c shown in SEQ ID NO:7.