CN1814792A - Real-time quantitative PCR detecting human DDIT3 transcript kit - Google Patents

Abstract

This invention relates to a real-time quantitative PCR(RQ-PCR) reagent box for quickly testing the copy numbers of man DDIT3 gene transcript, which tests the cDNA specimen by a real-time quantitative PCR test technology to accurately determine the copy numbers of DDIT3 gene transcript in the specimen. The box can be used in expression of DDIT3 in the specimen of marrow and peripheral blood of acute and chronic leukaemia and marrow hyperplasia abnormal syndrome patients

Description

Real-time quantitative PCR detecting human DDIT 3 transcript kit

Technical field

The invention belongs to biological technical field, relate to a kind of new DDIT3 gene transcripts fast quantification PCR detection kit, make the mensuration of DDIT3 transcript more quick, make things convenient for, and can accurate quantification.

Background technology

Tumour is one of principal disease that threatens the human life, is ascendant trend year by year at present.Proto-oncogene activates and the cancer suppressor gene afunction is the important molecule basis that causes tumor development.Various cell injury all can cause dna damage and genomic instability as ultraviolet ray, ionizing rays and many gene cytotoxic drugs; the DNA securing system plays crucial effects in keeping the integrity of genetic material; can start the DNA securing system after cell sustains damage repairs damaged dna; and this process need makes cell-cycle arrest and mitotic division stop to provide the time by activating G1/S and G2/M " reference mark ", relates to transcription factors such as P53, Rb, WT1 and P21.If these transcription factor generation dysfunctions, damaged cell just lacks time enough damage dna is repaired, the genome damage will be delivered to daughter cell after mitotic division, along with the continuous accumulation of gene damage, cell finally vicious transformation will take place.

Dna damage inductive transcription factor 3 (DDIT3) is a new transcription factor of discovered in recent years, is bringing into play important regulation at aspects such as inducing cell Cycle Arrest, promotion cytodifferentiation and apoptosis.The textural anomaly that has the DDIT3 gene in many tumour patients.DDIT3 is positioned at karyomit(e) 12q13, and the normal generation with multiple soft tissue neoplasm such as malignant fibrous histiocytoma, glioblastoma and glioblastoma multiforme of the amplification in this site is closely related.And involve the transposition t (12 in this site; 16) (q13; P11) and t (12; 22) (q13; Q12) then cause the formation of TLS/FUS-DDIT3 and EWS/DDIT3 fusion gene, cause myxoid liposarcoma and mucoid/round cell liposarcoma to take place.DDIT3 is expressed in the melanoma and significantly reduces than benign nevus, and its expression level and melanotic tumor patient's survival time is closely related.And the present C/EBP ζ transcript of discovering CML patient also obviously reduces.Therefore, detecting DDIT3 expression of gene level helps the diagnosis of tumour patient and prognosis to judge.

The DDIT3 transcript detects main by polymerase chain reaction (Polymerase Chain Reaction, PCR), the ultimate principle of this technology is to design a complementary Oligonucleolide primers respectively at fragment two ends to be amplified, under the effect of hot resistant DNA polymerase, external be raw material with dNTPs, with the determined nucleic acid is that template increases repeatedly, can make up to a million times of micro-purpose nucleic acid amplifications, again with product electrophoresis on sepharose, with ethidium bromide staining, observations on the gel imaging instrument, round pcr have been widely used in the molecular diagnosis of various infectious diseases and malignant tumour.At present PCR method mainly contains qualitative and two kinds of detection by quantitative: qualitative detection is mainly used nest-type PRC, its advantage is very sensitive, but its shortcoming is only can show the positive, negative findings, does not have concrete numerical value, can not observe its dynamic change and carry out the prognosis judgement according to numerical value, and complex operation; Quantivative approach is a competitive PCR, is on the basis of qualitative PCR, increases jointly with the dilution confidential reference items of difference (the contrast nucleic acid of modification) and sample, makes typical curve with the extent of dilution and the result of confidential reference items, judges the amount of purpose nucleic acid in the sample; Though competitive PCR can be accurately quantitative, operate more loaded down with trivial detailsly, be difficult to clinically routine and carry out.

Along with the development of technology, produced again real-time quantitative PCR (real-time quantitative PCR, RQ-PCR).The release of TaqMan probe RQ-PCR technology and respective detection instrument such as PE5700, PE7700 overcomes the deficiency of qualitative PCR and competitive quantitative PCR effectively, can be quick, easy, specifically the DDIT3 transcript is carried out accurate quantification.

Summary of the invention

Test kit of the present invention comprises: PCR damping fluid, dNTPs, MgCl ₂, specificity T aqMan probe, Auele Specific Primer, Taq enzyme, standard substance and reference substance.

Auele Specific Primer divides upstream primer and downstream primer:

The upstream primer sequence is: 5 '-GGAGCTGGAAGCCTGGTATG-3 ',

The downstream primer sequence is: 5 '-GCTCTGGGAGGTGCTTGT-3 ',

Specific probe sequence is: 5 '-FAM-TCTTCACCACTCTTGACCCTGCTTCTCT-TAMRA-3 '

The standard substance sequence is:

GATCCAACTG CAGAGATGGC AGCTGAGTCA TTGCCTTTCT CCTTTGGGAC

ACTGTCCAGC TGGGAGCTGG AAGCCTGGTA TGAGGACCTG CAAGAGGTCC

TGTCTTCAGA TGAAAATGGG GGTACCTATG TTTCACCTCC TGGAAATGAA

GAGGAAGAAT CAAAAATCTT CACCACTCTT GACCCTGCTT CTCTGGCTTG

GCTGACTGAG GAGGAGCCAC AACCAGCAGA GGTCACAAGC ACCTCCCAGA

GCCCTCACTC TCCAGATTCC AGTCAGAGCT CCCTGGCTCA GGAGGAAGAG

GAGGAAGACC AAGGGAGAAC CAGGAAACGC AAACAGAGTG GTCATTCCCC

AGCCCGGGCT GGAAAGCAGC GCATGAAGGA GAAAGAACAG GAGAATGAAA

GGAAAGTGGC ACAGCTAGCT GAAGAGAATG AACGGCTCAA GCAGGAAATC

GAGCGCCTGA CCAGGGAAGT AGAGGCGACT CGCCGAGCTC TGATTGACCG

AATGGTGAAT CTGCACCAAG CATGAACAAT T

Reference substance is divided into positive control and negative control, and positive control is Normocellular DDIT3 cDNA sample, and negative control is the aseptic double-distilled water of no DDIT3.

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

The present invention has set up the method for utilizing TaqMan real-time quantitative PCR technology for detection DDIT3 to express, and patient and normal control sample after testing, shows that this method is practical.Because what present method adopted is advanced TaqMan fluorogenic probe hybridzation quantitative PCR technique, can carry out accurate quantification to detecting sample; And because use is complementary probe, make the specificity of pcr amplification greatly improve, reduced the false positive rate of qualitative PCR amplification; In addition, be the stopped pipe operation because present method adopts, do not need PCR aftertreatments such as electrophoresis and gel imaging, the caused pollution of post-processing operation of qualitative and competitive quantitative PCR is greatly reduced, and make that to shorten work detection time more easy.

Test kit using method of the present invention:

Each detection all should be set up positive control and negative control.Standard substance are 10 with the aseptic deionized water dilution ⁸～10 ²Copy/μ l.

Augmentation detection: on quantitative real time PCR Instrument, carry out cumulative volume 25 μ l, wherein 13.5 μ l aseptic double-distilled waters, 2.5 μ lPCR damping fluids, 4 μ l MgCl ₂, each 1 μ l of Auele Specific Primer, 1 μ l TaqMan probe, 1U Taq archaeal dna polymerase and 1 μ l test sample (standard substance, cDNA to be measured, positive control or negative control).Reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 15s, 60 ℃ of 1min totally 45 circulations then according to the typical curve that is obtained, calculate the amount (copy number/μ l) of DDIT3 transcript in the sample to be measured.

Description of drawings

Fig. 1 detects (10 for the real-time quantitative PCR standard substance ⁸～10 ²Copy/μ l).

Fig. 2 is the real-time quantitative 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 PCR method detects primer, probe design and the standard substance preparation of DDIT3

One, material

Trizol reagent is available from U.S. Gibco company, and MMLV reversed transcriptive enzyme, RNAsin are available from American I nvitrogen company, and dNTPs, Taq archaeal dna polymerase are available from U.S. MBI company, and the PE5700 quantitative real time PCR Instrument is a U.S. Perkin Elmer company product.

Two, primer and probe design and synthetic

(Genbank accession number NM_004083) is template with the DDIT3 full length cDNA sequence, use primer and the TaqMan probe sequence of Primer Express2.0 software (U.S. PE company) design RQ-PCR, and use Primer Premier5.0 software (U.S. PremierBiosoft company) designed sequence is estimated, therefrom select best of breed.

RQ-PCR upstream primer sequence is: 5 '-GGAGCTGGAAGCCTGGTATG-3 ',

The downstream primer sequence is: 5 '-GCTCTGGGAGGTGCTTGT-3 ',

The TaqMan probe sequence is: 5 '-FAM-TCTTCACCACTCTTGACCCTGCTTCTCT-TAMRA-3 ', primer and probe are synthetic by Shanghai Bo Ya company.

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

The downstream primer sequence is: 5 '-CCGAATTCTTCATGCTTGGTGCAGA-3 ', give birth to worker company by Shanghai and synthesize.

Three, examination criteria product preparation:

Get normal people's marrow and separate mononuclearcell through Ficoll liquid, the aseptic washing of RPMI1640 2 times, centrifugal collecting cell adds Trizol reagent and extracts total RNA, get the total RNA of 2 μ g, after in 40 μ l reaction systems, it being carried out reverse transcription with six random primers and MMLV, increase at the enterprising performing PCR of PE5700 type PCR instrument with standard substance PCR upstream and downstream primer, condition is 95 ℃ of pre-sex change 5min, 35 circulations of 94 ℃ of 45 seconds, 58 ℃ 45 seconds, 72 ℃ 60 seconds coamplifications are then extended rearmounted 4 ℃ of preservations in 5 minutes in 72 ℃ at last.

The PCR product is cloned into the pMD18-T carrier through the gel electrophoresis separation and purification with the T-A cloning, and positive colony is identified through order-checking.Standard substance are the pMD18-T carrier that carries positive colony, and total length 3223bp measures its content and is diluted to 10 ⁹Copy/μ l puts-20 ℃ of preservations.

Embodiment 2

Fluorescence quantitative PCR method detects the application of DDIT3 transcript

One, sample detects:

Acute myeloid leukemia (AML) the patient bone marrow prepare that 20 examples are made a definite diagnosis through morphology and cytogenetics, use Ficoll liquid and separate mononuclearcell, centrifugal collecting cell extracts total RNA with adding Trizol after the PBS washing, get the total RNA of 2 μ g, after in 40 μ l reaction systems, it being carried out reverse transcription with six random primers and MMLV, increase at the enterprising performing PCR of PE5700 type PCR instrument with RQ-PCR upstream and downstream primer, condition is 95 ℃ of pre-sex change 5min, 45 circulations of 94 ℃ of 15 seconds, 60 ℃ 1 minute coamplifications then.Add standard product examine survey simultaneously and make typical curve.Measurement result is handled according to typical curve through instrument and is calculated the DDIT3 expression amount that detects sample.

Two, result:

(1) typical curve:

The standard substance detected result is referring to Fig. 1, and typical curve is referring to Fig. 2.

(2) sample detection:

10 routine normal controls and 10 routine AML patient's bone marrow prepare detected results are as follows:

Numbering	Sex	Diagnosis	Age (year)	bcr/abl
						C T	Quantitatively (copy/μ l)
				1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	The male men and women of woman men and women man men and women men and women woman men and women			Normal normal AML-M2 AML-M2 AML-M1 AML-M1 AML-M5 AML-M1 AML-M2 AML-M4 AML-M3 AML-M6	51 24 79 65 56 45 45 53 59 42 66 62 21 35 47 42 40 57 36 57	24.82 22.85 23.25 22.89 23.03 22.28 24.63 25.78 23.13 21.1 25.24 29.71 27.12 25.54 26.2 26.7 26.86 28.9 27.25 26.71	2.73×10 5 1.02×10 6 7.78×10 5 9.90×10 5 9.02×10 5 1.00×10 6 3.10×10 5 1.44×10 5 8.44×10 5 3.00×10 6 2.06×10 5 1.04×10 4 5.90×10 4 1.69×10 5 1.08×10 5 7.80×10 4 7.02×10 4 1.80×10 4 5.41×10 4 7.75×10 4

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 (7)

1. the rapid quantitative detection reagent box of a real-time quantitative PCR detecting human DDIT 3 gene transcripts is characterized in that: PCR damping fluid, dNTPs, MgCl ₂, specificity T aqMan probe, Auele Specific Primer, Taq enzyme, standard substance and reference substance.

2. according to right 1, reaction system is 1 * PCR damping fluid, 0.3 μ M Auele Specific Primer, 0.2mM dNTPs, 4.0mMMgCl ₂, 0.2 μ M TaqMan probe, 1U Taq enzyme.

3. according to right 1 described test kit, it is characterized in that: the upstream and downstream specific primer sequence is 5 '-GGAGCTGGAAGCCTGGTATG-3 ' and 5 '-GCTCTGGGAGGTGCTTGT-3 '.

4. according to right 1 described test kit, it is characterized in that: specific probe is a fluorescence labeling probe, and its sequence is 5 '-FAM-TCTTCACCACTCTTGACCCTGCTTCTCT-TAMRA-3 '.

5. according to right 1 described test kit, it is characterized in that: standard substance concentration is 10 ⁹Copy/μ l, contained DDIT3 sequence is:

GATCCAACTG CAGAGATGGC AGCTGAGTCA TTGCCTTTCT CCTTTGGGAC

ACTGTCCAGC TGGGAGCTGG AAGCCTGGTA TGAGGACCTG CAAGAGGTCC

TGTCTTCAGA TGAAAATGGG GGTACCTATG TTTCACCTCC TGGAAATGAA

GAGGAAGAAT CAAAAATCTT CACCACTCTT GACCCTGCTT CTCTGGCTTG

GCTGACTGAG GAGGAGCCAC AACCAGCAGA GGTCACAAGC ACCTCCCAGA

GCCCTCACTC TCCAGATTCC AGTCAGAGCT CCCTGGCTCA GGAGGAAGAG

GAGGAAGACC AAGGGAGAAC CAGGAAACGC AAACAGAGTG GTCATTCCCC

AGCCCGGGCT GGAAAGCAGC GCATGAAGGA GAAAGAACAG GAGAATGAAA

GGAAAGTGGC ACAGCTAGCT GAAGAGAATG AACGGCTCAA GCAGGAAATC

GAGCGCCTGA CCAGGGAAGT AGAGGCGACT CGCCGAGCTC TGATTGACCG

AATGGTGAAT CTGCACCAAG CATGAACAAT T

6. according to right 1 described test kit, it is characterized in that: reference substance divides negative contrast and positive control, and wherein negative control is the aseptic double-distilled water of no DDIT3, and positive control is Normocellular DDIT3cDNA sample.

7. according to right 1 described test kit, it is characterized in that: the PCR that utilizes designed primer of this test kit and probe to carry out carries out the qualitative and detection by quantitative of DDIT3.

Images