CN110305938B

CN110305938B - Primer pair and detection method for quantitatively detecting DNA residual quantity of CHO host cells

Info

Publication number: CN110305938B
Application number: CN201810259308.6A
Authority: CN
Inventors: 徐丽娟; 袁婀娜; 陶铜静; 韩进
Original assignee: Tasly Pharmaceutical Group Co Ltd
Current assignee: Tasly Pharmaceutical Group Co Ltd
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2023-02-17
Anticipated expiration: 2038-03-27
Also published as: CN110305938A

Abstract

The invention relates to a primer pair and a detection method for quantitatively detecting the residual quantity of CHO host cell DNA. The fluorescent quantitative PCR is to mark and track the PCR product through fluorescent dye and calculate the initial concentration of the sample template to be detected, wherein, the sequence of the specific primer pair is shown in SEQ ID NO. 15, and the sequence of the reverse primer is shown in SEQ ID NO. 16.

Description

Primer pair and detection method for quantitatively detecting DNA residual quantity of CHO host cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer pair and a detection method for quantitatively detecting DNA residual quantity of CHO cells (Chinese Hamster Ovary cells).

Background

CHO cells were isolated from adult female hamster ovary, dr. Theodore t. Puck, university of colorado, 1957, are epithelial-adherent cells, and are a cell line widely used in biotechnology. The cell has immobility, can be passaged for more than one hundred generations, and is a cell widely used in bioengineering. In addition, the CH0 cell has the advantage of being used in genetic engineering, belongs to fibroblast (fibroplast), is a non-secretory cell, rarely secretes CHO endogenous protein, and is very beneficial to the separation and purification of target protein. Can form active dimer (such as interleukin 2), has glycosylation function (such as EPO), and CHO is an ideal host for expressing complex biomacromolecule. CHO cells have become the most important expression or production system for biotechnological drugs, and this situation will continue and its proportion tends to increase year by year. The CHO cell large-scale culture technology and the bioreactor engineering thereof can be widely applied to research, development and industrial production of biotechnology products such as antibodies, gene recombinant protein drugs, virus vaccines and the like.

At present, recombinant protein drugs, antibody drugs, vaccines and other products in biological products are produced by expression of CHO cells after continuous passage, and DNA fragments of the CHO cells of host cells can still remain in the products although strict purification processes are carried out. The residual DNA fragments of host cells in biological products may carry viruses or oncogenes, and have potential oncogenic and infectious risks, so that the limit requirements of drug regulatory departments in various countries on DNA impurities are very strict. The DNA residual in the host cells must meet the guidelines set by the World Health Organization (WHO), european pharmacopoeia, the U.S. Food and Drug Administration (FDA) and other regulatory agencies. The DNA residue of the host cells is generally specified in the recombinant drug quality standard, for example, the DNA residue of each human dose is not more than 100pg. In the production, purification and preparation process, the detection of DNA residue becomes an important control element.

The host cell adopted by the recombinant human prourokinase (Pro-UK) for injection is a CHO cell, so that the production process of the product also has the problem of residual DNA fragments of the host cell.

At present, the foreign detection of the DNA residue of host cells generally adopts quantitative PCR (quantitative PCR, Q-PCR) technology, SYBRgreen dye method or probe labeling method. The domestic detection generally adopts 2 methods, namely a DNA probe hybridization method and a fluorescent staining method, which are recorded in an appendix IXB' external DNA residue determination method of 2010 version in Chinese pharmacopoeia (three), but the existing method has low detection sensitivity, large consumed stock solution sample amount and long operation time, and the method is a semi-quantitative detection method, can only judge whether the DNA amount is less than 100pg according to the judgment result of naked eyes, and cannot exactly obtain the specific level value of the DNA content.

Disclosure of Invention

The invention aims to provide a method for detecting DNA residues of CHO cells in biological products, which comprises a novel primer pair.

The invention relates to a method for participating in fluorescent quantitative PCR detection reaction and detecting residual CHO cell DNA in biological products by utilizing the novel primer pair. The fluorescent quantitative PCR is to mark and track the PCR product through a fluorescent dye or a fluorescent-marked specific probe, monitor the reaction process in real time on line, analyze the product by combining with corresponding software, and calculate the initial concentration of a sample template to be detected.

Wherein the primer is the key to the PCR specific reaction, the specificity of the PCR product depends on the extent of the complementarity of the primer and the template DNA, and the principle of designing the primer is as follows:

(1) length of the primer: 15-30bp, usually about 20 bp.

(2) Primer amplification span: preferably 200-500bp, and fragments up to 10kb can be amplified under specific conditions.

(3) Primer base: the content of G + C is preferably 40-60%, the amplification effect is poor when the content of G + C is too small, and a non-specific band is easy to appear when the content of G + C is too large. ATGC is preferably randomly distributed, avoiding the formation of strings of more than 5 purine or pyrimidine nucleotides.

(4) The secondary structure in the primer is avoided, the complementation between two primers, especially the complementation of the 3' end is avoided, otherwise, a primer dimer is formed, and a non-specific amplification band is generated.

(5) The bases at the 3' end of the primer, particularly the last and penultimate bases, should be strictly matched to avoid PCR failure due to unpaired end bases.

(6) The primers may or may be provided with suitable cleavage sites, preferably suitable cleavage sites for the target sequence to be amplified, which may be advantageous for cleavage analysis or molecular cloning.

(7) Specificity of the primers: the primers should have no significant homology to other sequences in the nucleic acid sequence database.

The amount of primers: the concentration of each primer is preferably 0.1 to 1. Mu. Mol or 10 to 100pmol, with the lowest primer amount giving the desired result, and higher primer concentrations may cause mismatches and non-specific amplification and increase the chance of dimer formation between primers.

To this end, the present invention provides a novel primer pair comprising a forward primer and a reverse primer, wherein the forward primer binds to positions 130-146 of the test sequence; wherein the reverse primer is combined with 266 th to 282 th positions of the test sequence, and the length of an amplification product obtained by amplification of the primer pair is 153bp;

wherein the test sequence is a nucleotide sequence which has more than 85 percent of sequence similarity with the sequence shown in SEQ ID NO. 1.

In a preferred embodiment, the test sequence has 98% sequence similarity to the sequence shown in SEQ ID NO 1.

In a preferred embodiment, the test sequence is set forth in SEQ ID NO 4.

In a particular embodiment of the present invention,

the forward primer is selected from: SEQ ID NO 5/7/9/11/13/15/17/19/21/23,

the reverse primer is selected from: SEQ ID NO 6/8/10/12/14/16/18/20/22/24.

Preferably, the forward primer is shown as SEQ ID NO. 15, and the reverse primer is shown as SEQ ID NO. 16.

The primer pair, especially the preferable primer pair, is obtained by screening, and the screening process is as follows:

H. through an NCBI website, a target product sequence is inquired, a primer pair P is designed, and PCR amplification is carried out on a target product.

I. And (4) carrying out sequence determination on the amplified product to obtain a test sequence.

J. BLAST alignment is carried out on the test sequence and the target product, and sequence similarity is determined.

K. Several pairs of primers P' were designed to amplify the test sequences.

And L, carrying out a dissolution curve test on the amplification product, and screening a primer pair with a specific amplification peak and no other non-specific impurity peak for subsequent tests.

And M, carrying out amplification curve and Ct value test on the amplification product at different annealing temperatures, and screening out a primer pair with the lowest Ct average value for subsequent test.

And N, performing standard substance linear test on the screened primer pair P', and screening a primer pair with excellent linear result.

Wherein the step G comprises the following steps:

(1) Diluting a target product standard substance by a plurality of concentration points;

(2) Amplifying different concentration points of the target product standard by using the screened primers, establishing a standard curve and calculating amplification efficiency;

(3) Screening standard curve R ² Not less than 0.98, and the linear range of the concentration of the amplification efficiency (E) between 90 percent and 110 percent is 10000pg/2 mu L-0.01 pg/2 mu L.

Wherein, in the primer pair P, the forward primer is shown as SEQ ID NO. 2, and the reverse primer is shown as SEQ ID NO. 3.

The number of the primer pairs P' is 10, and the forward primers: 5/7/9/11/13/15/17/19/21/23, reverse primer: SEQ ID NO 6/8/10/12/14/16/18/20/22/24.

The invention is characterized in that the novel primer pair is used for carrying out fluorescence quantitative PCR on a biological product sample to be detected and detecting the content of a PCR amplification product, thereby calculating the content of DNA residue in the biological product sample.

Wherein, the fluorescent quantitative PCR reaction system is

Premix Ex Taq II (Tli RNaseH Plus) (2X) 12.5. Mu.L, forward primer (F, 10. Mu.M/. Mu.L) 1. Mu.L, reverse primer (R, 10. Mu.M/. Mu.L) 1. Mu.L, H ₂ 0.5. Mu.L, 2. Mu.L of DNA template, and 25. Mu.L of total volume.

The reaction conditions are as follows: (1) pre-denaturation: 30sec at 95 ℃;

(2) fluorescence collection: amplifying for 40 cycles at 95 ℃ and 1min,52 ℃ and 1min and 72 ℃ for 1min, and collecting fluorescence signals after each cycle is finished;

(3) dissolution curve analysis: the temperature of 65 ℃ was gradually increased to 95 ℃ and 0.5 ℃ was increased every 0.5sec, and the fluorescence signal was collected. The beneficial effects of the invention are illustrated below by the instructions for use of the commercially available kit and the experimental data of the invention:

the invention has the following beneficial effects: the primer pair related by the invention can be effectively used for the specific detection of the DNA residue of CHO host cells in biological products, thereby evaluating the key steps of DNA removal in the production process of the biological products and achieving the aim of whole-process control. Compared with the detection method using a commercially available kit and a digoxigenin-labeled probe hybridization method, the detection method in the embodiment 2 of the invention greatly improves the detection efficiency and reduces the detection cost.

Drawings

FIG. 1, melting curves of primers P1 to P10 for amplifying CHO cell genomic DNA

FIG. 2 shows the amplification curves of primers P1 to P10 for CHO cell genomic DNA amplification

FIG. 3, amplification curve of P2 primer for amplifying CHO cell DNA standard

FIG. 4 shows a standard curve established at a concentration point of 10000 pg/2. Mu.L-0.01 pg/2. Mu.L for P2 primer amplification CHO cell DNA standard substance

FIG. 5 shows a standard curve established at a concentration point of 10000 pg/2. Mu.L-1 pg/2. Mu.L for P2 primer amplification of CHO cell DNA standard substance

FIG. 6, amplification curve of P6 primer for amplifying CHO cell DNA standard

FIG. 7 shows a standard curve established at a concentration point of 10000 pg/2. Mu.L-0.01 pg/2. Mu.L for P6 primer amplification CHO cell DNA standard substance

FIG. 8, P6 primer amplification curves of E.coli genomic DNA, CHO cell DNA standard and CHO engineered cell DNA

Detailed Description

EXAMPLE 1 screening method of primer set for detecting CHO genomic DNA of recombinant human prourokinase (Pro-UK) host cell

By NCBI website, CHO cell 18s rRNA series: genbank Number NR _045132.1 (SEQ ID NO: 1), design primer pair P, forward primer: f:5'-TCCTTTGGTCGCTCGCTCCT-3' (SEQ ID NO: 2); the reverse primer R:5'-TTCGCTCTGGTCCGTCTTGC-3' (SEQ ID NO: 3) was used for PCR amplification of CHO engineered cell genomic DNA for production of recombinant human prourokinase for injection (Pro-UK).

And (3) sending the PCR amplification product to Shanghai Meiji biological medicine science and technology limited company for sequencing to obtain a test sequence, wherein the sequencing result is shown as SEQ ID NO. 4.

Comparing the sequence of SEQ ID NO. 4 with NCBI website, the comparison result shows that the test sequence of SEQ ID NO. 4 has 98% sequence similarity with the sequence of SEQ ID NO. 1, and the test sequence obtained by amplification can be determined to be CHO cell genome sequence.

Using the test sequence SEQ ID NO:4, a primer pair P' (P1 to P10) was designed as shown in Table 1.

TABLE 1 primer pairs P1-P10 sequence Listing

The primers are screened out for further testing by performing dissolution curve testing, amplification curve testing and Ct value testing on 10 pairs of designed primers.

The results of the dissolution curve tests of P1-P10 are shown in figure 1.

The result shows that the 10 pairs of primers have only one specific amplification peak in the dissolution curve at a certain annealing temperature, and no other non-specific impurity peaks, so that the specificity of the primers is passed, and the CHO cell DNA can be specifically detected.

The amplification curves of P1-P10 at different annealing temperatures are shown in FIG. 2, and the Ct values are shown in Table 2.

TABLE 2 Ct values for Q-PCR amplification at different annealing temperatures

From the amplification curve and Ct value results, the following results can be obtained:

(1) Comparing the detection results of Ct values of Q-PCR amplification of 10 pairs of primers at different annealing temperatures, and screening P2 and P6 with the lowest Ct mean value for subsequent experiments (due to the fact that the Ct value is higher, the standard curve can not be detected at low concentration, and the amplification efficiency is influenced).

(2) According to the fact that the P2 primer can be amplified under the annealing temperature gradient of 55.0-65.0 ℃, the annealing temperature with the highest amplification efficiency is 61.4 ℃, and the optimal annealing temperature for the primer P2Q-PCR amplification is 61.4 ℃; according to the fact that the P6 primer can be amplified under the annealing temperature gradient of 55.0-65.0 ℃, the annealing temperature with the highest amplification efficiency is 52.0 ℃, and the optimal annealing temperature for the amplification of the primer P6Q-PCR is 52.0 ℃.

The selected P2 and P6 primers were further subjected to a linear test.

The CHO cell DNA national standard (batch No. 270026-201101) is diluted to 10000pg/2 μ L-0.01 pg/2 μ L, totaling 7 concentration points.

CHO cell DNA national standard (batch number: 270026-201101) is diluted to 10000pg/2 mu L-0.01 pg/2 mu L, 7 concentration points are counted, P2 and P6 primers are applied for amplification, a standard curve is established, and the amplification efficiency is calculated.

The test results of the P2 primer are shown in the attached figures 3-5, and the results show that the P2 primer is applied to determine the DNA national standard (batch number: 270026-201101) of the CHO cells and conforms to the standard curve R ² Not less than 0.98, and the linear range of the concentration of the amplification efficiency (E) between 90 percent and 110 percent is 10000pg/2 mu L-1 pg/2 mu L.

The test results of the P6 primer are shown in the attached figures 6 and 7, and the results show that the P6 primer is used for measuring the DNA national standard (batch number: 270026-201101) of the CHO cells and conforms to the standard curve R ² Not less than 0.98, the linear range of the concentration of 90-110 percent of the amplification efficiency (E) is 10000pg/2 mu L-0.01 pg/2 mu L, and the linear test result of the P6 primer is obviously superior to that of the P2 primer.

Through the experiment, a primer pair P6 capable of specifically amplifying CHO genome DNA is screened, a forward primer is shown as a sequence in SEQ ID NO. 15, a reverse primer is shown as a sequence in SEQ ID NO. 16, and the forward primer is combined with 130 th to 146 th sites of a test sequence (SEQ ID NO. 4); the reverse primer is combined with 266-282 th sites of the test sequence, and the length of the amplified product obtained by the amplification of the primer pair is 153bp.

Example 2 methodological validation of the CHO cell DNA content determination method

The residual amount of DNA in CHO host cells was determined using P6 primer set (forward primer F is shown in SEQ ID NO:15 and reverse primer R is shown in SEQ ID NO: 16).

The reaction system is

Premix Ex Taq II(Tli RNaseHPlus) (2X) 12.5. Mu.L, forward primer (F, 10. Mu.M/. Mu.L) 1. Mu.L, forward primer (R, 10. Mu.M/. Mu.L) 1. Mu.L, H ₂ 0.5 muL, 2 muL of DNA template, and 25 muL of total volume;

the reaction conditions are as follows: (1) pre-denaturation: 30sec at 95 ℃;

(3) dissolution curve analysis: the temperature of 65 ℃ was gradually increased to 95 ℃ and 0.5 ℃ was increased every 0.5sec, and the fluorescence signal was collected.

(1) Attribute validation

Q-PCR fluorescent signal using Escherichia coli (CMCC 44102) DNA as template has no specific amplification curve, while the Q-PCR fluorescent signal using CHO engineering cell genome DNA and CHO cell DNA content determination national standard as template has specific amplification curve, as shown in FIG. 8. The method established by the invention can be used for specifically amplifying the DNA of the CHO cell, and can not be used for amplifying the DNA of other microorganisms, thereby meeting the requirement of specificity.

(2) Linear verification

Standard Curve established with national standards was used for the determination 10 ⁴ pg/2μL～10 ^-2 pg/2. Mu.L concentration CHO cell DNA content. The detection results of 6 independent experiments are shown in Table 3, the Ct value and the logarithm value of the template DNA concentration have good linear relation, and the correlation coefficient R ² Not less than 0.98, and the amplification efficiency is up to 90-110%.

TABLE 3CHO cell genomic DNA amplification efficiency

Number of tests	Range of curves	Efficiency of amplification (E)	R ²	SLOPE
					For the first time	10000pg/2μL～0.01pg/2μL	95.4	0.996	-3.438
For the second time	10000pg/2μL～0.01pg/2μL	96.5	0.999	-3.410
					The third time	10000pg/2μL～0.01pg/2μL	96.1	0.999	-3.418
Fourth time	10000pg/2μL～0.01pg/2μL	97.0	1.000	-3.395
					Fifth time	10000pg/2μL～0.01pg/2μL	94.1	0.998	-3.473
The sixth time	10000pg/2μL～0.01pg/2μL	94.9	0.999	-3.449

(3) Accuracy verification

After standard DNA with 5 concentration (10000 pg/1000pg/100pg/10pg/1 pg) is added in a purified intermediate of Puyoug (namely, recombinant human prourokinase for injection), DNA extraction is carried out, then Q-PCR quantitative detection is carried out, and the recovery rates are respectively as follows: 94%, 120%, 65%, -6%, -14%, see table 4, 10000pg, 1000pg and 100pg meet the requirement of 50% -150% recovery rate. The DNA level of the Puck purified intermediate is about 150pg/100 mu L, and the accuracy of adding standard DNA into 10pg and 1pg cannot reach the requirement of 50-150% of recovery rate due to the difference of quantitative results caused by extraction.

Table 4 puyouk purified intermediate recovery

(4) Precision verification

The coefficient of variation (CV%) between groups of the same sample, which was measured 6 times in parallel, was 16% and 18%, and the coefficient of variation (CV%) between groups of the same sample, which was measured by different examiners, was 17%. The results are shown in Table 5. All meet the requirement that the precision standard CV percent of methodology is less than or equal to 25 percent.

TABLE 5 coefficient of variation of purified Puyouk intermediates

The method for detecting the DNA residue of the CHO host cell in the Puyou Ke intermediate is verified by methodology, and the specificity, linearity, accuracy and precision of the method are determined to meet the requirements of verification standards.

EXAMPLE 3 Effect of different concentrations of protein in samples on the accuracy of the assay results

By using the detection method disclosed by the invention, a sample containing high-concentration protein (the protein concentration is 26 mg/mL) and a sample containing medium-concentration protein (the protein concentration is 13 mg/mL) are respectively detected, the recovery rates of the detection results are 82% and 102%, and the requirements of 50% -150% of the recovery rates are met. The recovery rate results of the two samples are compared, and the results show that the samples contain proteins with different concentrations to influence the detection result, and the higher the protein concentration is, the process of extracting DNA in the samples can be interfered, and the loss rate is increased.

Example 4 comparison between different detection methods

A commercial kit for quantitative determination of DNA residues of CHO cell culture products and the method (SYBR Green Q-PCR method) established by the invention are adopted to simultaneously detect 10 batches of purified Puyou Ke intermediates, and the detection results are shown in Table 6. The coefficient of variation (CV%) between the detection results of the two methods is 4.72-29.82%. The method established by the invention is comparable to the detection result of the commercial kit, and the difference is not obvious.

TABLE 6 variation coefficient of different detection methods for detecting puyouk purified intermediates

Through the above test verification, the commercial kit and the detection method of the present invention are compared as shown in table 7.

TABLE 7 comparison of commercially available kits with the detection methods of the invention

The result shows that the detection result is comparable to the detection result of a commercial kit, and the kit can be effectively used for the specific detection of the DNA residue of the CHO host cell in the Puyouk (recombinant human prourokinase for injection) intermediate product, thereby evaluating the key step of DNA removal in the Puyouk production process and achieving the purpose of whole-process control. Compared with a probe hybridization method using a commercial kit and a digoxin marker, the method established by the method greatly improves the detection efficiency and reduces the detection cost.

Sequence listing

<110> Shanghai Tianshi drug industry Co., ltd

<120> primer pair for quantitatively detecting DNA residue of CHO host cell and detection method

<160>24

<210>1

<211>1877

<212>rRNA

<213> Chinese hamster (Cricetulus barabensis)

<220>

<221>CRICETULUS GRISEUS 18S RIBOSOMAL RNA(RN18S)

<222>

<223>

<400>1

tccctacctg gttgatcctg ccagtagcat atgcttgtct caaagattaa gccatgcatg 60

tctaagtacg cacggccggt acagtgaaac tgcgaatggc tcattaaatc agttatgggg 120

ttcctttggt cgctcgctcc tctcctactt ggataactgt ggtaattcta gagctaatac 180

atgccgacgg gcgctgaccc ccttcgcggg ggggatgcgt gcatttatca gatcaaaacc 240

aacccggtca gcttccttcc cggctccggc cgggggggcg ggcgccggcg gctttggtga 300

ctctagataa cctcgggccg atcgcacgcc ccccgtggcg gcgacgaccc attcgaacgt 360

ctgccctatc aactttcgat ggtagtcgat gtgcctacca tggtgaccac gggtgacggg 420

gaatcagggt tcgattccgg agagggagcc tgagaaacgg ctaccacatc caaggaaggc 480

agcaggcgcg caaattaccc actcccgacc cggggaggta gtgacgaaaa ataacaatac 540

aggactcttt cgaggccctg taattggaat gagtccactt taaatccttt aacgaggatc 600

cattggaggg caagtctggt gccagcagcc gcggtaattc cagctccaat agcgtatatt 660

aaagttgctg cagttaaaaa gctcgtagtt ggatcttggg agcgggcggg cggtccgccg 720

cgaggcgagt caccgcccgt ccccgcccct tgcctctcgg cgccccctcg atgctcttag 780

ctgagtgtcc cgcggggccc gaagcgttta ctttgaaaaa attagagtgt tcaaagcagg 840

cccgagccgc ctggataccg cagctaggaa taatggaata ggaccgcggt tctattttgt 900

tggttttcgg aactgaggcc atgattaaga gggacggccg ggggcattcg tattgcgccg 960

ctagaggtga aattcttgga ccggcgcaag acggaccaga gcgaaagcat ttgccaagaa 1020

tgttttcatt aatcaagaac gaaagtcgga ggttcgaaga cgatcagata ccgtcgtagt 1080

tccgaccata aacgatgccg actggcgatg cggcggcgtt attcccatga cccgccgggc 1140

agcttccggg aaaccaaagt ctttgggttc cggggggagt atggttgcaa agctgaaact 1200

taaaggaatt gacggaaggg caccaccagg agtggagcct gcggcttaat ttgactcaac 1260

acgggaaacc tcacccggcc cggacacgga caggattgac agattgatag ctctttctcg 1320

attccgtggg tggtggtgca tggccgttct tagttggtgg agcgatttgt ctggttaatt 1380

ccgataacga acgagactct ggcatgctaa ctagttacgc gacccccgag cggtcggcgt 1440

cccccaactt cttagaggga caagtggcgt tcagccaccc gagattgagc aataacaggt 1500

ctgtgatgcc cttagatgtc cggggctgca cgcgcgctac actgactggc tcagcgtgtg 1560

cctaccctac gccggcaggc gcgggtaacc cgttgaaccc cattcgtgat ggggatcggg 1620

gattgcaatt attccccatg aacgaggaat tcccagtaag tgcgggtcat aagcttgcgt 1680

tgattaagtc cctgcccttt gtacacaccg cccgtcgcta ctaccgattg gatggtttag 1740

tgaggccctc ggatcggccc cgccggggtc ggcccacggc cctggcggag cgctgagaag 1800

acggtcgaac ttgactatct agaggaagta aaagtcgtaa caaggtttcc gtaggtgaac 1860

ctgcggaagg atcatta 1877

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>2

Tcctttggtc gctcgctcct 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

Ttcgctctgg tccgtcttgc 20

<210>4

<211>835

<212>RNA

<213> Chinese hamster (Cricetulus barabensis)

<220>

<223>

<400>4

Actgtggtat tctagagcta tacatgccga cgggcgctga cccccttcgc gggggggatg 60

cgtgcattta tcagatcaaa accaacccgg tcagcttcct tcccggctcc ggccgggggg 120

gcgggcgccc caggctttgg tgactctaga taacctcggg ccgatcgcac gccccccgtg 180

gcggcgacga cccattcgaa cgtctgccct atcaactttc gatggtagtc gatgtgccta 240

ccatggtgac cacgggtgac ggggaacagt gaaaccgtga ctgagaggga gcctgagaaa 300

cggctaccac atccaaggaa ggcagcaggc gcgcaaatta cccactcccg acccggggag 360

gtagtgacga aaaataacaa tacaggactc tttcgaggcc ctgtaattgg aatgagtcca 420

ctttaaatcc tttaacgagg atccattgga gggcaagtct ggtgccagca gccgcggtaa 480

ttccagctcc aatagcgtat attaaagttg ctgcagttaa aaagctcgta gttggatctt 540

gggagcgggc gggcggtccg ccgcgaggcg agtcaccgcc cgtccccgcc ccttgcctct 600

cggcgccccc tcgatgctct tagctgagtg tcccgcgggg cccgaagcgt ttactttgaa 660

aaaattagag tgttcaaagc aggcccgagc cgcctggata ccgcagctag gaataatgga 720

ataggaccgc ggttctattt tgttggtttt cggaactgag gccatgatta agagggacgg 780

ccgggggcat tcgtattgcg ccgctagagg tgaaattctt ggaccggcgc aagacg 836

<210>5

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

Tctgccctat caactttc 18

<210>6

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>6

Ttttcgtcac tacctccc 18

<210>7

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>7

Gaaacggcta ccacatcca 19

<210>8

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>8

Caccagactt gccctcca 18

<210>9

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>9

Taagtgcggg tcataagc 18

<210>10

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>10

Ccatccaatc ggtagtagc 19

<210>11

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>11

Gtaagtgcgg gtcataag 18

<210>12

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>12

Catccaatcg gtagtagc 18

<210>13

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>13

Cgcggttcta ttttgttggt 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>14

Agtcggcatc gtttatggtc 20

<210>15

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>15

Ccaggctttg gtgactc 17

<210>16

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>16

Agtcacggtt tcactgt 17

<210>17

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>17

Cggacaggat tgacagattg 20

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>18

Caaatcgctc caccaactaa 20

<210>19

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>19

Gaccataaac gatgccgact 20

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>20

Agacaaatcg ctccaccaac 20

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>21

Tgaggccatg attaagaggg 20

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>22

Agtcggcatc gtttatggtc 20

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>23

Aaacggctac cacatccaag 20

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>24

Cctccaatgg atcctcgtta 20

Claims

1. A primer pair for quantitatively detecting the DNA residue of a CHO host cell comprises a forward primer and a reverse primer, and is characterized in that the forward primer is shown as SEQ ID NO. 15, and the reverse primer is shown as SEQ ID NO. 16.

2. The method for screening the primer set according to claim 1, comprising the steps of:

A. inquiring a target product sequence through an NCBI website, designing a primer pair P, and performing PCR amplification on a target product;

B. performing sequence determination on the amplification product to obtain a test sequence;

C. BLAST comparison is carried out on the test sequence and a target product, and sequence similarity is determined;

D. designing a plurality of primer pairs P' to amplify the test sequence;

E. carrying out a dissolution curve test on the amplification product, and screening a primer pair with a specific amplification peak and no other non-specific impurity peak to carry out a subsequent test;

F. carrying out amplification curve and Ct value tests on the amplification product at different annealing temperatures, and screening out a primer pair with the lowest Ct average value to carry out subsequent tests;

G. performing standard linear test on the screened primer pair P', and screening a primer pair with excellent linear result;

wherein the step G comprises the following steps:

3. A method for quantitatively detecting the residual amount of CHO host cell DNA by using the primer pair of claim 1, which is characterized in that the primer pair participates in a fluorescent quantitative PCR detection reaction and detects the content of the residual CHO cell DNA in a biological product; the fluorescent quantitative PCR is to mark and track the PCR product through fluorescent dye and calculate the initial concentration of the sample template to be detected.

4. The detection method according to claim 3, wherein the fluorescent quantitative PCR reaction system is a total volume of 25. Mu.L, and wherein 12.5. Mu.L of 2 Xconcentration fluorescent premixed reagent, 1. Mu.L of forward primer F, 10. Mu.M/. Mu.L, 1. Mu.L of reverse primer R, 10. Mu.M/. Mu.L, and H are added ₂ 0.5. Mu.L, DNA template 2. Mu.L.

5. The assay according to claim 3, wherein the reaction conditions for the PCR assay are: (1) pre-denaturation: 30sec at 95 ℃; (2) fluorescence collection: amplifying for 40 cycles at 95 ℃ and 1min,52 ℃ and 1min and 72 ℃ for 1min, and collecting a fluorescence signal after each cycle is finished; (3) dissolution curve analysis: the temperature of 65 ℃ was gradually increased to 95 ℃ and 0.5 ℃ was increased every 0.5sec, and the fluorescence signal was collected.