CN115927651A - Primer and method for detecting swine-origin DNA - Google Patents
Primer and method for detecting swine-origin DNA Download PDFInfo
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Abstract
The invention provides a primer pair for detecting pig genome DNA, a detection reagent or a kit containing the primer pair and a method for detecting the pig genome DNA by using the primer pair, wherein the primer pair is specifically combined with a sequence shown in SEQ ID NO. 1. The PCR detection method using the primer pair is simple, convenient and quick to operate, high in sensitivity and capable of distinguishing interference DNAs of cows, CHO, vero, people, NS0, MDCK, E.coli, pichia pastoris, sf9 and the like.
Description
Technical Field
The present invention relates to the field of biological detection. Specifically, the invention relates to a primer and a detection method for detecting swine DNA residues in a biological material.
Background
The biological source extracellular matrix (ECM) is widely applied to the fields of regenerative medicine and tissue engineering medical treatment, has a three-dimensional structure with a natural form, is rich in collagen and growth factors, provides a microenvironment for inducing cell adhesion, proliferation and diffusion, and promotes tissues to recover normal structures and functions. The extracellular matrix of biological origin is mainly derived from human or mammalian tissue and organs, such as dermis, bladder, small intestine and pericardium, etc., and is a main raw material of acellular matrix due to extensive sources and high biocompatibility of porcine tissue and organs. The biological material composed of the pig origin acellular matrix is clinically used for repairing and regenerating tissues and organs such as blood vessels, skin, intestinal tracts, heart and the like, and medical appliance products comprise biological hernia repair patches, oral repair films, biological patches, wound dressing and the like.
The decellularization and sterilization treatment is used to remove cell components and harmful microorganisms in animal tissues, and these cell components and nucleic acid residues are the main cause of immunogenicity risk in patients at the time of implantation, and serious adverse effects such as inflammatory reaction, severe pain and noninfectious edema may be caused. Therefore, the evaluation of the acellular effect is one of the important measures for the quality safety control of the biological material, and the acellular result is mainly evaluated by quantitatively detecting the residual cell components, such as double-stranded DNA and the like, in the acellular matrix biological material at present. The national animal-derived medical instrument industry standard YY/T0771 and the like and the animal-derived medical instrument product registration technical review guide principles provide risk management requirements for medical instruments manufactured by using animal tissues or derivatives thereof.
The molecular biology technology based on nucleic acid, such as fluorescence quantitative PCR, can utilize a Taqman probe with a specific fluorescent marker to carry out quantitative detection and analysis on residual DNA in a sample, the probe with a fluorescent group continuously excites a fluorescent signal in a thermal cycle process, and the increase of products in the PCR process is reflected by monitoring the change of the accumulated fluorescent signal, so that the quantitative analysis on the target DNA is realized. The real-time fluorescent quantitative PCR detection technology has the advantages of good specificity, high accuracy, high speed and the like.
At present, the detection of pig-derived ingredients is mainly concentrated in the food fields of meat food, gelatin and the like, and the detection of residual DNA in medical instruments applied to animal-derived biomaterials is less; in addition, the target design of the pig-derived qPCR usually uses single copy genes or mitochondrial genes, and the copy number of the genes is low or is not constant in cells, which may cause quantitative deviation or false negative condition, and is not suitable for quantitative detection of trace residual DNA of biological materials. The number of times of the highly repetitive sequence is more than one hundred thousand in mammals such as pigs, and the highly repetitive sequence is respectively uniform and stable in each chromosome. Therefore, the fluorescence quantitative PCR method based on the highly repetitive sequence target can greatly improve the sensitivity, specificity and accuracy of detection. The quantitative qPCR detection method can be used for establishing a set of quantitative qPCR detection method for trace residual pig source DNA in biological materials, and effectively reduces and avoids potential safety hazards when the quantitative qPCR detection method is applied to clinical or medical and aesthetic medical instruments.
Disclosure of Invention
The invention aims to provide a primer pair for detecting pig source DNA residues in biological materials with high sensitivity and high specificity, and a detection reagent or a PCR kit containing the primer pair.
Another objective of the invention is to provide a method for detecting pig source DNA residue in biological materials or a PCR method by using the primer pair or the detection reagent provided by the invention.
In a first aspect, the present invention provides a primer pair for detecting porcine genomic DNA, the primer pair comprising a forward primer and a reverse primer, wherein the forward primer binds to positions 2-30, preferably positions 5-24 of the sequence shown in SEQ ID NO. 1 on the porcine genomic DNA; wherein the reverse primer is combined with the 120 th to 150 th positions, preferably 127 th to 143 th positions of the sequence shown in SEQ ID NO. 1 on the pig genome DNA; and the length of an amplification product obtained by amplification of the primer pair is 139-149bp.
In a preferred embodiment, the length of the forward primer and the reverse primer is 16 to 22bp; preferably 20bp.
In a preferred embodiment, the forward and reverse primers have Tm temperatures of 58 to 60 ℃ and the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is 2 ℃ or less.
In a specific embodiment, in the primer pair, the forward primer is shown as SEQ ID NO. 2, and the reverse primer is shown as SEQ ID NO. 3.
In a second aspect, the present invention provides a detection reagent comprising the primer pair of the first aspect.
In a preferred embodiment, the detection reagent further comprises a probe.
In a preferred embodiment, the probe is as set forth in SEQ ID NO 4.
In a third aspect, the present invention provides a detection kit, comprising the primer pair of the first aspect or the detection reagent of the second aspect, and optionally instructions for using the primer pair or the detection reagent to detect swine-derived DNA.
In specific embodiments, the detection reagent further comprises a probe.
In a preferred embodiment, the probe is as set forth in SEQ ID NO. 4.
In a specific embodiment, the forward primer of the primer pair is shown as SEQ ID NO. 2, the reverse primer is shown as SEQ ID NO. 3, and the probe is shown as SEQ ID NO. 4.
In a preferred embodiment, the detection sensitivity of the detection reagent is 10 fg/reaction.
In a fourth aspect, the present invention provides a method for detecting porcine genomic DNA, the method comprising: carrying out PCR on a sample to be detected by using the primer pair of the first aspect, the detection reagent of the second aspect or the detection kit of the third aspect, and detecting a PCR amplification product.
In a fifth aspect, the present invention provides a PCR kit comprising a container and a primer pair of the first aspect in the container.
In a preferred embodiment, the length of the forward primer and the reverse primer is 16 to 22bp; preferably 20bp.
In a preferred embodiment, the forward and reverse primers have Tm temperatures of 58 to 60 ℃ and the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is 2 ℃ or less.
In a preferred embodiment, the kit further comprises a probe.
In a preferred embodiment, the probe is as set forth in SEQ ID NO 4.
In a preferred embodiment, the detection sensitivity of the detection reagent is 10 fg/reaction.
In a preferred embodiment, the kit further comprises a standard control.
In a sixth aspect, the present invention provides a PCR method comprising the steps of:
amplifying the target product in a PCR detection system using the primer pair of the first aspect.
In a preferred embodiment, the length of the forward primer and the reverse primer is 16 to 22bp; preferably 20bp.
In a preferred embodiment, the forward and reverse primers have Tm temperatures of 58 to 60 ℃ and the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is 2 ℃.
In a preferred embodiment, the kit further comprises a probe.
In a preferred embodiment, the probe is as set forth in SEQ ID NO. 4.
In a seventh aspect, the present invention provides a use of the primer pair of the first aspect or the detection reagent of the second aspect or the detection kit of the third aspect, for detecting whether the pig genomic DNA is present in a test subject.
In a preferred embodiment, the test object is a porcine acellular matrix; acellular matrixes derived from porcine dermis, bladder, small intestine and pericardium are preferred, and include, but are not limited to, biological hernia repair patches, oral repair membranes, biological patches, wound dressings, collagen, pericardial biological valves, ophthalmologically implanted corneas, artificial skin, medical suture materials, and the like.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the amplification curves of the reference samples using the primer pairs SEQ ID NO. 2 and SEQ ID NO. 3.
FIG. 2 shows a standard curve for the reference, in which the primer pairs used are SEQ ID NO. 2 and SEQ ID NO. 3.
FIGS. 3-5 show the amplification curves at concentrations 3 fg/. Mu.L, 1 fg/. Mu.L and 0.2 fg/. Mu.L, respectively, in a sensitivity assay, using primer pairs SEQ ID NO. 2 and SEQ ID NO. 3.
Detailed Description
Through intensive and extensive research, the inventor unexpectedly finds that a primer designed aiming at a sequence shown as SEQ ID NO. 1 in a pig genome not only can detect pig source DNA residues with high sensitivity, but also can distinguish interfering DNAs of cattle sources and the like. The method is simple, convenient and quick to operate, and high in specificity and sensitivity. The present invention has been completed based on this finding.
Primer set of the present invention
The term "primer" as used herein has the meaning conventionally understood by those skilled in the art. The pig genome DNA specific primer is not designed aiming at the exogenous gene or the virus vector, but is designed aiming at the segment shown in SEQ ID NO. 1 on the pig genome DNA. In other words, the primer of the present invention can specifically bind to the region shown by SEQ ID NO. 1 on the pig genomic DNA.
In view of the teachings of the present invention and the general knowledge in the art, one skilled in the art will appreciate that a variety of primer pairs can be designed for the segment shown in SEQ ID NO. 1. Therefore, the primer set of the present invention is not limited to the primer set specifically obtained in the examples.
In a specific embodiment, the forward primer of the invention binds to positions 2-30, preferably positions 5-24 of the sequence shown in SEQ ID NO. 1; wherein the reverse primer is combined with the 120 th to 150 th positions, preferably 127 th to 143 th positions of the sequence shown in SEQ ID NO. 1, and the length of an amplification product obtained by amplifying the primer pair is 139-149bp.
In a preferred embodiment, the length of the forward primer and the reverse primer is 16 to 22bp; preferably 20bp.
In a preferred embodiment, the forward and reverse primers have Tm temperatures of 58 to 60 ℃ and the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is 2 ℃.
In a specific embodiment, in the primer pair of the present invention, the forward primer is shown as SEQ ID NO. 2, and the reverse primer is shown as SEQ ID NO. 3.
Probe needle
The term "primer" as used herein has the meaning conventionally understood by those skilled in the art, i.e., a short piece of single-stranded DNA or RNA fragment for detecting a nucleic acid sequence complementary thereto.
In view of the teachings of the present invention and the general knowledge in the art, those skilled in the art will understand that, knowing the primer pair, one can design a probe autonomously based on the template sequence between the forward primer and the reverse primer binding site and test the technical effect of the probe and primer pair. In specific embodiments, one of ordinary skill in the art can design probes specifically as desired, either in the liquid phase or immobilized on a solid phase; the binding may be performed before amplification or after amplification. Therefore, the probe of the present invention is not limited to the probe specifically disclosed in the examples. The primer set of the present invention is not limited to the use of the probe set specifically disclosed in the examples.
In a specific embodiment, the probe of the invention is shown in SEQ ID NO 4.
Detection reagent of the present invention
The invention also provides a detection reagent for detecting the pig genome DNA, the detection reagent comprises the primer pair and other components such as probes and the like required by PCR, such as Taq enzyme, dNTP, mg 2+ And so on.
In a specific embodiment, the detection reagent of the present invention comprises a forward primer represented by SEQ ID NO. 2, a reverse primer represented by SEQ ID NO. 3, and a probe represented by SEQ ID NO. 4.
In a specific embodiment, the detection sensitivity of the detection reagent of the present invention reaches 10 fg/reaction.
On the basis of the primer pair or the detection reagent, the invention further provides a method for detecting the pig genome DNA, which comprises the following steps: the primer pair or the detection reagent of the invention is used for carrying out PCR on a sample to be detected and detecting a PCR amplification product.
On the basis of the primer pair, the invention also provides a PCR kit, which comprises a container and the primer pair or the detection reagent of the invention positioned in the container.
In a specific embodiment, the PCR kit of the present invention further comprises other components necessary for performing PCR and instructions for using the kit for PCR detection. In a preferred embodiment, the kit further comprises a standard control.
On the basis of the primer pair, the invention also provides a PCR method for amplifying a target product by using the primer pair.
Biological material
The primer pair, the detection reagent or the detection kit can be used for detecting pig DNA residues in biological materials. The biomaterial described herein is a biologically derived extracellular matrix. In a preferred embodiment, the extracellular matrix of biological origin is a decellularized matrix of porcine origin. In specific embodiments, the porcine acellular matrix is derived from porcine acellular matrices of dermis, bladder, small intestine and pericardium, including but not limited to biological hernia repair patch, oral repair membrane, biological patch, wound dressing, collagen, pericardial bioprosthetic valve, ophthalmologic implanted cornea, artificial skin, medical suture material and the like.
The advantages of the invention include:
1. the primer pair or the detection reagent can detect the pig genome DNA with high sensitivity;
2. the primer pair or the detection reagent can distinguish interference DNAs of cattle, CHO, vero, human, NS0, MDCK, E.coli, pichia pastoris, sf9 and the like;
3. the detection method is simple, convenient and quick to operate, and high in specificity and sensitivity.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Examples
Materials and methods
1. The porcine genomic DNA reference was from cheng biotechnology limited, lazhou.
2. Sample pretreatment reagent
The sample pretreatment reagent is from Huzhou Shenke biological technology company Limited and comprises proteinase K, proteinase K buffer solution, magnetic beads, binding solution, glycogen, yeast tRNA, washing solution A, washing solution B and eluent.
3.QPCR detection System
The qPCR detection kit is from Huzhou Shenke biotechnology limited and comprises qPCR Reaction buffer, DNA diluent, primers, probes, DNA standard substances and internal quality control.
4. The instrument comprises the following steps: applied biosystems 7500 fluorescent quantitative PCR instrument.
Experimental procedures and procedures
1. Sample pretreatment
1) The sample was weighed accurately 5-10 mg and recorded, and placed in a 1.5mL DNase-free sterile centrifuge tube.
2) After the sample was treated as small as possible, 1000. Mu.L proteinase K buffer, 200. Mu.L proteinase K, and DEPC water were added to a final volume of 2000. Mu.L, respectively. And (4) digesting for 1h by using a metal bath at 56 ℃ until the sample to be detected is completely digested and no particle sample can be seen by naked eyes.
3) After digestion, 200. Mu.L of sample is added into 200. Mu.L of working binding solution, 9. Mu.L of glycogen and 0.2. Mu.L of yeast tRNA, and mixed evenly.
4) Adding 200. Mu.L of isopropanol and 30. Mu.L of magnetic beads, shaking and mixing for 5min, quickly centrifuging for 10s, and standing on a magnetic frame. After the solution was clarified and the beads were completely separated, the cap was gently opened and the supernatant carefully aspirated.
5) Adding 700 mu L of washing solution A, shaking and mixing uniformly, then quickly centrifuging for 10s, and standing on a magnetic frame. After the solution is clarified and the magnetic beads are completely separated, the supernatant is aspirated.
6) Adding 700 mu L of washing solution B, shaking and uniformly mixing, then quickly centrifuging for 10s, and standing on a magnetic frame. After the solution is clarified and the magnetic beads are completely separated, the supernatant is aspirated.
7) And rapidly centrifuging for 10s again, standing in a magnetic frame, and removing residual liquid completely after the magnetic beads are completely separated.
8) And opening the tube cover, drying at room temperature for 5-10 s, and removing residual ethanol. Care was taken during the drying process to avoid drying the beads too much so that the beads did not dissolve completely during elution.
9) Adding 150 mu L of eluent, slightly shaking to uniformly mix the magnetic beads and the eluent, carrying out water bath at 70 ℃ for 7min, shaking every 2min in the water bath process, and shaking and uniformly mixing for 2-3 times.
10 Fast centrifugation for 10s, resting on a magnetic rack, and after separation of the magnetic beads, transferring the supernatant to a new centrifuge tube.
2. Detection of
2.1 reference preparation
The reference substance is genomic DNA extracted from pig kidney cells, and the concentration is 100 ng/. Mu.L.
2.2 Standard Curve sample preparation
The reference sample was diluted 10-fold with a DNA diluent to prepare a standard solution of 3000 pg/. Mu.L, 300 pg/. Mu.L, 30 pg/. Mu.L, 3 pg/. Mu.L, 0.3 pg/. Mu.L, or 0.03 pg/. Mu.L.
2.3qPCR reaction System
Samples to be tested, 10 mul of samples without template control and standard curve are respectively taken and added into 20 mul of qPCR reaction solution, and 3 multiple wells are arranged. After the sample is added, the sample is quickly centrifuged for 3s in a short time and then placed in a qPCR instrument. Setting a reaction program: pre-denaturation at 95 ℃ for 10min; 15s at 95 ℃, 40s at 60 ℃,40 cycles; the reaction volume was 30. Mu.L.
2.4 Standard Curve preparation
After the amplification is finished, a threshold line is set, and the amplification efficiency, the slope, R2 of the standard curve and the detection value of each sample are read.
2.5 determination of results
a) Correlation coefficient R of standard curve equation 2 Should be larger than 0.990, the slope is between-3.1 and-3.8 (i.e. the amplification efficiency is between 83.3% and 110%).
b) The Ct value of the No Template Control (NTC) was not detected or was 35 or more.
Example 1 design of primer pairs and probes
The present inventors designed primer pairs and probes for detecting porcine residual DNA using the following target sequences:
1. target sequences
qPCR targeting sequences the following sequences in the pig genome were selected:
AGGGAGTTCCCATCGTGGCTCAGTGGTAACGAACCTGACTAGTATCCATGAGGATGCAGATTCAATCCCTGGCCTTNCTCAGTGGGTTAAGGATCCNGCATTGCTGTGAGNTGTGGTGTAGGTCNCAGATGCGGCTCGGATCCCGTGTTGCTGTGGCTGTGGTGTAGGCCGGCAGCTACAGCTCCGATTNGACCCCTAGCCTGGGAACCTCCATATGCCGCGGGTGNGGCCCT(SEQ ID NO:1)
2. design of primers and probes
Aiming at a sequence shown in SEQ ID NO. 1, the inventor designs the following primer pairs and probes:
a forward primer: AGTTCCCACGTGGCTCAGT (SEQ ID NO: 2);
reverse primer: GGATCCGAGCCGCATCT (SEQ ID NO: 3);
and (3) probe: FAM-ATCCATGATGAGGATGCAGATT-MGB (SEQ ID NO: 4).
Example 2 detection of Linear Range
The inventors examined the linear range of the primer pair of the present invention. The specific experimental procedures were as described in "experimental procedures and procedures" above.
The test result proves that the concentration range of the swine DNA detection is 30 ng/reaction-300 fg/reaction (see table 1), and the standard curve parameter R 2 :0.999, the amplification efficiency was 99.16% (as shown in FIG. 1, 2).
TABLE 1 mean values of amplified Ct values for standard curves
| Standard song | Mean value of Ct values |
| St1 (30 ng/reaction) | 13.39 |
| St2 (3 ng/reaction) | 16.70 |
| St3 (300 pg/reaction) | 20.09 |
| St4 (30 pg/reaction) | 23.48 |
| St5 (3 pg/reaction) | 26.78 |
| St6 (300 fg/reaction) | 30.07 |
Example 3 detection of specificity
DNA of cells of multiple species such as cattle, CHO, vero, human, NS0, MDCK, E.coli, pichia pastoris and Sf9 is selected to be used as an interference experiment, and interference DNA of 30 ng/reaction is used as a template to carry out qPCR amplification. The results show that no Ct value is detected in all 9 interfering DNAs (30 ng/reaction) (see Table 2), and the qPCR detection has better specificity.
TABLE 2.9 Ct value results for detection of interfering DNAs
| Interfering DNA | Ct value mean value for qPCR detection |
| Cattle | Undet. |
| CHO | Undet. |
| Vero | Undet. |
| Human being | Undet. |
| NS0 | Undet. |
| MDCK | Undet. |
| E.coli | Undet. |
| Pichia pastoris | Undet. |
| Sf9 | Undet. |
Example 4 determination of detection sensitivity
1. In this example, the inventors measured the sensitivity of the detection system of the present invention.
The pig genome DNA reference substance is respectively diluted to the concentration of 3 fg/mu L,1 fg/mu L and 0.2 fg/mu L, 8 wells of each concentration detection are repeated for 3 times of independent experiments, and the total 24 times of detection data are counted. The results showed that 24 measurements at 1 fg/. Mu.L all gave a detectable value and a clear amplification curve (see Table 3, FIGS. 3-5). The detection sensitivity can reach 1 fg/muL, namely 10 fg/reaction.
TABLE 3 results of 24-time sensitivity measurements
Example 5 detection of actual samples
In this example, the detection system of the present invention was used to quantitatively detect porcine DNA residues in absorbable biofilm of biomaterial samples and lyophilized porcine type I collagen products. The result shows that the detection value of the pig source DNA remained in the absorbable biomembrane extracted sample is lower than the quantitative range of the standard curve, and the residual amount of the pig source DNA in the pig type I collagen freeze-dried product is 0.2ng/mg (see table 4).
TABLE 4 actual sample test results
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A primer pair for detecting pig genome DNA, which comprises a forward primer and a reverse primer, wherein the forward primer is combined with 2 nd to 30 th, preferably 5 th to 24 th of a sequence shown as SEQ ID NO. 1 on the pig genome DNA; wherein the reverse primer is combined with the 120 th to 150 th positions, preferably 127 th to 143 th positions of the sequence shown in SEQ ID NO. 1 on the pig genome DNA; and the length of an amplification product obtained by amplification of the primer pair is 139-149bp.
2. The primer pair of claim 1, wherein in the primer pair, the forward primer is shown as SEQ ID NO. 2, and the reverse primer is shown as SEQ ID NO. 3.
3. A detection reagent comprising the primer pair of claim 1 or 2.
4. A test kit comprising the primer pair of claim 1 or 2 or the detection reagent of claim 3, and optionally further comprising instructions for using the primer pair or detection reagent to detect porcine-derived DNA.
5. The test kit of claim 4, wherein the test reagents further comprise a probe.
6. The detection kit of claim 5, wherein the forward primer of the primer pair is shown as SEQ ID NO. 2, the reverse primer is shown as SEQ ID NO. 3, and the probe is shown as SEQ ID NO. 4.
7. A method of detecting porcine genomic DNA, the method comprising: performing PCR on a sample to be tested by using the primer pair of claim 1 or 2, or the detection reagent of claim 3, or the detection kit of any one of claims 4 to 6, and detecting the PCR amplification product.
8. A PCR kit comprising a container and the primer pair of claim 1 or 2 in the container.
9. A PCR method comprising the steps of:
amplifying a target product in a PCR detection system using the primer set of claim 1 or 2.
10. Use of the primer pair of claim 1 or 2 or the detection reagent of claim 3 or the detection kit of any one of claims 4 to 6 for detecting the presence of porcine genomic DNA in a test subject.
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| CN202210869347.4A CN115927651A (en) | 2022-07-22 | 2022-07-22 | Primer and method for detecting swine-origin DNA |
| PCT/CN2023/108728 WO2024017382A1 (en) | 2022-07-22 | 2023-07-21 | Primer for detecting porcine dna and detection method |
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| WO2024017382A1 (en) * | 2022-07-22 | 2024-01-25 | 中国食品药品检定研究院 | Primer for detecting porcine dna and detection method |
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| US20090325175A1 (en) * | 2008-05-30 | 2009-12-31 | Wyeth | METHODs OF DETECTION AND QUANTIFICATION OF HOST CELL DNA CONTAMINATION OF PURIFIED PROTEINS |
| WO2012059102A2 (en) * | 2010-11-05 | 2012-05-10 | Leo Pharma A/S | A method of detecting contaminant dna in biological samples |
| US9512475B2 (en) * | 2012-01-16 | 2016-12-06 | Biocon Limited | Method of detecting residual genomic DNA and a kit thereof |
| CN103290105A (en) * | 2013-02-01 | 2013-09-11 | 四川农业大学 | Swine-derived component real-time fluorescent PCR detection method and primers |
| CN109468386A (en) * | 2018-12-14 | 2019-03-15 | 上海上药第生化药业有限公司 | A kind of kit, method, primer pair and probe and its application |
| CN112080553A (en) * | 2019-06-13 | 2020-12-15 | 苏州融析生物科技有限公司 | qPCR detection method for sheep and pig gene content in crude sheep heparin sodium |
| CN113493824A (en) * | 2020-12-30 | 2021-10-12 | 锦州奥鸿药业有限责任公司 | Real-time fluorescent PCR method for calf serum animal-derived component species detection |
| CN115927651A (en) * | 2022-07-22 | 2023-04-07 | 中国食品药品检定研究院 | Primer and method for detecting swine-origin DNA |
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| WO2024017382A1 (en) * | 2022-07-22 | 2024-01-25 | 中国食品药品检定研究院 | Primer for detecting porcine dna and detection method |
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