CN101215604B

CN101215604B - Kit for detecting human galactophore globin mRNA expression quantity and application thereof

Info

Publication number: CN101215604B
Application number: CN2007103038725A
Authority: CN
Inventors: 魏海明; 程民; 田志刚; 陈永艳
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2007-12-26
Filing date: 2007-12-26
Publication date: 2010-11-03
Anticipated expiration: 2027-12-26
Also published as: CN101215604A

Abstract

The invention discloses a reagent case of detecting the mammary globulin mRNA expression of a human body and an application. The mammary globulin mRNA expression of the human body can be accurately calibrated mammary globulin mRNA expression of the human body in the detected specimen. The reagent case and the process which are provided by the invention can be applied in clinics and scientific researches, can carry out quantitative analysis for the expression condition of hMAM mRNA in peripheral blood and marrow of tumor patients, and plays a significant role for judging the generation, metastasis and recurrence of mammary cancer and dynamically observing conditions. The invention will plays important role in the medical detection field.

Description

A kind of test kit and application thereof that detects human galactophore globin mRNA expression quantity

Technical field

The present invention relates to a kind of test kit and application thereof that detects human galactophore globin mRNA expression quantity.

Background technology

Mammary cancer is human modal a kind of malignant tumour, is one of women's major malignant tumor.Countries in the world are because of the difference of geographical environment, living habit, and the sickness rate of mammary cancer has very big-difference, and the most countries in North America and Northern Europe is the district occurred frequently, and some countries of South America and southern Europe are medium, and the most countries in Asia, Latin America and Africa is the low district of sending out.In the North America, developed country such as two Europe, the sickness rate of women with breast cancer occupies the first place of women's malignant tumour sickness rate.Estimate that according to American Cancer Society there is 120,000 mammary cancer new cases every year in the U.S., sickness rate is 72.2/10 ten thousand.Though China belongs to the low district of sending out of women with breast cancer in the world, the sickness rate of mammary cancer obviously increases in recent years.The sickness rate of China's each department mammary cancer is also inequality, and Shanghai, capital, Tianjin and coastland are the hotspots of China's mammary cancer.China's breast cancer incidence was 52,/10 ten thousand in 2006, occupied first in women's malignant tumour.

The methods of treatment of mammary cancer has multiple, as surgical resection, radiotherapy, chemotherapy etc.At present, excision is the first-selection in the mammary cancer comprehensive treatment, yet when the excision technology had reached suitable maturation and popularized, the height recurrence and the rate of transform after the mammary cancer excision had become the major cause that the obstruction patient with breast cancer improves lifetime.Breast cancer cell before the operation since nature or aggressive iatrogenic factors drop to peripheral blood and be transferred to outside the mammary gland with blood, form circulating tumor cell, but because examined method sensitivity and the lower restriction of specificity, this micro-cancer cells fails to be detected, these cancer cells arrive peripheral tissues and are in dormant state with blood, when patient's body's immunity descends, use the anti-immunosuppressor etc. of repelling as major operation or after transplanting, the cancer cells of dormancy just might be activated, turn back to mammary gland with blood circulation, and in mammary gland, recover propagation, thereby caused the recurrence of mammary cancer.The transfer of tumour is a great problem of clinical treatment, also is the lethal major reason of tumour.At present, the discovery of metastases and definite is mainly diagnosed according to iconography (X-ray sheet, CT, mr, PET etc.) or pathomorphism.In the imaging examination, focus or metastasis need form a certain size can be by instrument imaging and effectively resolution; Pathomorphism need be got suitable detection sample in checking, and has a great deal of observable tumour cell to exist.Therefore, no matter be imaging diagnosis or pathological diagnosis, the stage that all is late period after tumour forms and shifts, exists a large amount of malignant cells to accumulate, if in tumour the early stage of transfer takes place, can find and definite its transfer, then can the guiding clinical treatment design for scheme, and strong prognosis foundation is provided.Hematogenous spread is one of main path of metastases, so in the tumour patient peripheral blood, carry out tumour cell genetic expression, it is a kind of effective ways of understanding the metastases situation that the tumour-specific markers product detects, the diagnosis that tumour is formed and shifts has great help, but metastases take place early stage, tumour cell in the peripheral blood generally seldom, can't rely on the pathomorphism inspection fully, when not forming metastasis or metastasis and not reaching certain volume, imaging diagnosis is also powerless, therefore, the research that micro-tumour cell genetic expression in the circulation blood and some specificity products detect has caused scholars' extensive concern.

The quantitative assay of tumour-specific gene mRNA in the peripheral blood helps the early diagnosis of tumour, the judgement of the definite and prognosis of treatment plan.Along with the develop rapidly of Protocols in Molecular Biology and the widespread use in medical research thereof, become and may and have been attempted by micro-tumour cell in RT-polymerase chain reaction (RT-PCR) the technology for detection peripheral blood.

The real-time fluorescence quantitative PCR technology is meant in the PCR reaction system to add a specificity fluorescent probe in a pair of primer of adding, utilizes the fluorescent signal accumulation whole PCR process of monitoring in real time.Taqman fluorescent probe commonly used at present is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group, so detect less than fluorescence; In the pcr amplification process, 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal.In the pcr amplification process, detect first order fluorescence intensity after each loop ends, entire reaction just can obtain a working curve after finishing, and can carry out quantitative analysis to unknown template according to this curve.The real-time fluorescence quantitative PCR technology is a kind of accurately detection method of quantification of mrna, by expression the existing with the judgement circulating tumor cell of detecting the tumour-specific gene mRNA.

(Human mammaglobin, hMAM) gene utilize the mRNA differential display technique to separate by Watson MA etc. and draw human galactophore globin, be positioned at 11q12-q13, cDNA total length 503bp, wherein open reading frame 279bp, 93 amino acid of encoding, relative molecular mass 10.5kD.The experimental results shows that the hMAM gene is only expressed in adult's mammary gland, other histoorgans are not expressed, overexpression in primary breast cancer, and normal people's peripheral blood hMAMmRNA is negative; It is very unstable to be free on extracellular mRNA, very easily by the RNA enzymolysis, so in peripheral blood if record hMAM mRNA, then point out and have complete breast cancer cell in the circulation of blood.(Watson MA，FlemingTP.Mammaglobin，a mammary-specific member of the uteroglobin gene family，is overexpressed in human breast cancer.Cancer Res，1996，56(4)：860-865.Gal S，Fidler C，Lo YM，et al.Detection of mammaglobin mRNA in the plasmaof breast cancer patients.Ann N Y Acad Sci，2001，945：192-194.)。Therefore, hMAM mRNA is a monitoring index of haematogenous breast cancer cell preferably.

HMAM mRNA in detection patient with breast cancer's marrow and the blood is to judging the generation of mammary cancer, shift, recurrence, dynamic observing of the treatment effectiveness evaluation and the state of an illness is significant, but still there is not the high detection technique of suitable positive rate at present, for example auspicious grade of Han Zheng used the expression that common RT-PCR detects patients with mastocarcinoma peripheral blood hMAM mRNA, positive rate only is 30.8% (16/52), benign breast disease patient and healthy people's control group are all negative, and (Han Zheng is auspicious, Zhang Jingchuan .hMAM mRNA and CEA mRNART-PCR detect the micrometastasis of mammary cancer peripheral blood. Chinese clinical tumor, 2004,31 (21): 1235-1239.); Common RT-PCR detects can only carry out qualitative detection to the expression of patient with breast cancer's peripheral blood hMAM mRNA, can not carry out quantitative analysis.

Summary of the invention

The purpose of this invention is to provide a kind of test kit that detects human galactophore globin mRNA expression quantity.

The test kit of detection human galactophore globin mRNA expression quantity provided by the present invention contains a pair of primer and probe.

In the mentioned reagent box, the nucleotide sequence of the primer that described primer is right is a sequence 1 in the sequence table, and the nucleotide sequence of another primer is a sequence 2 in the sequence table.

In the mentioned reagent box, the nucleotide sequence of described probe is a sequence 3 in the sequence table.

Also can comprise in the described test kit and carry out the required reagent of real-time fluorescence quantitative RT-PCR, these reagent all can obtain from commercial channels.

The application of the test kit of above-mentioned detection human galactophore globin mRNA expression quantity also belongs to protection scope of the present invention.

The present invention also provides a kind of method that detects human galactophore globin mRNA expression quantity.

The method of above-mentioned detection human galactophore globin mRNA expression quantity is: utilize the test kit of above-mentioned detection human galactophore globin mRNA expression quantity to adopt real time fluorescence quantitative RT-RCR detection of human mammaglobin mRNA expression amount.

Test kit provided by the present invention and method, can accurately quantitative sample to be measured in human galactophore globin mRNA expression quantity.Test kit provided by the present invention and method can be used for clinical and scientific research in, can the expression of hMAM mRNA in the peripheral blood of tumour patient, the marrow be carried out qualitative and quantitative analysis, to judging generation, transfer, the recurrence of mammary cancer, estimate result of treatment and dynamic observe the state of an illness significant.The present invention will play a significant role at the medical science detection range.

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.

Description of drawings

Fig. 1 is the detected result of standard substance

Fig. 2 is the typical curve of standard substance

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The preparation of embodiment 1, human galactophore globin mRNA expression quantity detection kit

Each component is in the test kit: (one) cell pyrolysis liquid I; (2) cell pyrolysis liquid II; (3) RNA lavation buffer solution I; (4) RNA lavation buffer solution II; (5) water of no RNA enzyme; (6) RNA separator column; (7) collection tube; (8) PCR pipe; (9) DNA enzyme I storage liquid; (10) reverse transcription damping fluid; (11) dNTP mixture; (12) reversed transcriptive enzyme storage liquid; (13) PCR reaction solution and probe; (14) uracil dna glycosylase (UNG enzyme) storage liquid; (15) standard substance; (16) reference substance (comprising positive reference substance and negative control product).

The composition of above-mentioned each component or be prepared as follows:

(1) cell pyrolysis liquid I

Solute is the material of following final concentration: 0.1mol/L Tutofusin tris-hydrochloric acid, 0.1mol/L sodium-chlor and 0.05mol/L magnesium chloride.Solvent is a water.

Cell pyrolysis liquid I is under 25 ℃ of conditions, and the pH value is 7.6.

(2) cell pyrolysis liquid II

Solute is the material of following final concentration: 3mol/L guanidinium isothiocyanate, 2mol/L Guanidinium hydrochloride, 0.3mol/L sodium-acetate, 0.2% sodium lauryl sulphate.Solvent is a water.

Cell pyrolysis liquid II is under 25 ℃ of conditions, and the pH value is 5.2.

(3) RNA lavation buffer solution I

Solute is the material of following final concentration: 0.2mol/L sodium-acetate, 0.1mol/L sodium chlorate, 0.1mol/L Tutofusin tris-hydrochloric acid.Solvent is a water.

RNA lavation buffer solution I is under 25 ℃ of conditions, and the pH value is 7.6.

(4) RNA lavation buffer solution II

Solute is the material of following final concentration: 1.2mol/L Trisodium Citrate, 0.5mol/L Tutofusin tris-hydrochloric acid.Solvent is a water.

RNA lavation buffer solution II is under 25 ℃ of conditions, and the pH value is 7.5.

(5) water of no RNA enzyme

In deionized water, add diethylpyrocarbonate (DEPC) (available from Biobasic company), to final concentration be 0.05%, place after 10-12 hour for 22-25 ℃, 121 ℃ of autoclavings 20 minutes, 22-25 ℃ of placement is standby.

(6) RNA separator column

Purchase excellent brilliant biotechnology company limited in Anhui.

(7) collection tube

Purchase excellent brilliant biotechnology company limited in Anhui.

(8) PCR pipe

Purchase in U.S. Applied Biosystems (ABI) company.

(9) DNA enzyme I storage liquid

Solute is the material of following final concentration: 50mmol/L Tutofusin tris-hydrochloric acid, 25mmol/L Tutofusin tris-acetyl, 12.5mmol/L magnesium chloride, 5.5mmol/L calcium chloride, 25% glycerine, 0.5U/ μ lDNA enzyme I.Solvent is a water.

DNA enzyme I storage liquid is under 25 ℃ of conditions, and the pH value is 7.5.

(10) reverse transcription damping fluid

Solute is the material of following final concentration: 9.1 μ mol/L Oligo (dT) _12-18(TaKaRa), 3.6U/ μ l RNA enzyme inhibitors, 72.7mmol/L dithiothreitol (DTT) (Invitrogen), 182mmol/L Tutofusin tris-hydrochloric acid, 273mmol/L Repone K, 11mmol/L magnesium chloride.Solvent is a water.

The reverse transcription damping fluid is under 25 ℃ of conditions, and the pH value is 8.3.

(11) dNTP mixture

Contain dATP, dCTP, dGTP, dTTP, be its sodium salt-aqueous solution, under 25 ℃ of conditions, the pH value is 7.0-7.5, and four kinds of dNTP concentration final concentrations are 10mmol/L.

As dATP is deoxyadenosine triphosphate, and one or two in three hydrogen on the phosphoric acid replaced by sodium, just formed sodium salt.Other are several also to be that so promptly dNTP exists with sodium-salt form.

(12) reversed transcriptive enzyme storage liquid

Solute is the material of following final concentration: 20mmol/L Tutofusin tris-hydrochloric acid, 100mmol/L sodium-chlor, 0.1mmol/L ethylenediamine tetraacetic acid (EDTA), 1.0mmol/L dithiothreitol (DTT) (Invitrogen), 50% glycerine, 0.01%Nonidet p-40,200U/ μ l reversed transcriptive enzyme (Invitrogen).Solvent is a water.

Reversed transcriptive enzyme is under 25 ℃ of conditions, and the pH value is 7.5.

(13) PCR reaction solution and probe

1, PCR reaction solution

Solute is the material of following final concentration: 18.5mmol/L Tutofusin tris-hydrochloric acid, 2.78mmol/L magnesium chloride, 92.6mmol/L Repone K, 0.37 μ mol/L upstream and downstream primer, 0.1U/ μ l Taq enzyme (TaKaRa), 400nmol/L dATP, 400nmol/L dCTP, 400nmol/L dGTP, 400nmol/L dTTP.Solvent is a water.

The PCR reaction solution is under 25 ℃ of conditions, and the pH value is 8.3.

Above-mentioned primer is:

P1 (upstream primer): 5 '-TGAAGTTGCTGATGGTCCTCAT-3 ' (SEQ ID NO:1),

P2 (downstream primer): 5 '-TCAGTCTTAGACACTTGTGGATTGATT-3 ' (SEQ ID NO:2).

Primer dissolves in (consisting of of TE solution: 10mmol/L Tutofusin tris-hydrochloric acid, 1mmol/L ethylenediamine tetraacetic acid (EDTA), water) in the TE solution.

2, probe

Probe sequence is: 5 '-FAM-AGCACTGCTACGCAGGCTCTGGCT-TAMRA-3 ' (SEQ ID NO:3).

Probe dissolves in the TE solution (TE solution consist of 10mmol/L Tutofusin tris-hydrochloric acid, 1mmol/L ethylenediamine tetraacetic acid (EDTA), water), and the concentration of probe is 2 μ mol/L.

Above-mentioned primer and probe are that (the GenBank accession number is: NM_002411.1) be template with the hMAM full length cDNA sequence, use ABI7000 type real-time fluorescence quantitative PCR instrument accompanying software to analyze TaqMan primer and probe site, consider hMAM genomic dna sequence situation simultaneously, therefrom select best of breed to obtain.Above-mentioned primer and probe are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

(14) uracil dna glycosylase (UNG enzyme) storage liquid

Solute is the material of following final concentration: 50% glycerine, 30mmol/L Tutofusin tris-hydrochloric acid, 150mmol/L sodium-chlor, 1.0mmol/L ethylenediamine tetraacetic acid (EDTA), 1.0mmol/L dithiothreitol (DTT) (Invitrogen), 0.05%Tween20,1U/ μ l uracil dna glycosylase.Solvent is a water.

UNG enzyme storage liquid is under 25 ℃ of conditions, and the pH value is 7.5.

(15) standard substance

Standard substance are that final concentration is 2 * 10 ⁸The pMD18-hMAM121 recombinant plasmid of copy number/μ l, TE solution dissolving (TE solution consist of 10mmol/L Tutofusin tris-hydrochloric acid, 1mmol/L ethylenediamine tetraacetic acid (EDTA), water).

The dna sequence dna of the hMAM fragment of inserting in the standard substance (name is called hMAM121) is shown in SEQ ID NO:4 in the sequence table.

1, the extraction of cell total rna

1) with human breast carcinoma SK-BR-3 cell, (Gibco company) cultivates according to a conventional method with the DMEM perfect medium.

2) with cultured cells with 0.25% trypsin quality percentage composition) digestion, the 1000rpm centrifugal collecting cell is transferred in the 1.5mL centrifuge tube, every pipe about 1 * 10 ⁶Individual cell.

3) extract cell total rna, detailed process is as follows:

1. add 650 μ l cell pyrolysis liquid II in the sedimentary cell and (in cell pyrolysis liquid II, add 2 mercapto ethanol, add-on is that to add 20 μ l concentration be the 2 mercapto ethanol of 14.5mol/L to every 1mL cell pyrolysis liquid II, the packing 2 mercapto ethanol will carry out under stink cupboard), the abundant mixing of vortex oscillation.

2. 70% ethanol that adds 650 μ l again, the vortex oscillation mixing.

3. above-mentioned mixed solution (not comprising precipitation) is joined in the RNA separator column that is fixed on the 2mL collection tube, centrifugal 1 minute of 10000g discards flowing liquid and proceeds step operation 3..

4. add the water that 400 μ l do not have the RNA enzyme in DNA enzyme I storage liquid pipe, mixing, of short duration centrifugal collection are drawn 45 these liquid of μ l and directly are added on the RNA separator column filter membrane, and room temperature was placed 15 minutes.

5. draw 700 μ l RNA lavation buffer solution I and join on the RNA separator column and wash pillar, centrifugal 1 minute of 10000g discards the 2mL collection tube.

6. the RNA separator column is fixed on another new 2mL collection tube, add the diluted RNA lavation buffer solution of 500 μ l II (RNA lavation buffer solution II before use, every 3mL adds the 12mL dehydrated alcohol), centrifugal 1 minute of 10000g, discard effluent liquid, this collection tube is reusable.

7. again with the RNA lavation buffer solution II of 500 μ l washing pillar, centrifugal and discard effluent liquid.12000g is centrifugal 1 minute then, dries RNA separator column matrix.

8. posts transfer to the 1.5ml centrifuge tube of no RNA enzyme, add the water that 50 μ l do not have the RNA enzyme, guarantee that the water of the no RNA enzyme that adds directly is added on the base for post matter centrifugal 1 minute of 10000g, eluted rna.The centrifuge tube that RNA solution is housed is placed on the ice chest, standby.

2, reverse transcription reaction

To be mixed with inverse transcription reaction liquid (volume ratio of dNTP mixture, reversed transcriptive enzyme storage liquid, reverse transcription damping fluid is 2: 1: 11) in dNTP mixture and the reversed transcriptive enzyme storage liquid adding reverse transcription damping fluid, mixing, of short duration centrifugal, place on the ice chest standbyly, use the remaining inverse transcription reaction liquid in back in-20 ℃ of refrigerators, to preserve.

The reverse transcription system is: the water 2.5ul of inverse transcription reaction liquid 3.5ul, total rna solution 4.0ul, no RNA enzyme.

The reverse transcription reaction condition is: 42 ℃, and 15 minutes → 95 ℃, 5 minutes.

After reaction finishes, take out centrifuge tube and place on the ice chest.

3, pcr amplification

With the regular-PCR amplification kit of TaKaRa company, reference reagent box specification sheets is operated.

The PCR reaction system is: reverse transcription product 5ul, 10 * PCR damping fluid 5.0ul, dNTP mixture 4.0ul, each 2.0ul of upstream and downstream primer (P1, P2), Taq enzyme 0.5ul, water 31.5ul.

The PCR reaction conditions is: 94 ℃, 3 minutes → 94 ℃ again, 30 seconds earlier, and 60 ℃, 45 seconds, 72 ℃, 50 seconds, totally 35 circulations → last 72 ℃, 7 minutes.

4, the acquisition of hMAM121 amplified production

1) pcr amplification product is carried out 1.2% agarose gel electrophoresis, cut the sepharose that contains the 121bp target DNA fragment under ultraviolet lamp, after drying with paper handkerchief, chopping is weighed, and determines the volume of glue according to the ratio of 100mg=100 μ l.

2) reclaim test kit (available from the handsome Bioisystech Co., Ltd in Shanghai) with dna gel and reclaim amplified production

1. add DE-A liquid according to the ratio in the test kit specification sheets in reclaiming glue, 60 ℃ are heated to fusing fully.

2. add 0.5 times of volume in the DE-B of DE-A liquid liquid, mixing; Add Virahol, making its final concentration is 20%.

3. aforesaid liquid is changed in the preparation pipe, centrifugal 1 minute of 5500rpm abandons filtrate.

4. after adding 0.5mL damping fluid W1, centrifugal 1 minute of 5500rpm abandons filtrate.

5. add 0.7mL damping fluid W2, centrifugal 1 minute of 5500rpm abandons filtrate.

6. add 0.7mL damping fluid W2 again, centrifugal 1 minute of 5500rpm abandons filtrate; 12000rpm, centrifugal 1 minute, to dry filter membrane matrix.

7. will prepare pipe and place new 1.5mL centrifuge tube, central authorities add 25 μ l water at filter membrane, after room temperature leaves standstill 1 minute, and 12000rpm, centrifugal 1 minute eluted dna.

5, the clone of hMAM121 and evaluation

1) structure of recombinant vectors

1. in the 10 μ l systems, add 1 μ l T carrier (pMD18-T Vector) (TaKaRa), 4 μ l DNA samples add 5 μ l and connect damping fluid (100mmol/L Tutofusin tris-hydrochloric acid, 15mmol/L magnesium chloride, 1.0mmol/L Triphosaden, 20mmol/L dithiothreitol (DTT) (Invitrogen), 1U/ μ lDNA ligase enzyme.Connect damping fluid under 25 ℃ of conditions, the pH value is 7.5.), 16 ℃ were reacted 14 hours.

2. get 5 μ l and connect product, join in the 100 μ l bacillus coli DH 5 alpha competence bacteriums, ice bath is 40 minutes behind the mixing, places 42 ℃ of water heat-shockeds 90 seconds, ice bath 2 minutes.

3. the LB substratum 400 μ l that add antibiotic-free mix jolting after 45 minutes with the speed of 150rpm on 37 ℃ of shaking tables, liquid in the centrifuge tube is coated on the LA flat board equably, 37 ℃ keep flat 20 minutes after, be inverted and cultivated 12 hours.

2) evaluation of recombinant vectors

1. observe the bacterium colony on the LA flat board, the picking bacterium colony preferably of looking goes to respectively in the Boiling tube that 5mL LA substratum is housed at random, and in the rearmounted 37 ℃ of shaking tables of numbering, 250rpm is about jolting 8-14 hour, to OD ₆₀₀≈ 0.4.

2. get the 1.5mL culture to centrifuge tube, 4 ℃, centrifugal 30 seconds of 11000rpm abandons most supernatant liquor.

3. lysate I (the 500mmol/L glucose of 100 μ l precoolings, 25mmol/L Tris.Cl (pH8.0,25 ℃), 10mmol/L EDTA (pH8.0,25 ℃), water) resuspended precipitation adds new lysate II (200mmol/l NaOH, the 1%SDS for preparing of 200 μ l behind the thermal agitation mixing, water), behind the mixing, ice bath 3 minutes adds lysate III (the 3mol/L Potassium ethanoate of 150 μ l precoolings again; PH5.2,25 ℃), put upside down behind the mixing ice bath 3-5 minute; 4 ℃, 11000rpm is transferred to supernatant in another centrifuge tube after centrifugal 5 minutes.

4. add isopyknic phenol/chloroform (1: 1), vibrate back 4 ℃, centrifugal 2 minutes of 11000rpm is transferred to supernatant another centrifuge tube again.

5. add 3M NaAc solution and 2.5 times of dehydrated alcohols of 1/10 volume, behind the mixing, room temperature left standstill 10 minutes, and 4 ℃, 11000rpm abandons supernatant after centrifugal 10 minutes.

6. add 1mL 70% ethanol, the vibration rinsing after 4 ℃, centrifugal 2 minutes of 11000rpm; Discard supernatant liquid.

7. add a small amount of dehydrated alcohol, 4 ℃, centrifugal 2 minutes of 11000rpm, discard ethanol after, room temperature was inverted 10 minutes, added 30 μ l dissolved in distilled water plasmid DNA.

8. get an amount of plasmid DNA as template, under the guiding of primer P1 and P2, carry out PCR and identify, can amplify the positive cloned plasmids of 151bp dna fragmentation, thereby filter out the purpose bacterium colony.

3) evaluation of hMAM121 amplified production

1. a large amount of extractings of plasmid

A) get the bacterium liquid that 1mL contains the purpose bacterium colony, join in the 30mL LA substratum, shaking table is cultured to OD ₆₀₀Be about 0.6.

B) get the above-mentioned bacterium liquid of 25mL and be seeded in the 300mL LA substratum, 37 ℃, 250rpm cultivated 10-14 hour, and 4 ℃ then, 4500rpm, 20 minutes centrifugal receipts bacterium.

C) the resuspended thalline of STE (10mmol/L Tris-HCl (PH8.0,25 ℃), 0.1mol/L NaCl, 1mmol/L EDTA (PH8.0,25 ℃), water) of usefulness 200mL precooling, 4 ℃, 4500rpm, 20 minutes centrifugal receipts bacterium.

D) add 9mL Solution I (500mmol/L glucose, 25mmol/L Tris.Cl (pH8.0,25 ℃), 10mmol/L EDTA (pH8.0,25 ℃), water), mixing, add the 1mL freshly prepared N,O-Diacetylmuramidase of 10mmol/L Tris-HCl (pH8.0) (10mg/mL) again, add the freshly prepared Solution II of 10mL (200mmol/l NaOH, 1%SDS, water) again, mixing is 5-7 time gently, and room temperature left standstill 5 minutes.

E) add ice-cold SolutionIII (the 3mol/L Potassium ethanoate of 7.5mL; PH5.2,25 ℃), mixing gently, ice bath 10 minutes; 4 ℃, centrifugal 10 minutes of 11000rpm gets supernatant.

F) Virahol of adding 2/3 volume, mixing, room temperature left standstill 10 minutes, and centrifugal 10 minutes of 11000rpm abandons supernatant.

G) with 70% washing with alcohol precipitation, 8000rpm, centrifugal recovery in 15 minutes is with 2mL TE dissolution precipitation.

H) LiCl of adding 2mL 5mol/L, mixing, centrifugal 10 minutes of 10000g, supernatant discarded.

I) with 70% washing with alcohol precipitation, abandon supernatant, control dry liquids as far as possible, room temperature leaves standstill to drying.

J) add 300 μ l TER, dissolution precipitation is transferred in the 1. 5mL centrifuge tubes, 37 ℃ water-bath 1-2 hour.

K) add 300 μ l 1.6mol/L NaCl (containing 13% polyoxyethylene glycol), mixing, 4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant.

L) 250 μ l TE (pH8.0) dissolution precipitations are used phenol, phenol/chloroform, each extracting of chloroform respectively once.

M) get supernatant, add 60 μ l 10mol/L NH ₄The dehydrated alcohol of Ac and 2 times of volumes (or 95% ethanol), mixing, 4 ℃, centrifugal 5 minutes of 12000g abandons supernatant.

N) 120 μ l, 75% washing with alcohol precipitation, 4 ℃, centrifugal 10 minutes of 12000g abandons supernatant, controls dry liquids as far as possible, and room temperature leaves standstill to drying.

O) add 100 μ l tri-distilled water dissolution precipitations, ultraviolet spectrophotometer detects the concentration and the purity of plasmid down.

2. the sequencing of plasmid

The positive colony plasmid that contains target gene fragment is carried out sequencing with 377 sequenators of U.S. Applied Biosystems company, insertion fragment hMAM121 in the recombinant vectors has the dna sequence dna of SEQ IDNO:4 in the sequence table as a result, with this recombinant vectors called after pMD18-hMAM121.With pMD18-hMAM121 as standard substance.

3. the plasmid pMD18-hMAM121 that above-mentioned extracting is obtained is 2 * 10 with TE solution dissolving (TE solution consist of 10mmol/L Tutofusin tris-hydrochloric acid, 1mmol/L ethylenediamine tetraacetic acid (EDTA), water) to final concentration ⁸Copy number/μ l.

(16) reference substance

Positive reference substance is the total RNA that contains hMAM mRNA, and the negative control product are the total RNA of normal people's peripheral blood, and the RNA extracting method is with sample RNA extracting method (step 3 of embodiment 2).To extract to such an extent that two kinds of total RNA are 0.2ug/ul with water dissolution to the final concentration of no RNA enzyme respectively, get the above-mentioned RNA sample of 5ul and add in the 1ml dehydrated alcohol-20 ℃ of preservations.

With above-mentioned each component packing, be combined as the test kit of 10 person-portions, the amount of each component is in every box: cell pyrolysis liquid I1 bottle (30mL/ bottle), cell pyrolysis liquid II1 bottle (10mL/ bottle), RNA lavation buffer solution I1 bottle (10mL/ bottle), RNA lavation buffer solution II1 bottle (3mL/ bottle), 1 bottle in no RNA enzyme water (4mL/ bottle), the RNA separator column, collection tube, the PCR pipe, DNA enzyme I storage liquid 1 pipe (120ul/ pipe), reverse transcription damping fluid 1 pipe (33ul/ pipe), dNTP mixture 1 pipe (6.0ul/ pipe), reversed transcriptive enzyme 1 pipe (3.0ul/ pipe), PCR reaction solution 1 pipe (251.75ul/ pipe), UNG enzyme storage liquid 1 pipe (4.75ul/ pipe), probe 1 pipe (28.5ul/ pipe), standard substance 1 pipe (10ul/ pipe), positive reference substance 1 pipe (1.0ug/ pipe), negative control product 1 pipe (1.0ug/ pipe).

The detection of embodiment 2, human galactophore globin gene mRNA expression amount

Detect human galactophore globin gene mRNA expression amount in following experimental group and the control group sample with the test kit of 10 person-portions of embodiment 1 preparation.

Experimental group: the patient with breast cancer that 46 routine pathological diagnosis are made a definite diagnosis, wherein 13 routine clinical definites shift.

Control group: 10 routine patients with lung cancer, 4 routine patients with gastric cancer, 6 routine liver cancer patients, 18 routine benign breast disease patients and 10 routine normal healthy controls.

One, experiment is prepared

1, cell pyrolysis liquid I is diluted to cell pyrolysis liquid I diluent in 1: 9 ratio with the sterilization deionized water.

2, add 2 mercapto ethanol in cell pyrolysis liquid II, add-on is: adding 20 μ l concentration among every 1mL cell pyrolysis liquid II is the 2 mercapto ethanol of 14.5M, and the packing 2 mercapto ethanol will carry out under stink cupboard.

3, RNA lavation buffer solution II is before using, and every bottle adds the 12mL dehydrated alcohol.

4, the ethanolic soln 10mL of preparation 70%.

Two, sampling

Adopt experimenter's venous blood 3mL in aseptic centrifuge tube, use EDTA to make antithrombotics (1.44mg/mL whole blood).Should use immediately after the sample collection,, can preserve 1-2 hour at 4 ℃, but the time be unsuitable long, otherwise will influence measurement result if can not use immediately.Whole blood after treatment, the karyocyte that obtains can be preserved in-80 ℃ or liquid nitrogen.Obtain experimental group sample and control sample.

Three, the extraction of the cell total rna of experimental group sample and control sample

1) get fresh anticoagulated blood that the 3mL step 2 obtains and join the aseptic centrifuge tube of 50mL after, add the cell pyrolysis liquid I diluent of 15mL, vortex oscillation mixing again.

2) ice bath is after 15 minutes, mixing twice rapidly on the vortex oscillation device, and the solution becomes clarification shows red corpuscle cracking.If the hemocytometer of individual samples when perhaps ECR raises, can prolong ice bath time to 20 minute.

3) 4 ℃, centrifugal 10 minutes precipitations of 450g karyocyte, abandoning supernatant fully.

4) karyocyte of the cell pyrolysis liquid I diluent washing precipitation of usefulness 5mL, vortex oscillation is with complete suspension cell.

5) centrifugal 10 minutes of 4 ℃, 450g, and supernatant discarded fully once more.

6) add 1mL cell pyrolysis liquid I diluent in sedimentary karyocyte, vortex oscillation is transferred to it in aseptic 1.5mL centrifuge tube with complete suspension cell, centrifugal 10 minutes of 4 ℃, 450g, supernatant discarded fully.

7) in sedimentary karyocyte, add 650 μ l cell pyrolysis liquid II (having added 2 mercapto ethanol), the abundant mixing of vortex oscillation.

8) add 70% ethanol of 650 μ l again, the vortex oscillation mixing.May produce throw out this moment owing to alcoholic acid adds, but this can not influence the extraction of RNA.

9) above-mentioned mixed solution (not comprising precipitation) is joined (this post maximum binding capacity is 800 μ l in the RNA separator column that is fixed on the 2mL collection tube, so each add-on should not surpass 750 μ l), centrifugal 1 minute of 10000g discards flowing liquid and proceeds the operation of step 9.

10) add the water that 480 μ l do not have the RNA enzyme in DNA enzyme I storage liquid pipe, mixing, of short duration centrifugal collection are drawn 45 these liquid of μ l and directly are added on the RNA separator column filter membrane, and room temperature was placed 15 minutes.

11) absorption 700 μ l RNA lavation buffer solution I join on the RNA separator column and wash pillar, and centrifugal 1 minute of 10000g discards the 2mL collection tube.

12) the RNA separator column is fixed on another new 2mL collection tube, adds the RNA lavation buffer solution II that 500 μ l have diluted, centrifugal 1 minute of 10000g discards effluent liquid.This collection tube is reusable.

13) the RNA lavation buffer solution II washing RNA that has diluted with 500 μ l again separates to live, and is centrifugal and discard effluent liquid.12000g is centrifugal 1 minute then, to dry RNA separator column matrix.

14) the RNA separator column is transferred on the 1.5mL centrifuge tube of no RNA enzyme, the water of getting 50 μ l and do not have the RNA enzyme joins on the RNA separator column matrix 10000g, centrifugal 1 minute.The centrifuge tube that RNA is housed is placed on the ice chest, standby.Obtain experimental group sample RNA and control sample RNA.

Four, quantitative fluorescent PCR

1, the reverse transcription of experimental group sample RNA, control sample RNA, positive control and negative control

The centrifuge tube that reverse transcription damping fluid, dNTP mixture, reversed transcriptive enzyme are housed is taken out from-20 ℃ of refrigerators, place treat on the ice chest that it slowly melts after, of short duration centrifugal, dNTP mixture and reverse transcription damping fluid all added in the reversed transcriptive enzyme pipe be mixed with inverse transcription reaction liquid, mixing, of short duration centrifugal, place on the ice chest standbyly, use back residue inverse transcription reaction liquid in-20 ℃ of refrigerators, to preserve.

Reverse transcription system: the water 1.5ul of inverse transcription reaction liquid 3.5ul, total rna solution 5.0ul, no RNA enzyme.

The reverse transcription reaction condition is: 42 ℃, 15 minutes → 95 ℃, 5 minutes.

After reaction finishes, the reaction product taking-up is placed on the ice chest.Obtain experimental group sample reverse transcription product, control sample reverse transcription product, positive control reverse transcription product and negative control reverse transcription product.

2, the processing of standard substance

With standard substance stock solution (2 * 10 ⁸Copy number/ul) gradient dilution is 2 * 10 ⁷, 2 * 10 ⁶, 2 * 10 ⁵, 2 * 10 ⁴, 2 * 10 ³, 2 * 10 ², 2 * 10 ¹Copy number/ul.

3, quantitative fluorescent PCR

The centrifuge tube that PCR reaction solution, probe and UNG enzyme storage liquid are housed is taken out from-20 ℃ of refrigerators, place treat on the ice chest that it slowly melts after, of short duration centrifugal, probe and PCR reaction solution are all added in the UNG enzyme storage liquid pipe, mixing, of short duration centrifugal, place on the ice chest standbyly, use the back remaining liq in-20 ℃ of refrigerators, to preserve.In the enterprising performing PCR amplification of ABI company 7000 type quantitative PCR instrument.

The PCR reaction system is: template 5.0 μ l, PCR reaction solution 15.0 μ l, water 5.0 μ l.

Wherein, template is experimental group sample reverse transcription product or control sample reverse transcription product or positive control reverse transcription product or negative control reverse transcription product or standard substance.

The PCR reaction conditions is: 37 ℃, and 10 minutes → 95 ℃, 15 minutes → 95 ℃, 15 seconds → 60 ℃, 1 minute.Totally 50 circulations.

Five, the making of typical curve

Standard substance fluorescence quantitative PCR detection result as shown in Figure 1.X-coordinate is the cycle number of PCR reaction, and ordinate zou is the detected fluorescent value of detector, and on behalf of the starting template number, curve respectively be from left to right among the figure: 1 * 10 ⁸, 1 * 10 ⁷, 1 * 10 ⁶, 1 * 10 ⁵, 1 * 10 ⁴, 1 * 10 ³, 1 * 10 ²Copy number.

According to detected result drawing standard curve, typical curve as shown in Figure 2.X-coordinate is the logarithmic value of the initial copy number of standard substance, and ordinate zou is the Ct value.Log among Fig. 2 refers in particular to Log ₁₀

Six, detected result

Choose above-mentioned all samples to detect the hole, carry out corresponding analysis, draw the starting template number (M) of testing sample according to typical curve.Copy number N=M * 6.6 of hMAM mRNA in every milliliter of whole blood sample.

1) patient with breast cancer's detected result of having shifted of 13 routine clinical definites is all positive, and concrete outcome is as shown in table 1.

The detected result of the patient with breast cancer hMAM mRNA copy number that table 1 13 routine clinical definites have shifted

Patient's numbering	Sex	Age	Sample	HMAM mRNA value (copy number/milliliter whole blood)
Patient's numbering	Sex	Age	Sample	HMAM mRNA value (copy number/milliliter whole blood)	1 2 3 4 5	Woman woman woman woman woman	42 65 50 62 40	Peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood	3.2×10 ⁶ 1.4×10 ⁷ 5.7×10 ⁵ 7.1×10 ⁶ 1.8×10 ⁷

6 7 8 9 10 11 12 13

Woman woman woman woman woman woman woman woman

52 44 57 66 80 42 70 63

Peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood

1.2×10 ⁶ 9.3×10 ⁵ 1.7×10 ⁶ 2.7×10 ⁶ 4.3×10 ⁵ 6.2×10 ⁴ 8.1×10 ⁵ 5.2×10 ³

2) 10 examples are positive among the clinical patient with breast cancer who does not make a definite diagnosis transfer of 33 examples, and all the other 23 examples are negative.Positive findings is as shown in table 2.

The clinical positive test symbol of not making a definite diagnosis the patient with breast cancer hMAM mRNA copy number of transfer of table 2 33 examples

Patient's numbering	Sex	Age	Sample	HMAM value (copy number/milliliter whole blood)
Patient's numbering	Sex	Age	Sample	HMAM value (copy number/milliliter whole blood)	17 18 21 25 29 30 34 39 40 45	Woman woman woman woman woman woman woman woman woman woman	38 56 49 56 62 55 48 55 61 70	Peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood peripheral blood	6.5×10 ⁵ 4.4×10 ⁵ 1.3×10 ⁶ 2.0×10 ⁴ 5.0×10 ⁵ 2.7×10 ³ 8.2×103 3.6×10 ³ 6.4×10 ² 2.0×10 ⁵

3) control group: 10 routine patients with lung cancer, 4 routine patients with gastric cancer, 6 routine liver cancer patients, 18 routine benign breast disease patients and 10 routine normal healthy peoples are all negative, do not detect the expression of hMAM.

The above results shows with test kit of the present invention can carry out qualitative comparatively accurately and quantitative analysis to the expression of hMAM mRNA.

Sequence table

＜110〉China Science ﹠ Technology University

＜120〉a kind of test kit and application thereof that detects human galactophore globin mRNA expression quantity

<130>CGGNARY71692

<160>4

<210>1

<211>22

<212>DNA

＜213〉artificial sequence

<400>1

tgaagttgct gatggtcctc at 22

<210>2

<211>27

<212>DNA

＜213〉artificial sequence

<400>2

tcagtcttag acacttgtgg attgatt 27

<210>3

<211>24

<212>DNA

＜213〉artificial sequence

<400>3

agcactgcta cgcaggctct ggct 24

<210>4

<211>121

<212>DNA

＜213〉Genus Homo people (Homo sapiens)

<400>4

tgaagttgct gatggtcctc atgctggcgg ccctctccca gcactgctac gcaggctctg 60

gctgcccctt attggagaat gtgatttcca agacaatcaa tccacaagtg tctaagactg 120

a 121

Claims

1. test kit that detects human galactophore globin mRNA expression quantity, comprise a pair of primer and probe, described primer centering, the nucleotide sequence of a primer is a sequence 1 in the sequence table, the nucleotide sequence of another primer is a sequence 2 in the sequence table, and the nucleotide sequence of described probe is a sequence 3 in the sequence table.

2. test kit as claimed in claim 1 is characterized by: also comprise in the described test kit and carry out the required reagent of real-time fluorescence quantitative RT-PCR.