WO2024017382A1 - 检测猪源dna的引物及检测方法 - Google Patents

检测猪源dna的引物及检测方法 Download PDF

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WO2024017382A1
WO2024017382A1 PCT/CN2023/108728 CN2023108728W WO2024017382A1 WO 2024017382 A1 WO2024017382 A1 WO 2024017382A1 CN 2023108728 W CN2023108728 W CN 2023108728W WO 2024017382 A1 WO2024017382 A1 WO 2024017382A1
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primer
primer pair
seq
detection
present
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PCT/CN2023/108728
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English (en)
French (fr)
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魏利娜
吴婉欣
陈亮
宗伟英
李康凯
吴晓双
邵安良
段晓杰
范行良
陈丽媛
张潇
徐丽明
李静莉
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中国食品药品检定研究院
湖州申科生物技术股份有限公司
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Publication of WO2024017382A1 publication Critical patent/WO2024017382A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention relates to the field of biological detection. Specifically, the present invention relates to primers and detection methods for detecting pig-derived DNA residues in biological materials.
  • Biologically derived extracellular matrix is widely used in the fields of regenerative medicine and tissue engineering.
  • Naturally derived extracellular matrix has a natural three-dimensional structure, is rich in collagen and growth factors, and provides microorganisms that induce cell adhesion, proliferation and diffusion. environment to promote the organization's return to normal structure and function.
  • Biological extracellular matrix is mainly derived from human or mammalian tissues and organs, such as dermis, bladder, small intestine, pericardium, etc. Since pig tissues and organs are widely sourced and have high biocompatibility, they are the main raw materials for acellular matrix. .
  • Biomaterials composed of pig-derived acellular matrix are used clinically for the repair and regeneration of blood vessels, skin, intestines, heart and other tissues and organs.
  • Medical device products include biological hernia repair patches, oral repair membranes, biological patches, wound dressings, etc.
  • Decellularization and sterilization are used to remove cellular components and harmful microorganisms from animal tissues. These cellular components and nucleic acid residues are the main cause of immunogenicity risks for patients during implantation. In severe cases, they can lead to inflammatory reactions and severe pain. and non-infectious edema and other negative effects. Therefore, the evaluation of the decellularization effect is one of the important measures for quality and safety control of biomaterials.
  • the decellularization results are mainly evaluated through quantitative detection of residual cellular components, such as double-stranded DNA, in decellularized matrix biomaterials.
  • domestic industry standards for animal-derived medical devices such as YY/T0771 and the "Guiding Principles for Technical Review of Registration of Animal-derived Medical Device Products" both put forward risk management requirements for medical devices manufactured using animal tissues or their derivatives.
  • Molecular biology techniques based on nucleic acids can use specific fluorescently labeled Taqman probes to quantitatively detect and analyze residual DNA in samples.
  • the probes with fluorescent groups continuously excite fluorescent signals during the thermal cycle. Monitoring changes in accumulated fluorescence signals reflect the increase in product during PCR, thereby enabling quantitative analysis of target DNA.
  • Real-time fluorescence quantitative PCR detection technology has the advantages of good specificity, high accuracy and fast speed.
  • the detection of pig-derived ingredients is mainly concentrated in the fields of meat products, gelatin and other food products, and there is less detection of residual DNA in medical devices used in animal-derived biological materials; in addition, the target design of pig-derived qPCR usually uses single-copy genes or Mitochondrial genes have low copy numbers or are not constant in cells, which may lead to quantitative bias or false negatives, and are not suitable for quantitative detection of trace residual DNA in biological materials.
  • the number of repetitions of highly repetitive sequences in pigs and other mammals can reach more than 100,000 times, and they are evenly and stably distributed in each chromosome. Therefore, fluorescence quantitative PCR methods based on highly repetitive sequence targets can greatly improve the sensitivity, specificity and accuracy of detection. It can be used to establish a set of quantitative qPCR detection methods for trace residual pig-derived DNA in biological materials, effectively reducing and avoiding safety hazards in clinical or aesthetic medical devices.
  • the purpose of the present invention is to provide a primer pair for detecting porcine DNA residues in biological materials with high sensitivity and specificity, as well as a detection reagent or PCR kit containing the primer pair.
  • Another object of the present invention is to provide a method or PCR method for detecting pig-derived DNA residues in biological materials using the primer pairs or detection reagents provided by the present invention.
  • the present invention provides a primer pair for detecting pig genomic DNA
  • the primer pair includes a forward primer and a reverse primer, wherein the forward primer is bound to pig genomic DNA as shown in SEQ ID NO: 1 Positions 2-30 of the sequence, preferably positions 5-24; the reverse primer thereof binds to positions 120-150, preferably positions 127-143, of the sequence shown in SEQ ID NO: 1 on pig genomic DNA; and the The length of the amplification product amplified by the primer pair is 139-149 bp.
  • the length of the forward primer and reverse primer is 16 to 22 bp; preferably 20 bp.
  • the Tm temperature of the forward primer and the reverse primer is 58-60°C
  • the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is ⁇ 2°C.
  • the forward primer is as shown in SEQ ID NO:2
  • the reverse primer is as shown in SEQ ID NO:3.
  • the present invention provides a detection reagent, which includes the primer pair described in the first aspect.
  • the detection reagent further includes a probe.
  • the probe is as shown in SEQ ID NO:4.
  • the present invention provides a detection kit, which includes the primer pair described in the first aspect or the detection reagent described in the second aspect, and optionally also includes the use of the primer pair or detection reagent. Instructions for use in detecting pig DNA.
  • the detection reagent further includes a probe.
  • the probe is as shown in SEQ ID NO:4.
  • the forward primer of the primer pair is as shown in SEQ ID NO:2
  • the reverse primer is as shown in SEQ ID NO:3
  • the probe is as shown in SEQ ID NO:4.
  • the detection sensitivity of the detection reagent is 10 fg/reaction.
  • the present invention provides a method for detecting pig genomic DNA, which method includes: using the primer pair described in the first aspect or the detection reagent described in the second aspect or the detection kit described in the third aspect , perform PCR on the sample to be tested, and detect the PCR amplification product.
  • the present invention provides a PCR kit, which includes a container and the primer pair described in the first aspect located in the container.
  • the length of the forward primer and reverse primer is 16 to 22 bp; preferably 20 bp.
  • the Tm temperature of the forward primer and the reverse primer is 58-60°C
  • the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is ⁇ 2°C.
  • the kit also contains a probe.
  • the probe is as shown in SEQ ID NO:4.
  • the detection sensitivity of the detection reagent is 10 fg/reaction.
  • the kit also contains a standard control.
  • the present invention provides a PCR method, including the steps:
  • the primer pair described in the first aspect is used to amplify the target product.
  • the length of the forward primer and reverse primer is 16 to 22 bp; preferably 20 bp.
  • the Tm temperature of the forward primer and the reverse primer is 58-60°C
  • the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is ⁇ 2°C.
  • the kit also contains a probe.
  • the probe is as shown in SEQ ID NO:4.
  • the present invention provides the use of the primer pair described in the first aspect, the detection reagent described in the second aspect, or the detection kit described in the third aspect, for detecting the presence of porcine genomic DNA in the object to be tested. .
  • the test object is a porcine-derived acellular matrix; preferably acellular matrix derived from porcine dermis, bladder, small intestine, and pericardium, including but not limited to biological hernia repair patches and oral repair membranes. , biological patches, wound dressings, collagen, pericardial biological valves, ophthalmic corneal implants, artificial skin, medical suture materials, etc.
  • Figure 1 shows the amplification curve of the reference product using the primer pair SEQ ID NO:2 and SEQ ID NO:3.
  • Figure 2 shows the standard curve of the reference product using the primer pair SEQ ID NO:2 and SEQ ID NO:3.
  • Figures 3-5 show the amplification curves of the concentrations 3fg/ ⁇ L, 1fg/ ⁇ L and 0.2fg/ ⁇ L in the sensitivity assay respectively, where the primer pairs used are SEQ ID NO:2 and SEQ ID NO:3.
  • the inventor unexpectedly discovered that the primers designed for the sequence shown in SEQ ID NO: 1 in the pig genome can not only detect pig-derived DNA residues with high sensitivity, but also distinguish cattle origin, etc. of interfering DNA.
  • the method of the invention is simple and quick to operate, and has high specificity and sensitivity. On this basis, the present invention was completed.
  • the term "primer” as used herein has the meaning conventionally understood by those skilled in the art.
  • the porcine genomic DNA-specific primer of the present invention is not designed for the exogenous gene itself or the viral vector itself, but for the segment shown in SEQ ID NO: 1 on the porcine genomic DNA.
  • the primer of the present invention can specifically bind to the segment shown in SEQ ID NO: 1 on pig genomic DNA.
  • primer pairs of the present invention are not limited to the primer pairs specifically obtained in the examples.
  • the forward primer of the present invention is bound to positions 2-30, preferably 5-24, of the sequence shown in SEQ ID NO: 1; the reverse primer is bound to the sequence shown in SEQ ID NO: 1 Positions 120-150 of the sequence are shown, preferably positions 127-143, and the length of the amplification product amplified by the primer pair is 139-149 bp.
  • the length of the forward primer and reverse primer is 16 to 22 bp; preferably 20 bp.
  • the Tm temperature of the forward primer and the reverse primer is 58-60°C
  • the absolute value of the difference between the Tm of the forward primer and the Tm of the reverse primer is ⁇ 2°C.
  • the forward primer is as shown in SEQ ID NO:2
  • the reverse primer is as shown in SEQ ID NO:3.
  • primer used herein has the meaning conventionally understood by those skilled in the art, that is, a small single-stranded DNA or RNA fragment used to detect a nucleic acid sequence complementary to it.
  • probes of the present invention are not limited to the probes specifically disclosed in the examples.
  • primer pairs of the present invention are not limited to pairing with the probes specifically disclosed in the examples.
  • the probe of the present invention is shown in SEQ ID NO: 4.
  • the present invention also provides a detection reagent for detecting pig genomic DNA.
  • the detection reagent includes the primer pair of the present invention and probes and other components required to implement PCR, such as Taq enzyme, dNTPs, Mg 2+ and so on.
  • the detection reagent of the present invention includes the forward primer shown in SEQ ID NO:2, the reverse primer shown in SEQ ID NO:3, and the probe shown in SEQ ID NO:4.
  • the detection sensitivity of the detection reagent of the present invention reaches 10 fg/reaction.
  • the present invention further provides a method for detecting pig genomic DNA.
  • the method includes: using the primer pair or detection reagent of the present invention, performing PCR on the sample to be tested, and detecting the PCR amplification product.
  • the present invention also provides a PCR kit, which includes a container and the above-mentioned primer pair of the present invention or detection reagent located in the container.
  • the PCR kit of the present invention is also equipped with other required components for performing PCR and instructions for using the kit for PCR detection.
  • the kit also contains a standard control.
  • the present invention also provides a PCR method for amplifying a target product using the primer pair of the present invention.
  • the primer pair, detection reagent or detection kit of the present invention can be used to detect pig DNA residues in biological materials.
  • the biomaterials described herein are biogenic extracellular matrices.
  • the biologically derived extracellular matrix is a porcine derived acellular matrix.
  • the porcine-derived acellular matrix is derived from the acellular matrix of pig dermis, bladder, small intestine and pericardium, including but not limited to biological hernia repair patch, oral repair membrane, biological patch, wound surface Dressings, collagen, pericardial biological valves, ophthalmic corneal implants, artificial skin, medical suture materials, etc.
  • the primer pair or detection reagent of the present invention can detect pig genomic DNA with high sensitivity
  • the primer pair or detection reagent of the present invention can distinguish interfering DNA such as bovine, CHO, Vero, human, NSO, MDCK, E. coli, Pichia pastoris and Sf9;
  • the detection method of the present invention is simple and fast to operate, and has high specificity and sensitivity.
  • Porcine genomic DNA reference material comes from Huzhou Shenke Biotechnology Co., Ltd.
  • Sample pretreatment reagents were from Huzhou Shenke Biotechnology Co., Ltd., including proteinase K, proteinase K buffer, magnetic beads, binding solution, glycogen, yeast tRNA, washing solution A, washing solution B, and eluent.
  • the qPCR detection kit comes from Huzhou Shenke Biotechnology Co., Ltd. and contains qPCR Reaction buffer, DNA diluent, primers, probes, DNA standards, and internal quality control.
  • the reference product is genomic DNA extracted from pig kidney cells, with a concentration of 100ng/ ⁇ L.
  • DNA diluent to dilute the reference product 10 times in a gradient to prepare standard solution of 3000pg/ ⁇ L, 300pg/ ⁇ L, 30pg/ ⁇ L, 3pg/ ⁇ L, 0.3pg/ ⁇ L, and 0.03pg/ ⁇ L.
  • the correlation coefficient R 2 of the standard curve equation should be greater than 0.990, and the slope should be between -3.1 and -3.8 (that is, the amplification efficiency should be between 83.3% and 110%).
  • NTC no template control
  • the inventor used the following target sequences to design primer pairs and probes for detecting pig-derived residual DNA:
  • the qPCR target sequence selects the following sequences in the pig genome:
  • Reverse primer GGATCCGAGCCGCATCT (SEQ ID NO: 3);
  • the inventors examined the linear range of the primer pairs of the present invention.
  • the specific experimental process is as described in "Experimental Operations and Processes" above.
  • test results show that the concentration range of pig DNA detection ranges from 30ng/reaction to 300fg/reaction (see Table 1), the calibration parameter R2 : 0.999, and the amplification efficiency is 99.16% (shown in Figures 1 and 2).
  • the DNA of bovine, CHO, Vero, human, NS0, MDCK, E.coli, Pichia pastoris and Sf9 cells from multiple species was selected for interference experiments, and 30ng/reaction interference DNA was used as a template for qPCR amplification.
  • the results showed that none of the 9 interfering DNAs (30ng/reaction) had detected Ct values (see Table 2), and the qPCR detection had good specificity.
  • the inventor measured the sensitivity of the detection system of the present invention.
  • the porcine genomic DNA reference material was diluted to concentrations of 3fg/ ⁇ L, 1fg/ ⁇ L and 0.2fg/ ⁇ L respectively, and 8 replicate wells were tested at each concentration. Three independent experiments were repeated, and a total of 24 test data were collected. The results showed that the 24 detection results of 1fg/ ⁇ L all had detection values and obvious amplification curves (see Table 3, Figure 3-5). The detection sensitivity can reach 1fg/ ⁇ L or 10fg/reaction.
  • Porcine genomic DNA was used to prepare spiked samples with three different concentrations (300pg/ ⁇ L, 3pg/ ⁇ L, 0.06pg/ ⁇ L). The samples were extracted and tested by qPCR to calculate the spiked recovery rate. Each concentration was tested three times in duplicate. The results showed that the standard recovery rate was 78.8-96.7%, and the CV was 4.4-13.0% (see Table 4).
  • the precision includes repeatability and intermediate precision.
  • the repeatability was tested using a spiked sample of porcine genomic DNA configured with 3pg/ ⁇ L. The test was repeated 6 times, and the CV value of the test result was 13.5% (see Table 5).
  • the intermediate precision test uses pig genomic DNA to configure 3 samples of different concentrations (300pg/ ⁇ L, 3pg/ ⁇ L, 0.06pg/ ⁇ L). A total of 3 independent qPCR quantitative tests are performed on each sample by different experimenters, and each test is repeated. 3 times. The detection CV is 11.0 ⁇ 27.2% (see Table 6).
  • the detection system of the present invention is used to quantitatively detect pig-derived DNA residues in absorbable biofilms of biological material samples and freeze-dried porcine type I collagen.
  • the results showed that the detection value of residual pig-derived DNA in the absorbable biofilm extraction sample was lower than the quantitative range of the standard curve, and the residual amount of pig-derived DNA in the freeze-dried porcine type I collagen was 0.2ng/mg (see Table 7).

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Abstract

本发明提供了检测猪基因组DNA的引物对、包含本发明引物对的检测试剂或试剂盒以及利用所述引物对检测猪基因组DNA的方法，所述引物对特异性结合于SEQ ID NO:1所示序列。利用所述引物对的PCR检测方法操作简便快捷、灵敏度高、能区分牛、CHO、Vero、人、NS0、MDCK、E.coli、毕赤酵母和Sf9等干扰性DNA。

Description

检测猪源DNA的引物及检测方法 技术领域
本发明涉及生物检测领域。具体地说，本发明涉及检测生物材料中猪源DNA残留的引物及检测方法。
背景技术
生物源细胞外基质(ECM)广泛应用于再生医学与组织工程医疗领域，天然来源的细胞外基质具有自然形态的三维构造，富含胶原蛋白和生长因子，提供诱导细胞黏附，增殖和扩散的微环境，促进组织恢复正常结构和功能。生物源细胞外基质主要来源于人或哺乳动物的组织器官，如真皮，膀胱，小肠和心包膜等，由于猪的组织器官来源广泛且生物相容性高，是主要的脱细胞基质的原材料。猪源脱细胞基质组成的生物材料在临床用于血管、皮肤、肠道、心脏等组织器官的修复和再生，医疗器械产品包括生物疝修补片、口腔修复膜、生物补片、创面敷料等。
脱细胞和灭菌处理用于去除动物组织中的细胞成分和有害微生物，这些细胞成分和核酸残留是导致患者在植入时产生免疫原性风险的主要原因，严重的会导致炎症反应，剧烈疼痛和非感染性水肿等负面作用。因此对脱细胞效果的评估是进行生物材料质量安全控制的重要措施之一，目前主要通过定量检测脱细胞基质生物材料中残留的细胞成分，如双链DNA等来评估脱细胞结果。国内动物源性医疗器械行业标准YY/T0771等和《动物源性医疗器械产品注册技术审查指导原则》对使用动物组织或其衍生物制造的医疗器械均提出了风险管理要求。
基于核酸的分子生物学技术如荧光定量PCR可以利用特异性荧光标记的Taqman探针对样本中残留DNA进行定量检测分析，带有荧光基团的探针在热循环过程中不断激发荧光信号，通过监测积累的荧光信号的变化反映PCR过程中产物的增加，从而实现对目标DNA的定量分析。实时荧光定量PCR检测技术具有特异性好、准确性高和速度快等优势。
目前，对于猪源成分检测主要集中在肉类食品、明胶等食品领域，应用于动物源性生物材料的医疗器械中残留DNA检测较少；此外，猪源qPCR的靶标设计通常使用单拷贝基因或线粒体基因，这些基因的拷贝数低或在细胞中不恒定，可能会导致定量偏差或者出现假阴性情况，不适用于生物材料痕量残留DNA的定量检测。高度重复序列在猪等哺乳动物中的重复次数可到达十万次以上，且在各染色体中分别均匀稳定。因此，基于高度重复序列靶标的荧光定量PCR方法可以大大提高检测的灵敏度、特异性和准确性。可以用于建立一套生物材料中痕量残留猪源DNA定量qPCR检测方法，有效减少和避免应用于临床或医美医疗器械的安全隐患。
发明内容
本发明的目的是提供一种高灵敏度，高特异性地检测生物材料中猪源DNA残留的引物对，以及包含所述引物对的检测试剂或PCR试剂盒。
本发明的另一目的是提供利用本发明提供的引物对或检测试剂检测生物材料中猪源DNA残留的方法或PCR方法。
在第一方面，本发明提供一种检测猪基因组DNA的引物对，所述引物对包括正向引物和反向引物，其中所述正向引物结合于猪基因组DNA上SEQ ID NO:1所示序列的第2-30位，优选第5-24位；其中的反向引物结合于猪基因组DNA上SEQ ID NO:1所示序列的第120-150位，优选第127-143位；并且所述引物对所扩增获得的扩增产物的长度为139-149bp。
在优选的实施方式中，所述正向引物和反向引物的长度为16～22bp；优选20bp。
在优选的实施方式中，所述正向引物和反向引物的Tm温度为58～60℃，并且正向引物的Tm与反向引物的Tm的差值的绝对值≤2℃。
在具体的实施方式中，所述引物对中，所述正向引物如SEQ ID NO:2所示，所述反向引物如SEQ ID NO:3所示。
在第二方面，本发明提供一种检测试剂，所述检测试剂包含第一方面所述的引物对。
在优选的实施方式中，所述检测试剂还包含探针。
在优选的实施方式中，所述探针如SEQ ID NO:4所示。
在第三方面，本发明提供一种检测试剂盒，所述检测试剂盒包含第一方面所述的引物对或第二方面所述的检测试剂，还任选包含利用所述引物对或检测试剂检测猪源DNA的使用说明书。
在具体的实施方式中，所述检测试剂还包含探针。
在优选的实施方式中，所述探针如SEQ ID NO:4所示。
在具体的实施方式中，所述引物对中正向引物如SEQ ID NO:2所示，所述反向引物如SEQ ID NO:3所示，所述探针如SEQ ID NO:4所示。
在优选的实施方式中，所述检测试剂的检测灵敏度为10fg/反应。
在第四方面，本发明提供一种检测猪基因组DNA的方法，所述方法包括：利用第一方面所述的引物对或第二方面所述的检测试剂或第三方面所述的检测试剂盒，对待测样品进行PCR，并检测PCR扩增产物。
在第五方面，本发明提供一种PCR试剂盒，所述试剂盒中包括容器以及位于所述容器中的第一方面所述的引物对。
在优选的实施方式中，所述正向引物和反向引物的长度为16～22bp；优选20bp。
在优选的实施方式中，所述正向引物和反向引物的Tm温度为58～60℃，并且正向引物的Tm与反向引物的Tm的差值的绝对值≤2℃。
在优选的实施方式中，所述试剂盒中还装有探针。
在优选的实施方式中，所述探针如SEQ ID NO:4所示。
在优选的实施方式中，所述检测试剂的检测灵敏度为10fg/反应。
在优选的实施方式中，所述试剂盒中还装有标准品对照。
在第六方面，本发明提供一种PCR方法，包括步骤：
在一PCR检测体系中，利用第一方面所述的引物对扩增目标产物。
在优选的实施方式中，所述正向引物和反向引物的长度为16～22bp；优选20bp。
在优选的实施方式中，所述正向引物和反向引物的Tm温度为58～60℃，并且正向引物的Tm与反向引物的Tm的差值的绝对值≤2℃。
在优选的实施方式中，所述试剂盒中还装有探针。
在优选的实施方式中，所述探针如SEQ ID NO:4所示。
在第七方面，本发明提供第一方面所述的引物对或第二方面所述的检测试剂或第三方面所述的检测试剂盒的用途，用于检测待测对象中是否存在猪基因组DNA。
在优选的实施方式中，所述待测对象是猪源脱细胞基质；优选来源于猪的真皮、膀胱、小肠和心包膜的脱细胞基质，包括但不限于生物疝修补片、口腔修复膜、生物补片、创面敷料、胶原蛋白、心包生物瓣膜、眼科植入角膜、人工皮肤、医用缝合材料等。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了参考品的扩增曲线，其中利用的引物对是SEQ ID NO:2和SEQ ID NO:3。
图2显示了参考品的标准曲线，其中利用的引物对是SEQ ID NO:2和SEQ ID NO:3。
图3-5分别显示了灵敏度测定中浓度3fg/μL、1fg/μL和0.2fg/μL的扩增曲线，其中利用的引物对是SEQ ID NO:2和SEQ ID NO:3。
具体实施方式
经过深入而广泛的研究，本发明人出乎意料地发现针对猪基因组中的SEQ ID NO:1所示序列设计的引物，不仅能够高度灵敏地检测猪源DNA残留，还能区分牛源等等的干扰性DNA。本发明方法操作简便快捷、特异性和灵敏性高。在此基础上完成了本发明。
本发明的引物对
本文所用的术语“引物”具有本领域技术人员常规理解的意义。本发明的猪基因组DNA特异性引物不是针对外源基因本身或病毒载体本身设计，而是针对猪基因组DNA上SEQ ID NO:1所示区段设计的。换言之，本发明的引物可以特异性结合于猪基因组DNA上SEQ ID NO:1所示区段。
鉴于本发明的教导和本领域的公知常识，本领域技术人员应该理解，针对SEQ ID NO:1所示区段可以设计多种引物对。因此，本发明的引物对不限于实施例中具体得到的引物对。
在具体的实施方式中，本发明的正向引物结合于SEQ ID NO:1所示序列的第2-30位，优选第5-24位；其中的反向引物结合于SEQ ID NO:1所示序列的第120-150位，优选第127-143位，并且所述引物对所扩增获得的扩增产物的长度为139-149bp。
在优选的实施方式中，所述正向引物和反向引物的长度为16～22bp；优选20bp。
在优选的实施方式中，所述正向引物和反向引物的Tm温度为58～60℃，并且正向引物的Tm与反向引物的Tm的差值的绝对值≤2℃。
在具体的实施方式中，本发明的引物对中，所述正向引物如SEQ ID NO:2所示，所述反向引物如SEQ ID NO:3所示。
探针
本文所用的术语“引物”具有本领域技术人员常规理解的意义，即，一小段单链DNA或者RNA片段，用于检测与其互补的核酸序列。
鉴于本发明的教导和本领域的公知常识，本领域技术人员应该理解，在知晓引物对的前提下，本领域技术人员可以根据正向引物和反向引物结合位点之间的模板序列自主设计探针，并检测该探针与引物对的技术效果。在具体的实施方式中，本领域普通技术人员可根据需要具体设计探针，所述探针可以处于液相中，也可以固定于固相上；可以在扩增前结合，也可以在扩增后结合。因此，本发明的探针并不限于实施例中具体公开的探针。本发明的引物对也不限于与实施例中具体公开的探针配对使用。
在具体的实施方式中，本发明的探针如SEQ ID NO:4所示。
本发明的检测试剂
本发明还提供一种检测猪基因组DNA的检测试剂，所述检测试剂包含本发明的引物对以及探针等实施PCR所需的其它成分，例如Taq酶、dNTP、Mg2+等等。
在具体的实施方式中，本发明的检测试剂包含SEQ ID NO:2所示正向引物、SEQ ID NO:3所示反向引物、SEQ ID NO:4所示探针。
在具体的实施方式中，本发明检测试剂的检测灵敏度达到10fg/反应。
在本发明的引物对或检测试剂的基础上，本发明进一步提供一种检测猪基因组DNA的方法，所述方法包括：利用本发明的引物对或检测试剂，对待测样品进行PCR，并检测PCR扩增产物。
在本发明的引物对的基础上，本发明还提供一种PCR试剂盒，所述试剂盒中包括容器以及位于所述容器中的上述本发明引物对或检测试剂。
在具体的实施方式中，本发明的PCR试剂盒还装有用于实施PCR的其它所需成分以及使用该试剂盒作PCR检测的使用说明书。在优选的实施方式中，所述试剂盒中还装有标准品对照。
在本发明的引物对的基础上，本发明还提供利用本发明的引物对扩增目标产物的PCR方法。
生物材料
本发明的引物对、检测试剂或检测试剂盒可以用于检测生物材料中的猪DNA残留。本文所述的生物材料是生物源细胞外基质。在优选的实施方式中，所述生物源细胞外基质是猪源脱细胞基质。在具体的实施方式中，所述猪源脱细胞基质来源于猪的真皮、膀胱、小肠和心包膜的脱细胞基质，包括但不限于生物疝修补片、口腔修复膜、生物补片、创面敷料、胶原蛋白、心包生物瓣膜、眼科植入角膜、人工皮肤、医用缝合材料等。
本发明的优点包括：
1.本发明的引物对或检测试剂能够高度灵敏地检测猪基因组DNA；
2.本发明的引物对或检测试剂能够区分牛、CHO、Vero、人、NS0、MDCK、E.coli、毕赤酵母和Sf9等干扰性DNA；
3.本发明的检测方法操作简便快捷、特异性和灵敏性高。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(Cold Spring Harbor Laboratory Press，2001)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例
材料与方法
1.猪基因组DNA参考品来自湖州申科生物技术有限公司。
2.样本前处理试剂
样本前处理试剂来自湖州申科生物技术有限公司，包含蛋白酶K，蛋白酶K缓冲液，磁珠，结合液，糖原，酵母tRNA，洗涤液A，洗涤液B，洗脱液。
3.qPCR检测体系
qPCR检测试剂盒来自湖州申科生物技术有限公司，包含qPCR Reaction buffer，DNA稀释液，引物，探针，DNA标准品，内部质控。
4.仪器：Applied biosystems 7500荧光定量PCR仪。
实验操作及过程
1.样本前处理
1)精确称重样本5～10mg并记录，放进1.5mL DNase-free无菌离心管内。
2)将样本尽量处理成小碎片后，分别加入蛋白酶K缓冲液1000μL，蛋白酶K 200μL，加DEPC水至最终体积为2000μL。56℃金属浴消化1h，至待测样品完全消化，无肉眼可见颗粒样物为止。
3)消化后取200μL样本加入工作结合液200μL，糖原9μL，酵母tRNA 0.2μL，混匀。
4)加入200μL异丙醇，30μL磁珠，振荡混匀5min，快速离心10s，静置于磁力架。待溶液澄清，磁珠完全分离后，轻轻打开管盖，小心吸出上清。
5)加入700μL洗涤液A，振荡混匀，然后快速离心10s，静置于磁力架。待溶液澄清，磁珠完全分离后，吸出上清。
6)加入700μL洗涤液B，振荡混匀，然后快速离心10s，静置于磁力架。待溶液澄清，磁珠完全分离后，吸出上清。
7)再次快速离心10s，静置于磁力架，待磁珠完全分离后，将残余液体去除干净。
8)打开管盖在室温下干燥5s～10s，除去残留乙醇。在干燥过程中注意观察，勿让磁珠太干，以免洗脱时磁珠不会完全溶解。
9)加入150μL洗脱液，轻微震荡使磁珠和洗脱液混匀，70℃水浴7min，水浴过程中每2min震荡一次，振荡混匀2～3次。
10)快速离心10s，静置于磁力架，待磁珠分离后，转移上清液到新离心管中。
2.检测
2.1参考品准备
参考品是猪肾细胞提取的基因组DNA，浓度为100ng/μL。
2.2标准曲线样品准备
用DNA稀释液将参考品进行10倍梯度稀释，配制成3000pg/μL、300pg/μL、30pg/μL、3pg/μL、0.3pg/μL、0.03pg/μL的标准品溶液。
2.3qPCR反应体系
检测体系30μL：20μL qPCR反应液+10μL模板。
分别取待测样本，无模板对照和标准曲线样本10μL，加到20μL的qPCR反应液中，设置3个复孔。加样完成后，短时间快速离心3s后放入qPCR仪中。设置反应程序：95℃预变性10min；95℃15s，60℃40s，40个循环；反应体积30μL。
2.4标准曲线绘制
扩增结束后，设置阈值线，读取标准曲线的扩增效率、斜率、R2以及各样品的检测值。
2.5结果判定
a)标准曲线方程的相关系数R2应大于0.990，斜率在-3.1～-3.8(即扩增效率在83.3％～110％)。
b)无模板对照(NTC)的Ct值未检出或者≥35。
实施例1.引物对和探针的设计
本发明人利用以下靶标序列设计用于检测猪源残留DNA的引物对和探针：
1.靶标序列
qPCR靶向序列选择猪基因组中的以下序列：
2.引物和探针的设计
针对SEQ ID NO:1所示序列，本发明人设计了以下引物对和探针：
正向引物：AGTTCCCATCGTGGCTCAGT(SEQ ID NO:2)；
反向引物：GGATCCGAGCCGCATCT(SEQ ID NO:3)；
探针：FAM-ATCCATGAGGATGCAGATT-MGB(SEQ ID NO:4)。
实施例2.线性范围的检测
本发明人检测了本发明的引物对的线性范围。具体实验过程如上文“实验操作及过程”所述。
试验结果证明，猪源DNA检测的浓度范围在30ng/反应～300fg/反应(见表1)，标曲参数R2：0.999，扩增效率为99.16％(如图1，2所示)。
表1.标准曲线的扩增Ct值均值

实施例3.专属性的检测
选取多个物种细胞牛、CHO、Vero、人、NS0、MDCK、E.coli、毕赤酵母和Sf9的DNA作干扰实验，以30ng/反应的干扰DNA为模板进行qPCR扩增。结果显示9种干扰DNA(30ng/反应)均无检测Ct值(见表2)，qPCR检测具有较好的特异性。
表2.9种干扰DNA的检测Ct值结果
实施例4.检测灵敏度的测定
1.在本实施例中，本发明人测定了本发明的检测体系的灵敏度。
将猪基因组DNA参考品分别稀释到浓度3fg/μL，1fg/μL和0.2fg/μL，每个浓度检测8复孔，重复3次独立实验，统计共24次检测数据。结果显示1fg/μL的24次检测结果均有检测值和明显的扩增曲线(见表3，图3-5)。检测灵敏度能达到1fg/μL即10fg/反应。
表3.灵敏度24次检测数据结果

实施例5.准确性
使用猪基因组DNA配置3个不同浓度(300pg/μL，3pg/μL，0.06pg/μL)的加标样本，对样品进行提取并qPCR检测计算加标回收率。每个浓度重复检测3次。结果显示加标回收率为78.8～96.7％，CV为4.4～13.0％(见表4)。
表4.准确性检测结果
实施例6.精密度
精密度包含重复性和中间精密度，重复性使用猪基因组DNA配置3pg/μL的加标样本测试，重复检测6次，检测结果的CV值为13.5％(见表5)。中间精密度测试使用猪基因组DNA配置3个不同浓度(300pg/μL，3pg/μL，0.06pg/μL)样本，由不同实验人员对每个样品进行共3次独立qPCR定量检测，每次检测重复3次。检测CV为11.0～27.2％(见表6)。
表5.重复性检测结果

表6.中间精密度检测结果
实施例7.实际样本的检测
在本实施例中，利用本发明的检测体系对生物材料样本可吸收生物膜和猪I型胶原冻干品中猪源DNA残留进行定量检测。结果表明可吸收生物膜提取样本中残留猪源DNA检测值低于标曲的定量范围，猪I型胶原冻干品中猪源DNA的残留量为0.2ng/mg(见表7)。
表7.实际样本检测结果
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种检测猪基因组DNA的引物对，所述引物对包括正向引物和反向引物，其中所述正向引物结合于猪基因组DNA上SEQ ID NO:1所示序列的第2-30位，优选第5-24位；其中的反向引物结合于猪基因组DNA上SEQ ID NO:1所示序列的第120-150位，优选第127-143位；并且所述引物对所扩增获得的扩增产物的长度为139-149bp。
  2. 如权利要求1所述的引物对，其特征在于，所述引物对中，所述正向引物如SEQ ID NO:2所示，所述反向引物如SEQ ID NO:3所示。
  3. 一种检测试剂，所述检测试剂包含权利要求1或2所述的引物对。
  4. 一种检测试剂盒，所述检测试剂盒包含权利要求1或2所述的引物对或权利要求3所述的检测试剂，还任选包含利用所述引物对或检测试剂检测猪源DNA的使用说明书。
  5. 如权利要求4所述的检测试剂盒，其特征在于，所述检测试剂还包含探针。
  6. 如权利要求5所述的检测试剂盒，其特征在于，所述引物对中正向引物如SEQ ID NO:2所示，所述反向引物如SEQ ID NO:3所示，所述探针如SEQ ID NO:4所示。
  7. 一种检测猪基因组DNA的方法，所述方法包括：利用权利要求1或2所述的引物对或权利要求3所述的检测试剂或权利要求4-6中任一项所述的检测试剂盒，对待测样品进行PCR，并检测PCR扩增产物。
  8. 一种PCR试剂盒，所述试剂盒中包括容器以及位于所述容器中的权利要求1或2所述的引物对。
  9. 一种PCR方法，包括步骤：
    在一PCR检测体系中，利用权利要求1或2所述的引物对扩增目标产物。
  10. 权利要求1或2所述的引物对或权利要求3所述的检测试剂或权利要求4-6中任一项所述的检测试剂盒的用途，用于检测待测对象中是否存在猪基因组DNA。
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