CN102234688A - Method for quickly and quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA) - Google Patents

Abstract

The invention relates to a method for quantitatively detecting mammalian cell deoxyribose nucleic acid (DNA), which is used for detecting host cell DNA remained in a biological product which takes mammalian cells as matrixes. Multicopy genes in a mammalian cell genome are used as detection tag sequence templates, DNA polymerase, a molecular marker primer and molecular marker DNA are synthesized into a detection purpose sequence, and the detection purpose sequence is quantitatively analyzed. Compared with a molecular hybrid method, the method has the characteristics of short detection time (a detection process can be finished within two hours), stable detection results and high sensitivity.

Description

A kind of DNA in mammalian cells fast quantitative measurement method for detecting

Technical field

The present invention relates to a kind of novel gene group DNA detection by quantitative with method and test kit, belong to biological technical field.

Background of invention

Along with biotech development and health level improve, to various viral vaccines and therapeutic antibodies increase in demand, wherein cultivating the various products that produced with mammalian cell increases gradually, because various clones such as VERO, CHO, MDCK, PER.C6 etc. have oncogenic worry when high generation is cultivated, so according to Pharmacopoeia of People's Republic of China (in 2005 version) requirement, the product of each this type of cells produce of kind, its host cell DNA residual quantity all has concrete regulation, be no more than 100pg for every dose as purified rabies vaccine, so producing, research and development, all need to carry out the host cell DNA content detection in the quality arbitration process.

Used detection method is a random primer at present, and archaeal dna polymerase synthesizes random probe, and we find in production practice, this probe in detecting method improves at hybridization temperature, when being the specificity raising, sensitivity descends, and usually can not reach application purpose, detectability is in the nanogram level, have to increase the probe usage quantity, reduce hybridization temperature, the result, sensitivity has improved, but specificity descends, and cost improves; Another aspect needs 2.5 day time from synthesising probing needle to hybridizing to develop the color to finish, and the time is oversize.

Alu repeated sequence is a member of SINE family in the mammalian genes group, and 500,000 parts of copies are arranged approximately.That is to say just has an Alu sequence among average 4～6kb, account for people's gene group sequence 10.7%.Because the recognition sequence AGCT of restricted property endonuclease Alu in this dna sequence dna is so be called Alu repeated sequence.Typical people's gene group Alu sequence is about 300bp, by two homologies but differentiated subunit constitute.Restriction enzyme A luI can cut into it two sections of 130bp and 170bp, therefore it is named to be Alu sequence.Subunit derives from the 7SLRNA gene of deletion mutantion and point mutation.Connect by the intensive sequence of adenine nucleotide between two subunits.There is irrelevant 31bp to insert fragment in the subunit on the right, is called IH.Respectively there is a forward tumor-necrosis factor glycoproteins at the Alu sequence two ends, and end has a poly (A) tail.

Alu sequence generally is dispersed in distribution, and minority is bunch shape and distributes.Observe on the cytogenetics level, Alu repeated sequence concentrates in the most active chromosome segment of genetic transcription.In all known gene introns, nearly all found Alu sequence.

The identity average out to 87% of the concensus sequence between the Alu family different members (consensus sequence).The B1 tumor-necrosis factor glycoproteins that 50,000 parts of copies are arranged in the mouse genome approximately, long 130bp reaches 70%-80% with the homology of the subunit of Alu.

These characteristics of Alu sequence make it become and substitute the random probe hybridizing method and carry out one of optimal selection of genomic dna detection by quantitative.

Summary of the invention

The object of the invention is to provide a kind of mammalian genes group dna molecular quantitative detecting method and test kit of being used for.

The testing goal sequence is that copy number is higher than 1000 tumor-necrosis factor glycoproteins in genome among the present invention, wherein choose Alu repeated sequence and homologous tumor-necrosis factor glycoproteins thereof of optimum.

Implementation method of the present invention is:

Obtaining the tumor-necrosis factor glycoproteins of the host cell that detects, design and synthesize the molecule marker primer, by mixing the mark base, is template with DNA in the sample, synthesizes the aim sequence that contains molecule marker more than 2 kinds by the archaeal dna polymerase method that those skilled in the art know.For example primer is with biotin labeling, mix digoxin or fluorescein-labeled deoxynucleotide, so aim sequence promptly contains vitamin H-digoxigenin labeled or digoxin-fluorescein-labelled, because wherein mark 1 (vitamin H) is the relation more than 1 pair in aim sequence with mark 2 (as digoxin or fluorescein), therefore signal can amplify.

Mark 1 is used for fixing on solid phase carrier, as materials such as the plastics of pan coating mark 1 part (as avidin), polyester, nylon, glass, micas.

And can detect by the antibody of enzyme labelling for mark 2, be digoxin as mark 2, then can pass through the enzyme labelling DigiTAb, perhaps DigiTAb+enzyme labelling two anti-detections.If mark 2 is a fluorescein, then can directly detect by fluorescence detection device, perhaps chemiluminescent substance activates, and its signal can reflect DNA amount in the sample, by the preparation standard curve dna content in the sample is carried out detection by quantitative.

With primer, archaeal dna polymerase, damping fluid, (mark) thymus nucleic acid mixed solution, marker antibody or two anti-, Enzymology method detects substrate, fixingly is prepared into test kit with the part bag by orifice plate and detects.

Description of drawings

Fig. 1 aim sequence is synthetic;

Fig. 2 detection method;

Fig. 3 and random probe detectability be (analysis of VERO cell DNA) relatively

Specific embodiment

Following example is to be described in further detail the present invention, but content of the present invention is not to be defined in this.

1, primer is synthetic

From GENBANK, look into and get the cercopithecus aethiops Alu sequence, synthetic primer:

PBIOALU1：5’-BIOTIN-AGGCCGGGCGCAGTGGCTCA-3’

PBIOALU2：5’-BIOTIN-GGACTGCAGTGGCGCAATCTC-3’

2, sample DNA extracts

Get the VERO cell rabies vaccine, press scalar quantity and add 1 times of volume extracting solution (0.1mol/L NaOH, 0.1mol/LNaCl, 5g/L SDS), capable again phenol-chloroform extracting, the DNA post precipitation is dissolved in the 50 μ l TE damping fluids.

3, aim sequence is synthetic

Get 2 μ l examples, 2 products as template, get primer and be diluted to 10 μ M, press PBALU15 μ l, Taq enzyme 5U, 10 * PCR damping fluid, 5 μ l, dNTPs (10mMol/L) 4 μ l, digoxigenin labeled dig-11-Dutp (1mMol/L) 40 μ l, template 100ng, pure water add to 50 μ l.By 95 ℃ 5 minutes, then 95 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 10 minutes, add 100mM EDTA 5 μ l termination reactions then.

4, aim sequence detects

Example 3 reaction solns 1 μ l adds to be had in the micropore of 45 μ l hybridization solutions, places 10min in 37 ℃, washes plate 3 times with 37 ℃ PBS/T then, adds 50 μ l (1 * 10 ⁵U/L) anti-digoxin peroxidase enzyme connection thing is placed 10min for 37 ℃, discards liquid in the hole then, washes-plate 3 times with PBS/T, goes into 1 TMB solution and 1 0.3ml/L H more in addition ₂O ₂, lucifuge is placed 10min, adds 0.5mol/L H ₂SO ₄A is measured in the color development stopping reaction _450nm

Claims (7)

1. DNA in mammalian cells quantitative detecting method, being used for the mammalian cell is that the residual host cell DNA of biological products of matrix detects.Multi-copy gene is detection flag sequence template in the employing mammalian cell genome, and the synthetic testing goal nucleotide sequence of archaeal dna polymerase, molecular marker labeled primer and molecule marker thymus nucleic acid, and it is carried out the mark quantitative analysis.

According to the described multi-copy gene of claim 1 refer to these gene orders in genome sequence more than 1000 copies, be preferably Alu superfamily gene order, or the combination of this genoid more than a kind.

3. make up for the molecular marker that has simultaneously more than 2 kinds according to the described testing goal nucleic acid sequence identity of claim 1, wherein a kind of being used for is fixed on solid phase carrier to molecular marker, and another kind is used for the signal amplification detection.

4. be specially vitamin H, digoxin, fluorescein, chemiluminescent substance, can be used for the antigen of mark base according to the described molecular marker of claim 3; Combination between these marks is preferably vitamin H-digoxin, vitamin H-fluorescein combination.

5. by the molecular force between molecular marker-part the purpose nucleotide sequence is connected to solid phase carrier according to the described fixedly finger of claim 3, so that remove unmarked sequence.

6. refer to be incorporated into the part that has detectable label according to the described signal amplification detection of claim 3, be specially enzymic-labelled antibody, become to detect product, be convenient to detect by catalytic conversion to substrate being used for fixing another outer mark.

7. a test kit comprises described purpose nucleotide sequence of claim 1 and synthesis condition, and the described testing conditions of claim 6.

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