CN1195871C - Real time fluorescence quantitative RT-PCR detection human alpha-fetoprotein reagent box - Google Patents

Abstract

The present invention relates to a kit for detecting human alpha-fetal protein expressed by primary liver cancer with real-time fluorescent quantitative RT-PCR, which obtains cDNA through a reverse transcription (RT) mRNA sample. A real-time fluorescent quantitative PCR detection technology is also combined, which can accurately and quantitatively detect the mRNA expression quantity of AFP of a specimen. The kit can be used for detecting the AFP expression of specimens, such as the peripheral blood, tumor tissues, hydrothorax, ascites, pucture fluid, bone marrow, etc., of patients of clinics and scientific research for assisting diagnosis, guiding treatment and prompting prognosis.

Description

The real time fluorescent quantitative RT-polymerase chain reaction detects the test kit of human a-fetoprotein

Technical field

The invention belongs to biological technical field, relate to quantitative RT-PCR detecting kit, relate generally to a kind of real time fluorescence quantitative RT-RCR detection of human alpha-fetoprotein test kit, be a kind ofly to obtain cDNA by reverse transcription (RT) mRNA sample, again in conjunction with the real-time fluorescence quantitative PCR detection technique, can detect first embryo fetoprotein (alpha fetal protein, the test kit of mRNA expression amount AFP) in the sample by accurate quantification.

Background technology

The transfer of malignant tumour is a great problem of clinical treatment, also is the lethal major reason of malignant tumour.Find and make diagnosis early, the clinical treatment of tumour is selected that very important meaning is arranged, can greatly improve patient's survival rate.The discovery of metastases at present and definite mainly by clinical and pathological data, is diagnosed according to iconography (X-ray sheet, CT, mr, PET etc.) or pathomorphism.But make in the imaging examination and can find focus, need a certain size metastasis formation, can be by the instrument imaging and effectively differentiate; Make on the pathomorphism whether judge malignant change of cell, then must get suitable detection sample, and have a considerable amount of observable tumour cells to exist.On this point, no matter be imaging diagnosis or pathological diagnosis, the stage that all is suitable late period after tumour forms and shifts, has had a large amount of malignant cells to accumulate, if take place to shift early stage in tumour, on pathology, can be observed or metastasis is just found before forming, and determine to shift, then can the guiding clinical treatment design for scheme, and strong prognosis foundation is provided.

It is one of main path of tumour diffusion that the blood road shifts, therefore in patient's peripheral blood, carry out tumour cell genetic expression detection, the tumour-specific markers product detects is an efficient manner understanding the metastases situation, so the diagnosis that the detection of tumor marker (tumormarker) forms and shifts tumour has great help.But metastases take place early stage, tumour cell in the peripheral blood usually seldom, can't rely on the pathomorphism inspection fully, the content of tumour specific antigen also seldom simultaneously, also be difficult to detect by enzyme-labeled immunity adsorption analysis (ELISA) or radioimmunoassay methods such as (RIA) commonly used, therefore, the research to micro-tumour cell genetic expression in the circulation blood and the detection of some specificity products has caused people's extensive concern.

Tumor incidence and mortality ratio are high at present, and some malignant tumour also has the trend that rises.If can be to metastases patient early discovery, the formulation of further treatment plan there is very important meaning, be one of curative ratio important channel of improving tumour.Development to China's medical and health care system has epochmaking social effect and economic implications.Therefore, we are devoted to develop malignant tumour and shift early detection and diagnostic techniques, to solve a great problem of clinical treatment.

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.RT-PCR has than incomparable highly sensitive of methods such as pathology or ELISA/RIA and relative high specific, for the detection of micro-tumour cell in the peripheral blood has brought hope, but therefore also some unavoidable false positive problems under cover in the behind of highly sensitive, high specific have also hindered this new and high technology applying in clinical detection to a certain extent.

AFP is a primary hepatocarcinoma cell-specific tumors of higher related antigen.Therefore detect the expression that has or not AFP mRNA in the samples such as patient's peripheral blood, ascites pleural fluid, puncture fluid, marrow and specimens from pri, can provide strong evidence to the diagnosis and the treatment of tumour, can also offer help the transfer of tumour and recurrence situation and observation of curative effect.What the detection of AFP mRNA was adopted is qualitative RT-PCR method in the past, and from technical standpoint, though qualitative PCR is significantly improved from the traditional methods such as ELISA of remolding sensitivity, but its specificity is relatively poor relatively, and can't accurate quantification.The release of TaqMan fluorescent quantitative PCR technique and respective detection instrument makes that people can be under the situation of utilizing the PCR high sensitivity, the PCR product is carried out real-time accurate quantification to be detected, not only solve the quantitative problem of PCR, but also improved the specificity that detects greatly.

Summary of the invention

Test kit of the present invention comprises: reverse transcription reaction liquid (RT reaction solution), moloneys mouse leukemia virus reverse transcriptase (M-MLV reversed transcriptive enzyme), RNA enzyme inhibitors (Rnasin), PCR reaction solution, hot resistant DNA polymerase (Taq archaeal dna polymerase), standard substance, reference substance.

Wherein reverse transcription (RT) reaction solution contains the water (DEPC-H that diethylpyrocarbonate is handled ₂O), moloneys mouse leukemia virus reverse transcriptase (M-MLV-RT) damping fluid, oligomerization (dT) _15-18(Oligo (dT) _15-18), dNTPs.

Wherein the PCR reaction solution contains PCR damping fluid, MgCl ₂, dNTPs, detection be with primer, fluorescent probe.

Detection divides upstream primer and downstream primer with primer:

The upstream primer sequence is: 5 '-ACCTCGTCGGAGCTGATGG-3 '

The downstream primer sequence is: 5 '-TGGCCTCCTGTTGGCATATG-3 '.

The fluorescent probe sequence is: 5 '-FAM-TGGCGAGGGAGCGGCTGACATT-TAMRA-3 '.The standard substance sequence is:

CGAAGCTGCG?GCCTCTTCCA?GAAACTAGGA?GAATATTACT?TACAAAATGC?GTTTCTCGTT

GCTTACACAA?AGAAAGCCCC?CCAGCTGACC?TCGTCGGAGC?TGATGGCCAT?CACCAGAAAA

ATGGCAGCCA?CAGCAGCCAC?TTGTTGCCAA?CTCAGTGAGG?ACAAACTATT?GGCCTGTGGC

GAGGGAGCGG?CTGACATTAT?TATCGGACAC?TTATGTATCA?GACATGAAAT?GACTCCAGTA

AACCCTGGCG?TTGGCCAGTG?CTGCACTTCT?TCATATGCCA?ACAGGAGGCC?ATGCTTCAGC

AGCTTGGTGG?TGGATGAAAC?ATATGTCCCT?CCTGCATTCT?CTGATGACAA?GTTCATTTTC

CATAAGGATC?TGTGCCAAGC?TCAGGGTGTA?GCGCTGCAAA?CGATGAAGCA?AGAGTTTCTC

ATTAACCTTG?TGAAGCAAAA?GCCACAAATA?ACAGAGGAAC?AACTTGAGGC?TGTCATTGCA

GATTTCTCAG GCCTGTTGGA GAAATGCTGC CAAGGCCA reference substance is divided into positive reference substance and negative control product, and negative control is the RNA sample of no AFPmRNA, and positive control is the RNA sample that AFPmRNA is arranged.

This test kit is stored in-20 ℃, reduces multigelation as far as possible.

The present invention has set up the method for utilizing TaqMan technology for detection AFP to express, and patient's sample after testing, shows that this method is practical.Because present method has adopted the pcr amplification technology, the detection sensitivity that makes AFP express improves greatly, and because the application of fluorescent probe makes its specificity also improve greatly, reduce the false positive rate of conventional pcr amplification, made us can in few sample, obtain enough information.Primer, probe and detected result that present method is designed can provide reliable foundation for the exploitation of fluorescent quantificationally PCR detecting kit.

In this test item, we have adopted present state-of-the-art specificity fluorescent probe hybridization quantitative PCR detection technique, reduce false positive as far as possible and disturb under the prerequisite that keeps hypersensitivity.Before the real-time fluorescence quantitative PCR detection technique occurs, no matter people are direct PCR or competitive PCR quantitatively to pcr template, basically all to pass through PCR product electrophoresis, again with electrophoresis result machine picture processing as calculated, determine what of final PCR product amount according to the brightness of electrophoretic band, or the PCR product of the tape label mode with ELISA detected, infer the amount of starting template more thus, but these methods in fact all belong to the sxemiquantitative level, even because PCR condition optimization, the unstable of the operation of electrophoresis and subsequent step still can be brought influence to interpretation of result, thereby influences quantitative this purpose.Appearance along with the real-time fluorescence quantitative PCR technology, people can accomplish the accurate quantification to pcr template veritably, this high sensitivity that has quantitatively not only kept conventional PCR, and because specificity fluorescent probe hybridization The Application of Technology makes the specificity of detected gene improve greatly.

Test kit using method of the present invention:

Each detection all should be set up positive control and negative control.Standard substance are 1 * 10 with the aseptic deionized water dilution ²-1 * 10 ⁹Copy/ml.

Reverse transcription: cumulative volume 25 μ l, 5 minutes RNA to be measured of 2 μ l70 ℃ of pre-sex change wherein, 20.75 μ lRT reaction solutions, 1.0 μ l M-MLV reversed transcriptive enzymes, 1.25 μ l Rnasin.In 37 ℃ of water-bath 10-15 minutes.

Augmentation detection: on quantitative real time PCR Instrument, carry out cumulative volume 50 μ l, 47.0 μ l PCR reaction solutions wherein, 2.5 μ l test sample (reverse transcription product, standard substance, positive or negative contrast), 0.5 μ l Taq archaeal dna polymerase.Reaction conditions: 95 ℃ of pre-sex change 5 minutes, 95 ℃ 30 seconds, 62 ℃ 30 seconds, 72 ℃ totally 40 circulations in 30 seconds, 72 ℃ were extended 10 minutes, and put 4 ℃.According to the typical curve that is obtained, calculate the amount (copy number/ml) of AFP in each sample to be measured.

Description of drawings

Fig. 1 detects for the real-time fluorescence quantitative RT-PCR standard substance.

Fig. 2 is the real-time fluorescence quantitative RT-PCR typical curve.

Embodiment

The present invention is described further with specific embodiment in conjunction with the accompanying drawings.Should be appreciated that these embodiment only are used for illustration purpose, and are not used in the restriction scope of the invention.

Embodiment 1 fluorescence quantitative RT-RCR method detects the expression of AFP

One, material: Trizol reagent and restriction enzyme be all available from U.S. LTI/Gibco company, pGEM-T-Easy cloning system, MMLV reversed transcriptive enzyme, Taq archaeal dna polymerase, Oligo (dT) _15-18Purchase white U.S. Promega company, sequencing reagent, 377 type sequenators, 2400 type PCR instrument and 7700 type quantitative PCR instrument are U.S. Perkin Elmer company product.

Two, primer and probe design and synthetic: (GenBank accession number M17303) is template with the AFP full length cDNA sequence, uses Primer Express ^TM(V1.0, U.S. Perkin Elmer company) software analysis TaqMan primer and probe site, and, therefrom select best of breed according to considering AFP genomic dna sequence situation simultaneously.

Standard substance PCR upstream primer sequence is: 5 '-CGAAGCTGCGGCCTCTTC-3 ',

Downstream primer is: 5 '-CCTGGCCTTGGCAGCAT-3 ',

Detect and be with PCR upstream primer sequence: 5 '-ACCTCGTCGGAGCTGATGG-3 ',

The downstream primer sequence is: 5 '-TGGCCTCCTGTTGGCATATG-3 ', give birth to worker company by Shanghai and synthesize.The fluorescent probe sequence is: 5 '-FAM-TGGCGAGGGAGCGGCTGACATT-TAMRA-3 ', precious biotech company is synthetic by Dalian.

Three, examination criteria product preparation: the fresh surgical sample of Patients with Primary is liquid nitrogen cryopreservation immediately, and extracts total RNA with Trizol reagent after grinding in mortar under liquid nitrogen exists, and gets 1g RNA, uses oligo (dT) in the 25l total reaction volume _15-18For after primer carries out reverse transcription to it, increase at the enterprising performing PCR of 2400 type PCR instrument with standard substance upstream and downstream primer, condition is 95 ℃ of sex change in 5 minutes, and 95 ℃ 30 seconds, 62 ℃ 30 seconds, 72 ℃ were carried out 30 cyclic amplifications in 30 seconds, at last in 72 ℃ extend 10 minutes rearmounted 4 ℃.The PCR product promptly inserts the pGEM-T-Easy cloning vector with cloning system after electrophoresis detection, and with positive colony through sequence verification.The EcoRI enzyme is cut the back and is reclaimed the 540bp fragment, is standard substance.Measure concentration and be converted into (copy number/volume).The result:

Through order-checking, above-mentioned standard product conform to expection fully, the standard substance fragment sequence of recovery following (comprising EcoRI site, two ends):

GAATTC

CCAGTCCCAT?CTCAGCATGA?AAGAGTCTTT?GGAGCACACT?CTAGAGGAGA?CCAAGGCCCG

TTACAGCAGC?CAGTTAGCCA?ACCTCCAGTC?GCTGTTGAGC?TCTCTGGAGG?CCCAACTGAT

GCAGATTCGG?AGTAACATGG?AACGCCCGAA?CAACGAATAC?CATATCCTTC?TTGACATAAA

GACTCGACTT?GAACAGGAAA?TTGCTACTTA?CCGCCGCCTT?CTGGAAGGAG?AAGACGTAAA

AACTACAGAA?TATCAGTTAA?GCACCCTGGA?AGAGAGAGAT?ATAAAGAAAA?CCACCAAGAT

TAAGACAGTC?GTGCAAGAAG?TAGTGGATGG?CAAGGTCGTG?TCATCTGAAG?TCAAAGAGGT

GGAAGAAAAT?ATCTAAATAG?CTACAGAAGG?AGATGCTGCT?GAGGTTTTGA?AAGAAATTTG

GCTATAATCT?TATCTTTGCT?CCCTGCAAGA?AATCAGCCAT?AAGAAAGCAC?TATTAATACT

CTGCAGTGAT?TAGAAGGGGT?GGGGTG? GAATTC

Embodiment 2 fluorescence quantitative RT-RCR methods detect the application of AFP

One, sample detects:

The malignant tumor patient specimens from pri that 6 examples are made a definite diagnosis through pathology is extracted cell total rna with Trizol reagent behind the collecting cell, gets 1 gRNA, uses oligo (dT) in the 25l total reaction volume _15-18For after primer carries out reverse transcription to it, use downstream primer in the enterprising performing PCR amplification of PE company 7700 type quantitative PCR instrument with detection, condition is 95 ℃ of sex change in 5 minutes, and 95 ℃ 30 seconds, 62 ℃ 30 seconds, 72 ℃ were carried out 40 cyclic amplifications in 30 seconds, at last in 72 ℃ extend 10 minutes rearmounted 4 ℃.Add standard product examine survey simultaneously and make typical curve.Measurement result is handled according to typical curve through instrument and is calculated the AFP expression amount that detects sample.

Two, sample detection result

The standard substance detected result is seen Fig. 1.The typical curve that detects is seen Fig. 2.

6 routine patient tissue detection result of specimen are as follows:

The patient numbers sex age sample AFP value (the sample AFP value of copy number/ml) (copy number/ml)

1 male 28 tumor tissues 2.3 * 10 ⁸Margin tissue 6.0 * 10 ³

2 male 42 tumor tissues 8.5 * 10 ⁹Margin tissue 6.0 * 10 ⁷

3 women 54 tumor tissues 3.0 * 10 ⁷Margin tissue 4.3 * 10 ²

4 male 49 tumor tissues 1.4 * 10 ⁵Margin tissue 5.0 * 10 ²

5 male 57 tumor tissues 5.4 * 10 ⁸Margin tissue 8.4 * 10 ⁵

6 male 54 tumor tissues 3.6 * 10 ⁷Margin tissue 5.1 * 10 ⁵

The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (2)

1. the test kit of the human a-fetoprotein that detects of a real time fluorescent quantitative RT-polymerase chain reaction, contain reverse transcription reaction liquid, moloneys mouse leukemia virus reverse transcriptase, RNA enzyme inhibitors, pcr reaction solution, hot resistant DNA polymerase, standard substance, reference substance, it is characterized in that: reverse transcription reaction liquid contains water, moloneys mouse leukemia virus reverse transcriptase damping fluid, the oligomerization (dT) that diethylpyrocarbonate is handled _15-18, dNTPs, polymerase chain reaction liquid contains pcr buffer, MgCl ₂, dNTPs, detection with upstream primer, detect with downstream primer, fluorescent probe, wherein:

Detect and be with the upstream primer sequence

5′-ACCTCGTCGGAGCTGATGG-3′，

Detect and be with the downstream primer sequence

5′-TGGCCTCCTGTTGGCATATG-3′，

The fluorescent probe sequence is

5′-FAM-TGGCGAGGGAGCGGCTGACATT-TAMRA-3′，

The standard substance sequence is

CGAAGCTGCG?GCCTCTTCCA?GAAACTAGGA?GAATATTACT?TACAAAATGC?GTTTCTCGTT

GCTTACACAA?AGAAAGCCCC?CCAGCTGACC?TCGTCGGAGC?TGATGGCCAT?CACCAGAAAA

ATGGCAGCCA?CAGCAGCCAC?TTGTTGCCAA?CTCAGTGAGG?ACAAACTATT?GGCCTGTGGC

GAGGGAGCGG?CTGACATTAT?TATCGGACAC?TTATGTATCA?GACATGAAAT?GACTCCAGTA

AACCCTGGCG?TTGGCCAGTG?CTGCACTTCT?TCATATGCCA?ACAGGAGGCC?ATGCTTCAGC

AGCTTGGTGG?TGGATGAAAC?ATATGTCCCT?CCTGCATTCT?CTGATGACAA?GTTCATTTTC

CATAAGGATC?TGTGCCAAGC?TCAGGGTGTA?GCGCTGCAAA?CGATGAAGCA?AGAGTTTCTC

ATTAACCTTG?TGAAGCAAAA?GCCACAAATA?ACAGAGGAAC?AACTTGAGGC?TGTCATTGCA

GATTTCTCAG?GCCTGTTGGA?GAAATGCTGC?CAAGGCCA。

2. real time fluorescent quantitative RT-polymerase chain reaction as claimed in claim 1 detects the test kit of human a-fetoprotein, it is characterized in that: reference substance is divided into negative control product and positive reference substance, negative control is the RNA sample of no human a-fetoprotein mRNA, and positive reference substance is the RNA sample that human a-fetoprotein mRNA is arranged.