CN102839173B - 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 to carry out prognosis evaluation in early days in the HIV infection, 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) be 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, have conservative property, sequential and the tissue specificity of height, 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.

Acquired immune deficiency syndrome (AIDS) is global serious infectious diseases, serious threat people's health and the development that influences economic society.After HIV infected, progression of disease speed difference generally entered the AIDS stadium through 8-10, is 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, rapid progress person ratio is higher in the man male sex 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: other indexs such as CD4+T cell quantity, virus load, immune activation.The CD4+T cell quantity is the target cell that HIV infects, can effectively reflect immune status, but its detection needs flow cytometer, instrument and corresponding reagent are expensive, the technical requirements height is not suitable for generally carrying out, and rapid progress person and typical progress person infect early stage at HIV, CD4+T cell quantity difference is remarkable inadequately, is difficult for the progression of disease 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, to the equipment requirements height, need professional's analysis, sample is preferably selected fresh specimens for use, stores transportation inconvenience, is difficult to promote.

Studies show that in recent years, it is closely related with miRNA that HIV infects, thus miRNA can influence progression of disease by acting on virus itself or acting on immunity system, so the diagnosis that makes progress that catches also may have corresponding effect to HIV.Infect the more and effect of relevant miRNA kind with HIV and differ, there are some researches show that infecting back miRNA at HIV generally reduces, the miR-17/92 gene cluster can suppress HIV infection etc.Though carried out some researchs 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 progression of disease, 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 peripheral blood mononuclear cell of HIV infection is compared typical progress person and is obviously reduced, can predict the progression of disease 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, described diagnostic reagent is by detecting the content of miR-200c in subject's peripheral blood mononuclear cell, and infect typical progress person's mean level (ML) with HIV and compare to 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 peripheral blood mononuclear cell.In a specific embodiment, use the method for quantitative PCR to detect miR-200c content in subject's peripheral blood mononuclear cell.

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

(1) peripheral blood mononuclear cell 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 described peripheral blood mononuclear cell total RNA extraction reagent that sequence is the External Control-1 of 5 '-CAACCUCCUAGAAAGA-3 ' (SEQ ID NO:2); Described RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of 5 '-UGAGCAACGCGAACAA-3 ' (SEQ ID NO:3); Comprise in the described RT-PCR reagent sequence be 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4) RT-Primer(wherein, V is A or C or G, N is A or T or C or G); Comprise in the described 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 peripheral blood mononuclear cell

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 in contrast, density gradient centrifugation is collected peripheral blood mononuclear cell, 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 peripheral blood mononuclear cell, extract total RNA, add 1 μ l(20nM in per 250 μ l peripheral blood mononuclear cell) sequence is that External Control-1(Shanghai of 5 '-CAACCUCCUAGAAAGA-3 ' (SEQ ID NO:2) is given birth to worker's biotechnology company limited and synthesized) monitor the extraction quality of RNA in the peripheral blood mononuclear cell.The total RNA that extracts uses Thermo NanoDrop2000c to measure concentration.

2. the miRNA in the three-step approach detection by quantitative peripheral blood mononuclear cell

(1) add the polyA tail:

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

(annotate: the RNA volume of adding is determined 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 instrument (Thermo) and hatched 1 hour.

(2) RT-PCR obtains the cDNA strand:

I. add 0.5 μ l(0.5ng/ μ l in the reaction solution that obtains to (1)) to be that worker's biotechnology company limited is given birth in RT-Primer(Shanghai of 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4) synthetic for sequence), 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 are 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 Beijing Quanto Biotechnology Co., Ltd.

Green PCR Master Mix.）

Wherein, the sequence of general reverse primer UPM-short and UPM-long is respectively 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:5) and 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 paraffin oil fluid-tights.

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

Vi. draw melting curve, check the specificity of 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 peripheral blood mononuclear cell 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 peripheral blood mononuclear cell significantly descends than typical progress person.Single factor Cox risk regression analysis and K-M survival analysis show that miR-200c can infect the progression of disease of early prediction infection after 1 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

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Claims (4)

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

   (1) peripheral blood mononuclear cell total RNA extraction reagent,

   (2) RNA adds polyA reagent,

   (3) RT-PCR reagent,

   (4) quantitative PCR reagent;

   Wherein, comprise in the described RT-PCR reagent that sequence is the RT-Primer of SEQ ID NO:4; Comprise in the described quantitative PCR reagent that sequence is the catch specificity forward primer of progress mark miR-200c of the HIV of SEQ ID NO:7, and sequence is respectively general reverse primer UPM-short and the UPM-long of SEQ ID NO:5 and 6.
2. 2. the described diagnostic kit of claim 1, wherein said HIV catch the sequence of progress molecular marker miR-200c shown in SEQ ID NO:1.
3. 3. the described diagnostic kit of claim 1 wherein, comprises in the described peripheral blood mononuclear cell total RNA extraction reagent that sequence is the External Control-1 of SEQ ID NO:2.
4. 4. the described diagnostic kit of claim 1, wherein, described RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of SEQ ID NO:3.