CN110540988A - A compound amplification system for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood and the primer combination used therefor - Google Patents

A compound amplification system for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood and the primer combination used therefor Download PDF

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CN110540988A
CN110540988A CN201910822152.2A CN201910822152A CN110540988A CN 110540988 A CN110540988 A CN 110540988A CN 201910822152 A CN201910822152 A CN 201910822152A CN 110540988 A CN110540988 A CN 110540988A
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赵一霞
孙启凡
季安全
胡胜
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Institute of Forensic Science Ministry of Public Security PRC
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Abstract

the invention discloses a composite amplification system for identifying whether body fluid to be detected is non-blood, menstrual blood or peripheral blood and a primer combination used by the same. The primer combination consists of 12 DNA molecules shown in a sequence 1 to a sequence 12 in a sequence table. The primer combination is adopted to construct a composite amplification system, so that whether the body fluid to be detected is non-blood, menstrual blood or peripheral blood can be identified, and the accuracy is higher. The invention provides accurate scientific basis for determining case property, conviction and sentencing, and has important application value.

Description

一种鉴定待测体液为非血液、月经血还是外周血的复合扩增 体系及其使用的引物组合A compound amplification method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood The system and the primer combinations used

技术领域technical field

本发明属于法医技术学领域,具体涉及一种鉴定待测体液为非血液、月经血还是外周血的复合扩增体系及其使用的引物组合。The invention belongs to the field of forensic technology, and in particular relates to a compound amplification system for identifying whether a body fluid to be tested is non-blood, menstrual blood or peripheral blood and a primer combination used therefor.

背景技术Background technique

通过鉴别案发现场体液斑迹的组织来源,可推断样本组织来源及其真实犯罪活动,从而为案件定性及现场重建提供重要信息,为法庭科学提供重要的科学证据。案件现场常见的体液包括外周血、月经血、唾液、精液、阴道分泌液等。其中,外周血和月经血虽然均含有大量红细胞,但与外周血相比,月经血中还含有大量的子宫内膜碎片、子宫颈粘液和阴道分泌物等混合物。现场血液类检材是属于外周血还是月经血是非常重要的法庭科学问题,往往对案件定性及重建案发现场、提供法庭证据具有十分重要的意义;尤其是针对性侵案件、女性失踪案以及某些伤害案中,女性受害者称因受暴力攻击出现血尿,在这种情况下,需要确定尿液中的血细胞来源是否存在月经血污染。因此,月经血与外周血的两种血液类体液间的鉴别区分仍是法医学体液鉴别的重点及难点。传统的血痕鉴定方法主要是生化和免疫学分析法,即利用血液中的血红蛋白及其衍生物进行鉴别,但此类方法不仅费时费力,而且非常消耗检材,容易出现假阳性或假阴性结果。从犯罪现场中获取的微量检材亟需一种更为灵敏、准确的鉴定方法进行鉴别。By identifying the tissue source of body fluid stains at the crime scene, the source of sample tissue and its real criminal activities can be inferred, thereby providing important information for case characterization and scene reconstruction, and important scientific evidence for forensic science. Common bodily fluids at the scene of the case include peripheral blood, menstrual blood, saliva, semen, and vaginal secretions. Among them, although both peripheral blood and menstrual blood contain a large number of red blood cells, compared with peripheral blood, menstrual blood also contains a large number of mixtures such as endometrial fragments, cervical mucus, and vaginal secretions. Whether the on-site blood samples belong to peripheral blood or menstrual blood is a very important forensic scientific issue, which is often of great significance to the characterization of the case, reconstruction of the scene of the case, and provision of court evidence; especially for rape cases, cases of missing women and In some injury cases, female victims claim to have hematuria due to violent assault, in which case it is necessary to determine whether the source of blood cells in the urine is contaminated with menstrual blood. Therefore, the distinction between menstrual blood and peripheral blood, two blood-like body fluids, is still the focus and difficulty of forensic body fluid identification. Traditional bloodstain identification methods are mainly biochemical and immunological analysis methods, that is, using hemoglobin and its derivatives in blood for identification, but such methods are not only time-consuming and laborious, but also consume a lot of sample materials, and are prone to false positive or false negative results. A more sensitive and accurate identification method is urgently needed for the identification of trace samples obtained from crime scenes.

大量研究表明,基于分子生物学水平的mRNA标记检测有望成为体液斑迹组织属性来源鉴定的有效方法。该方法依赖于不同体液中mRNA的独特表达谱来鉴定体液,具有特异性强、灵敏度高、只需要微量检材等优势,并且兼容当前的DNA/RNA共提技术,即从生物检材中同时提取DNA和mRNA,其中DNA用于多态分型,mRNA用于组织来源鉴定,既可提高工作效率,又使微量检材得到充分利用。欧洲DNA分型小组的研究表明,在没有RNA检测经验的实验室中依然可以用mRNA分析方法成功鉴定血迹,将不同体液混合后,进行RT-PCR复合扩增,成功获得各体液组织特异性的mRNA基因位点检测结果,并对体液来源进行了准确判断。A large number of studies have shown that the detection of mRNA markers based on molecular biology is expected to become an effective method for the identification of the source of tissue properties of body fluid stains. This method relies on the unique expression profiles of mRNA in different body fluids to identify body fluids. It has the advantages of strong specificity, high sensitivity, and requires only a small amount of sample material. It is also compatible with the current DNA/RNA co-extraction technology, that is, simultaneous extraction from biological samples Extract DNA and mRNA, in which DNA is used for polymorphic typing, and mRNA is used for tissue source identification, which can not only improve work efficiency, but also make full use of trace samples. Research by the European DNA Typing Group has shown that in laboratories with no experience in RNA detection, mRNA analysis can still be used to successfully identify blood stains. After mixing different body fluids, RT-PCR multiplex amplification is performed, and the specificity of each body fluid tissue is successfully obtained. mRNA gene locus detection results, and accurate judgment on the source of body fluids.

发明内容Contents of the invention

本发明的目的是鉴定待测体液为非血液、月经血还是外周血。The purpose of the present invention is to identify whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood.

本发明首先保护引物组合,可包括引物组合甲和引物组合乙。The present invention first protects the primer combination, which may include primer combination A and primer combination B.

所述引物组合甲可包括引物1、引物2、引物3和引物4;The primer combination A may include primer 1, primer 2, primer 3 and primer 4;

所述引物1可为序列表中的序列1所示的单链DNA分子;The primer 1 can be a single-stranded DNA molecule shown in sequence 1 in the sequence listing;

所述引物2可为序列表中的序列2所示的单链DNA分子;The primer 2 can be a single-stranded DNA molecule shown in sequence 2 in the sequence listing;

所述引物3可为序列表中的序列3所示的单链DNA分子;The primer 3 can be a single-stranded DNA molecule shown in sequence 3 in the sequence listing;

所述引物4可为序列表中的序列4所示的单链DNA分子。The primer 4 can be a single-stranded DNA molecule shown in sequence 4 in the sequence listing.

所述引物组合乙可包括引物5、引物6、引物7和引物8;The primer combination B may include primer 5, primer 6, primer 7 and primer 8;

所述引物5可为序列表中的序列5所示的单链DNA分子;The primer 5 can be a single-stranded DNA molecule shown in sequence 5 in the sequence listing;

所述引物6可为序列表中的序列6所示的单链DNA分子;The primer 6 can be a single-stranded DNA molecule shown in sequence 6 in the sequence listing;

所述引物7可为序列表中的序列7所示的单链DNA分子;The primer 7 can be a single-stranded DNA molecule shown in sequence 7 in the sequence listing;

所述引物8可为序列表中的序列8所示的单链DNA分子。The primer 8 can be a single-stranded DNA molecule shown in sequence 8 in the sequence listing.

所述引物1、所述引物3、所述引物5和所述引物7均用荧光标记。The primer 1, the primer 3, the primer 5 and the primer 7 are all labeled with fluorescent light.

所述引物1、所述引物3、所述引物5和所述引物7的5’末端均用荧光标记。The 5' ends of the primer 1, the primer 3, the primer 5 and the primer 7 are all fluorescently labeled.

所述荧光标记具体可为FAM标记。The fluorescent label can specifically be a FAM label.

所述引物组合甲具体可由所述引物1、所述引物2、所述引物3和所述引物4组成。The primer combination A may specifically consist of the primer 1, the primer 2, the primer 3 and the primer 4.

所述引物组合乙具体可由所述引物5、所述引物6、所述引物7和所述引物8组成。The primer combination B may specifically consist of the primer 5, the primer 6, the primer 7 and the primer 8.

所述引物组合具体可由所述引物组合甲和所述引物组合乙组成。The primer combination may specifically consist of the primer combination A and the primer combination B.

本发明还保护所述引物组合甲。The present invention also protects the primer combination A.

本发明还保护所述引物组合乙。The present invention also protects the primer combination B.

上述任一所述引物组合还可包括引物组合丙;所述引物组合丙可包括引物9、引物10、引物11和引物12;Any of the primer combinations described above may also include primer combination C; the primer combination C may include primer 9, primer 10, primer 11 and primer 12;

所述引物9可为序列表中的序列9所示的单链DNA分子;The primer 9 can be a single-stranded DNA molecule shown in sequence 9 in the sequence listing;

所述引物10可为序列表中的序列10所示的单链DNA分子;The primer 10 can be a single-stranded DNA molecule shown in sequence 10 in the sequence listing;

所述引物11可为序列表中的序列11所示的单链DNA分子;The primer 11 can be a single-stranded DNA molecule shown in sequence 11 in the sequence listing;

所述引物12可为序列表中的序列12所示的单链DNA分子。The primer 12 can be a single-stranded DNA molecule shown in sequence 12 in the sequence listing.

所述引物组合丙具体可由所述引物9、所述引物10、所述引物11和所述引物12组成。The primer combination C may specifically consist of the primer 9, the primer 10, the primer 11 and the primer 12.

所述引物组合具体可由所述引物组合甲、所述引物组合乙和所述引物组合丙组成。The primer combination may specifically consist of the primer combination A, the primer combination B and the primer combination C.

上述任一所述引物组合甲还可包括所述引物组合丙。Any of the above-mentioned primer combination A may also include the primer combination C.

所述引物组合甲具体可由所述引物1、所述引物2、所述引物3、所述引物4和所述引物组合丙组成。The primer combination A may specifically consist of the primer 1, the primer 2, the primer 3, the primer 4 and the primer combination C.

上述任一所述引物组合乙还可包括所述引物组合丙。Any of the above-mentioned primer combinations B may also include the primer combination C.

所述引物组合乙具体可由所述引物5、所述引物6、所述引物7、所述引物8和所述引物组合丙组成。The primer combination B may specifically consist of the primer 5, the primer 6, the primer 7, the primer 8 and the primer combination C.

上文中,引物1、引物2、引物3、引物4、引物5、引物6、引物7、引物8、引物9、引物10、引物11和引物12的摩尔比可为0.01:0.01:0.01:0.01:4.0:4.0:5.0:5.0:0.2:0.2:1.0:1.0。Above, the molar ratio of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11 and primer 12 can be 0.01:0.01:0.01:0.01 :4.0:4.0:5.0:5.0:0.2:0.2:1.0:1.0.

本发明还保护一种复合扩增体系,包括上述任一所述引物组合、或、上述任一所述引物组合甲、或、上述任一所述引物组合乙。The present invention also protects a composite amplification system, comprising any of the above-mentioned primer combinations, or, any of the above-mentioned primer combination A, or, any of the above-mentioned primer combination B.

所述复合扩增体系具体可由上述任一所述引物组合组成。The multiplex amplification system can be specifically composed of any one of the primer combinations described above.

所述复合扩增体系具体可由上述任一所述引物组合甲组成。The multiplex amplification system can be specifically composed of any one of the above-mentioned primer sets A.

所述复合扩增体系具体可由上述任一所述引物组合乙组成。The multiplex amplification system can be specifically composed of any one of the above-mentioned primer combinations B.

上述复合扩增体系中,所述引物1、所述引物2、所述引物3和所述引物4在所述复合扩增体系中的浓度为0.01μM。所述引物5和所述引物6在所述复合扩增体系中的浓度为4.0μM。所述引物7和所述引物8在所述复合扩增体系中的浓度为5.0μM。所述引物9和所述引物10在所述复合扩增体系中的浓度为0.2μM。所述引物11和所述引物12在所述复合扩增体系中的浓度为1.0μM。In the above multiple amplification system, the concentration of the primer 1, the primer 2, the primer 3 and the primer 4 in the multiple amplification system is 0.01 μM. The concentration of the primer 5 and the primer 6 in the multiplex amplification system is 4.0 μM. The concentration of the primer 7 and the primer 8 in the multiplex amplification system is 5.0 μM. The concentration of the primer 9 and the primer 10 in the multiplex amplification system is 0.2 μM. The concentration of the primer 11 and the primer 12 in the multiplex amplification system is 1.0 μM.

上述复合扩增体系中,所述复合扩增体系还可包括进行PCR扩增反应所需的试剂;所述“进行PCR扩增反应所需的试剂”不包括PCR扩增反应所需的引物。In the above composite amplification system, the composite amplification system may also include reagents required for PCR amplification reaction; the "reagents required for PCR amplification reaction" does not include primers required for PCR amplification reaction.

所述“进行PCR扩增反应所需的试剂”可包括DNA聚合酶、dNTP和Mg2+中的至少一种。所述DNA聚合酶在所述复合扩增体系中的浓度可为0.5U/μL。所述dNTP在所述复合扩增体系中的浓度可为0.2mmol/L(即dATP、dTTP、dCTP和dGTP的浓度均为0.2mmol/L)。所述Mg2+在所述复合扩增体系中的浓度可为1.5mmol/L。The "reagents required for PCR amplification reaction" may include at least one of DNA polymerase, dNTP and Mg 2+ . The concentration of the DNA polymerase in the multiplex amplification system may be 0.5U/μL. The concentration of the dNTP in the multiplex amplification system may be 0.2mmol/L (that is, the concentrations of dATP, dTTP, dCTP and dGTP are all 0.2mmol/L). The concentration of the Mg 2+ in the multiple amplification system may be 1.5mmol/L.

所述复合扩增体系具体可由上述任一所述引物组合和进行PCR扩增反应所需的试剂组成。The multiplex amplification system may be specifically composed of any one of the primer combinations described above and reagents required for PCR amplification reactions.

所述复合扩增体系具体可由上述任一所述引物组合甲和进行PCR扩增反应所需的试剂组成。The multiplex amplification system can be specifically composed of any one of the above-mentioned primer sets A and reagents required for PCR amplification reaction.

所述复合扩增体系具体可由上述任一所述引物组合乙和进行PCR扩增反应所需的试剂组成。The multiplex amplification system can be specifically composed of any one of the above-mentioned primer combinations B and reagents required for PCR amplification reaction.

本发明还保护含有上述任一所述引物组合的试剂盒甲;所述试剂盒甲用于鉴定待测体液为非血液、月经血还是外周血。The present invention also protects the kit A containing any one of the above-mentioned primer combinations; the kit A is used for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood.

本发明还保护含有上述任一所述引物组合甲的试剂盒乙;所述试剂盒乙用于鉴定待测体液为血液还是非血液。The present invention also protects the kit B containing any one of the above-mentioned primer combinations A; the kit B is used to identify whether the body fluid to be tested is blood or non-blood.

本发明还保护含有上述任一所述引物组合乙的试剂盒丙;所述试剂盒丙用于鉴定待测血液为月经血还是外周血。The present invention also protects the kit C containing any one of the above-mentioned primer combinations B; the kit C is used to identify whether the blood to be tested is menstrual blood or peripheral blood.

上述任一所述复合扩增体系、试剂盒甲、试剂盒乙或试剂盒丙的制备方法也属于本发明的保护范围。The preparation method of any one of the above multiplex amplification systems, kit A, kit B or kit C also belongs to the scope of protection of the present invention.

上述任一所述复合扩增体系、试剂盒甲、试剂盒乙或试剂盒丙的制备方法,可包括将上述任一所述引物组合、上述任一所述引物组合甲或上述任一所述引物组合乙中的各条引物单独包装的步骤。The preparation method of any of the above-mentioned composite amplification systems, kit A, kit B or kit C may include combining any of the above-mentioned primer combinations, any of the above-mentioned primer combination A or any of the above-mentioned The step of individually packaging each primer in primer combination B.

下述X1)或X2)或X3)也属于本发明的保护范围。The following X1) or X2) or X3) also belong to the protection scope of the present invention.

X1)上述任一所述引物组合、或、上述任一所述复合扩增体系(尤其指包括上述任一所述引物组合的复合扩增体系)在鉴定待测体液为非血液、月经血还是外周血中的应用。X1) Any of the above-mentioned primer combinations, or, any of the above-mentioned composite amplification systems (especially referring to the composite amplification system including any of the above-mentioned primer combinations) is useful in identifying whether the body fluid to be tested is non-blood, menstrual blood or Application in peripheral blood.

X2)上述任一所述引物组合甲、或、上述任一所述复合扩增体系(尤其指包括上述任一所述引物组合甲的复合扩增体系)在鉴定待测体液为血液还是非血液中的应用。X2) Any of the above-mentioned primer combination A, or, any of the above-mentioned composite amplification systems (especially referring to the above-mentioned composite amplification system including any of the above-mentioned primer combination A) in identifying whether the body fluid to be tested is blood or non-blood in the application.

X3)上述任一所述引物组合乙、或、上述任一所述复合扩增体系(尤其指包括上述任一所述引物组合乙的复合扩增体系)在鉴定待测血液为月经血还是外周血中的应用。X3) Any of the above-mentioned primer combinations B, or, any of the above-mentioned composite amplification systems (especially referring to the composite amplification system including any of the above-mentioned primer combinations B) is useful in identifying whether the blood to be tested is menstrual blood or peripheral application in blood.

本发明还保护S1)或S2)或S3)。The invention also protects S1) or S2) or S3).

S1)一种鉴定待测体液为非血液、月经血还是外周血的方法,可包括如下步骤:以待测体液的cDNA为模板,分别采用引物对1、引物对2、引物对3和引物对4进行PCR扩增,得到PCR扩增产物;所述引物对1由所述引物1和所述引物2组成;所述引物对2由所述引物3和所述引物4组成;所述引物对3由所述引物5和所述引物6组成;所述引物对4由所述引物7和所述引物8组成;然后进行如下判断:S1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, using primer pair 1, primer pair 2, primer pair 3 and primer pair respectively 4. Perform PCR amplification to obtain a PCR amplification product; the primer pair 1 is composed of the primer 1 and the primer 2; the primer pair 2 is composed of the primer 3 and the primer 4; the primer pair 3 is composed of the primer 5 and the primer 6; the primer pair 4 is composed of the primer 7 and the primer 8; and then judged as follows:

如果采用所述引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用所述引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,则该待测体液为或疑似为月经血;If the PCR amplification product obtained by using the primer pair 1 contains a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 contains a DNA fragment with a size of 159bp, and the primer pair 3 is used to obtain If the PCR amplification product contains a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using the primer pair 4 contains a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be menstrual blood;

如果采用所述引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用所述引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,则该待测体液为或疑似为外周血;If the PCR amplification product obtained by using the primer pair 1 contains a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 contains a DNA fragment with a size of 159bp, and the primer pair 3 is used to obtain If the PCR amplification product does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using the primer pair 4 does not contain a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be peripheral blood;

如果采用所述引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,采用所述引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,则该待测体液为或疑似为非血液。If the PCR amplification product obtained by using the primer pair 1 does not contain a DNA fragment with a size of 243bp, and the PCR amplification product obtained with the primer pair 2 does not contain a DNA fragment with a size of 159bp, use the primer pair If the PCR amplification product obtained in 3 does not contain a DNA fragment with a size of 126 bp, and the PCR amplification product obtained with the primer pair 4 does not contain a DNA fragment with a size of 272 bp, then the body fluid to be tested is or is suspected to be non-blood .

S2)一种鉴定待测体液为血液还是非血液的方法,可包括如下步骤:以待测体液的cDNA为模板,分别采用所述引物对1和所述引物对2进行PCR扩增,得到PCR扩增产物;然后进行如下判断:S2) A method for identifying whether the body fluid to be tested is blood or non-blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, respectively using the primer pair 1 and the primer pair 2 to perform PCR amplification to obtain PCR Amplify the product; then judge as follows:

如果采用所述引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,则待测体液为或疑似为血液;If the PCR amplification product obtained by using the primer pair 1 contains a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 contains a DNA fragment with a size of 159bp, then the body fluid to be tested is or is suspected to be for blood;

如果采用所述引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,则该待测体液为或疑似为非血液。If the PCR amplification product obtained by using the primer pair 1 does not contain a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 does not contain a DNA fragment with a size of 159bp, then the body fluid to be tested Is or is suspected to be non-blood.

S3)一种鉴定待测血液为月经血还是外周血的方法,可包括如下步骤:以待测血液的cDNA为模板,分别采用所述引物对3和所述引物对4进行PCR扩增,得到PCR扩增产物;然后进行如下判断:S3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the blood to be tested as a template, respectively using the primer pair 3 and the primer pair 4 to perform PCR amplification to obtain PCR amplification product; then judge as follows:

如果采用所述引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,则该待测血液为或疑似为月经血;If the PCR amplification product obtained by using the primer pair 3 contains a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using the primer pair 4 contains a DNA fragment with a size of 272bp, then the blood to be tested is or Suspected menstrual blood;

如果采用所述引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,则该待测血液为或疑似为外周血。If the PCR amplification product obtained by using the primer pair 3 does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using the primer pair 4 does not contain a DNA fragment with a size of 272bp, then the blood to be tested Is or is suspected to be peripheral blood.

本发明还保护T1)或T2)或T3)。The invention also protects T1) or T2) or T3).

T1)一种鉴定待测体液为非血液、月经血还是外周血的方法,可包括如下步骤:以待测体液的cDNA为模板,分别采用所述引物对1、所述引物对2、所述引物对3、所述引物对4、所述引物对5和所述引物对6进行PCR扩增,得到PCR扩增产物;然后进行如下判断:T1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, using the primer pair 1, the primer pair 2, and the The primer pair 3, the primer pair 4, the primer pair 5 and the primer pair 6 carry out PCR amplification to obtain a PCR amplification product; then judge as follows:

如果采用所述引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用所述引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为月经血;If the PCR amplification product obtained by using the primer pair 1 contains a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 contains a DNA fragment with a size of 159bp, and the primer pair 3 is used to obtain The PCR amplified product contains a DNA fragment with a size of 126bp, the PCR amplified product obtained by using the primer pair 4 contains a DNA fragment with a size of 272bp, and the PCR amplified product obtained with the primer pair 5 contains a DNA fragment with a size is a 250bp DNA fragment, and the PCR amplification product obtained by using the primer pair 6 contains a 291bp DNA fragment, the body fluid to be tested is or is suspected to be menstrual blood;

如果采用所述引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用所述引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为外周血;If the PCR amplification product obtained by using the primer pair 1 contains a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 contains a DNA fragment with a size of 159bp, and the primer pair 3 is used to obtain The PCR amplification product does not contain a DNA fragment with a size of 126bp, the PCR amplification product obtained by using the primer pair 4 does not contain a DNA fragment with a size of 272bp, and the PCR amplification product obtained by using the primer pair 5 Contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using the primer pair 6 contains a DNA fragment with a size of 291bp, then the body fluid to be tested is or is suspected to be peripheral blood;

如果采用所述引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,采用所述引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为非血液。If the PCR amplification product obtained by using the primer pair 1 does not contain a DNA fragment with a size of 243bp, and the PCR amplification product obtained with the primer pair 2 does not contain a DNA fragment with a size of 159bp, use the primer pair 3 The PCR amplification product obtained does not contain a DNA fragment with a size of 126bp, the PCR amplification product obtained by using the primer pair 4 does not contain a DNA fragment with a size of 272bp, and the PCR amplification product obtained by using the primer pair 5 If the product contains a DNA fragment with a size of 250 bp, and the PCR amplification product obtained by using the primer pair 6 contains a DNA fragment with a size of 291 bp, then the body fluid to be tested is or is suspected to be non-blood.

T2)一种鉴定待测体液为血液还是非血液的方法,可包括如下步骤:以待测体液的cDNA为模板,分别采用所述引物对1、所述引物对2、所述引物对5和所述引物对6进行PCR扩增,得到PCR扩增产物;然后进行如下判断:T2) A method for identifying whether the body fluid to be tested is blood or non-blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, using the primer pair 1, the primer pair 2, the primer pair 5 and the primer pair respectively. Described primer is carried out PCR amplification to 6, obtains PCR amplification product; Then carry out following judgment:

如果采用所述引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为血液;If the PCR amplification product obtained by using the primer pair 1 contains a DNA fragment with a size of 243bp, and the PCR amplification product obtained by using the primer pair 2 contains a DNA fragment with a size of 159bp, and the primer pair 5 is used to obtain The PCR amplification product contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using the primer pair 6 contains a DNA fragment with a size of 291bp, then the body fluid to be tested is or is suspected to be blood;

如果采用所述引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用所述引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则该待测体液为或疑似为非血液。If the PCR amplification product obtained by using the primer pair 1 does not contain a DNA fragment with a size of 243bp, and the PCR amplification product obtained with the primer pair 2 does not contain a DNA fragment with a size of 159bp, use the primer pair If the PCR amplification product obtained in 5 contains a DNA fragment with a size of 250 bp, and the PCR amplification product obtained with the primer pair 6 contains a DNA fragment with a size of 291 bp, then the body fluid to be tested is or is suspected to be non-blood.

T3)一种鉴定待测血液为月经血还是外周血的方法,可包括如下步骤:以待测血液的cDNA为模板,分别采用所述引物对3、所述引物对4、所述引物对5和所述引物对6进行PCR扩增,得到PCR扩增产物;然后进行如下判断:T3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the blood to be tested as a template, using the primer pair 3, the primer pair 4, and the primer pair 5 respectively Carry out PCR amplification with described primer pair 6, obtain PCR amplification product; Then carry out following judgment:

如果采用所述引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则该待测血液为或疑似为月经血;If the PCR amplification product obtained by using the primer pair 3 contains a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using the primer pair 4 contains a DNA fragment with a size of 272bp, and the primer pair 5 is used to obtain If the PCR amplification product contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using the primer pair 6 contains a DNA fragment with a size of 291bp, then the blood to be tested is or is suspected to be menstrual blood;

如果采用所述引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用所述引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,采用所述引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用所述引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则该待测血液为或疑似为外周血。If the PCR amplification product obtained by using the primer pair 3 does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained with the primer pair 4 does not contain a DNA fragment with a size of 272bp, use the primer pair If the PCR amplification product obtained in 5 contains a DNA fragment with a size of 250 bp, and the PCR amplification product obtained with the primer pair 6 contains a DNA fragment with a size of 291 bp, then the blood to be tested is or is suspected to be peripheral blood.

上述任一所述待测体液可为外周血、月经血、唾液、精液或阴道分泌液。Any of the aforementioned body fluids to be tested can be peripheral blood, menstrual blood, saliva, semen or vaginal secretions.

上述任一所述待测血液可为外周血或月经血。Any of the blood to be tested above can be peripheral blood or menstrual blood.

上述任一所述血液可为外周血或月经血。Any of the blood mentioned above may be peripheral blood or menstrual blood.

上述任一所述非血液可为唾液、精液或阴道分泌液。Any of the aforementioned non-blood can be saliva, semen or vaginal fluid.

上述任一所述PCR扩增产物可通过毛细管电泳检测。Any of the aforementioned PCR amplification products can be detected by capillary electrophoresis.

在本发明的一个实施例中,从河南地区的80个志愿者(年龄在18-35周岁)中获得20份外周血样本、20份唾液样本、20份精液样本、20份月经血样本和20份阴道分泌液样本。采用本发明提供的复合扩增体系对80个样本进行鉴定,结果如下:20份外周血样本中,75%以上的外周血样本可以检测到HBB基因、HBA基因、GAPDH基因和ACTB基因的表达,且任何外周血样本均没有MMP7基因和MMP10基因的表达;20份月经血样本中,75%以上的月经血样本可以检测到HBA基因、HBB基因、MMP7基因和GAPDH基因的表达,50%-75%的月经血样本可以检测到MMP10基因和ACTB基因的表达;20份精液样本中,75%以上的精液样本可以检测到GAPDH基因和ACTB基因的表达,且任何精液样本均没有HBB基因、HBA基因、MMP7基因和MMP10基因的表达;20份唾液样本中,50%-75%的唾液样本可以检测到GAPDH基因和ACTB基因的表达,且任何唾液样本均没有HBB基因、HBA基因、MMP7基因和MMP10基因的表达;20份阴道分泌液样本中,50%以上的阴道分泌液样本可以检测到GAPDH基因和ACTB基因的表达,1份阴道分泌液样本可以检测到MMP10基因的表达,2份阴道分泌液样本可以检测到MMP7基因的表达。结果表明,本发明提供的复合扩增体系可以鉴定待测体液为血液、外周血还是月经血,还可以鉴定待测血液为外周血还是月经血,均具有较高的准确性。本发明为明确案件性质和定罪量刑等提供准确的科学依据,具有重要的应用价值。In one embodiment of the present invention, 20 peripheral blood samples, 20 saliva samples, 20 semen samples, 20 menstrual blood samples and 20 A sample of vaginal fluid. The multiple amplification system provided by the present invention was used to identify 80 samples, and the results were as follows: among the 20 peripheral blood samples, the expression of HBB gene, HBA gene, GAPDH gene and ACTB gene could be detected in more than 75% of the peripheral blood samples, And there is no expression of MMP7 gene and MMP10 gene in any peripheral blood sample; in 20 menstrual blood samples, more than 75% of menstrual blood samples can detect the expression of HBA gene, HBB gene, MMP7 gene and GAPDH gene, 50%-75 The expression of MMP10 gene and ACTB gene can be detected in % of menstrual blood samples; the expression of GAPDH gene and ACTB gene can be detected in more than 75% of the 20 semen samples, and there is no HBB gene, HBA gene in any semen sample , MMP7 gene and MMP10 gene expression; in 20 saliva samples, GAPDH gene and ACTB gene expression could be detected in 50%-75% of the saliva samples, and any saliva sample had no HBB gene, HBA gene, MMP7 gene and MMP10 gene Gene expression; in 20 vaginal fluid samples, the expression of GAPDH gene and ACTB gene can be detected in more than 50% of vaginal fluid samples, the expression of MMP10 gene can be detected in 1 vaginal fluid sample, and the expression of MMP10 gene can be detected in 2 vaginal fluid samples The sample can detect the expression of MMP7 gene. The results show that the complex amplification system provided by the invention can identify whether the body fluid to be tested is blood, peripheral blood or menstrual blood, and can also identify whether the blood to be tested is peripheral blood or menstrual blood, all of which have high accuracy. The invention provides accurate scientific basis for clarifying the nature of the case, conviction and sentencing, etc., and has important application value.

附图说明Description of drawings

图1为单重PCR扩增特异性检测。Figure 1 is a single-plex PCR amplification specific detection.

图2为多重PCR扩增特异性检测。Figure 2 is the multiplex PCR amplification specific detection.

具体实施方式Detailed ways

以下的实施例便于更好地理解本发明,但并不限定本发明。The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

下述实施例中的实验方法,如无特殊说明,均为常规方法。The experimental methods in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

以下实施例中的定量试验,均设置三次重复实验,结果取平均值。Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

下述实施例中,所有引物均有sangon公司合成。miRNeasy Mini Kit为德国Qiagen公司的产品。III First-Strand Synthesis System为美国Invitrogen公司的产品。HotstarTaq Polymerase为德国Qiagen公司的产品。Hi-Di甲酰胺、分子量内标LIZ600和3500XL遗传分析仪均为美国Applied Biosystems公司的产品。MastercyclerNexus PCR仪为德国Eppendorf公司的产品。Nanodrop 2000c紫外分光光度计为美国ThermoScientific公司的产品。In the following examples, all primers were synthesized by Sangon Company. miRNeasy Mini Kit is a product of Qiagen, Germany. III First-Strand Synthesis System is a product of Invitrogen Corporation of the United States. HotstarTaq Polymerase is a product of Qiagen, Germany. Hi-Di formamide, molecular weight internal standard LIZ600 and 3500XL genetic analyzer are all products of Applied Biosystems, USA. The MastercyclerNexus PCR instrument is a product of Eppendorf, Germany. Nanodrop 2000c ultraviolet spectrophotometer is a product of American ThermoScientific Company.

实施例1、基于6个基因的复合扩增体系的制备Embodiment 1, the preparation of the compound amplification system based on 6 genes

一、特异表达基因的筛选1. Screening of specifically expressed genes

本发明的发明人通过查阅文献获得大量候选的在外周血、月经血、唾液、精液、阴道分泌液中特异表达的基因,综合考虑各基因的序列结构,最终筛选获得4个特异表达基因(分别为HBA基因、HBB基因、MMP7基因、MMP10基因)和两个管家基因(分别为GAPDH基因和ACTB基因)。The inventors of the present invention obtained a large number of candidate genes specifically expressed in peripheral blood, menstrual blood, saliva, semen, and vaginal secretions by consulting the literature, comprehensively considered the sequence structure of each gene, and finally screened and obtained 4 specifically expressed genes (respectively) HBA gene, HBB gene, MMP7 gene, MMP10 gene) and two housekeeping genes (GAPDH gene and ACTB gene respectively).

二、基于6个基因的复合扩增体系的制备2. Preparation of multiple amplification system based on 6 genes

依据各基因的核苷酸序列及扩增片段长度范围(在100bp-300bp之间),合理选取不同扩增片段长度以区分各个基因,然后制备基于6个基因的复合扩增体系。具体步骤如下:According to the nucleotide sequence of each gene and the length range of the amplified fragment (between 100bp-300bp), different amplified fragment lengths were reasonably selected to distinguish each gene, and then a multiple amplification system based on 6 genes was prepared. Specific steps are as follows:

1、采用miRNeasy mini kit提取月经血的总RNA,然后用Nanodrop 2000c紫外分光光度计进行定量,同一样品重复三次计算平均值。1. The total RNA of menstrual blood was extracted with miRNeasy mini kit, and then quantified with a Nanodrop 2000c UV spectrophotometer. The same sample was repeated three times to calculate the average value.

2、完成步骤1后,取月经血的总RNA,按照III First-Standsynthesissystem说明书的方法进行逆转录,得到月经血的cDNA。2. After completing step 1, take the total RNA of menstrual blood and follow the The method of III First-Standsynthesissystem instruction manual is carried out reverse transcription, obtains the cDNA of menstrual blood.

逆转录反应体系为20μL,由200ng月经血的总RNA、1μL dNTP(浓度为2.5mmol/L)、1μL oligo(dT)(浓度为50μM)、2μL 10×RT Buffer、4μL Mg2+(浓度为25mmol/L)、2μL DTT(浓度为0.1mol/L)、1μL SuperScriptTMⅢRT(浓度为200U/μL)、1μLRNaseOUTTM(浓度为40U/μL)和去核酸酶的水组成。oligo(dT)、10×RT Buffer、SuperScriptTMⅢRT和RNaseOUTTM均为III First-Stand synthesis system中的组件。The reverse transcription reaction system was 20 μL, consisting of 200ng total RNA of menstrual blood, 1 μL dNTP (concentration: 2.5 mmol/L), 1 μL oligo( dT ) (concentration: 25mmol/L), 2μL DTT (concentration: 0.1mol/L), 1μL SuperScriptTMⅢRT (concentration: 200U/μL), 1μL RNaseOUTTM (concentration: 40U/μL) and nuclease-free water. oligo(dT), 10×RT Buffer, SuperScriptTMⅢRT and RNaseOUTTM are all Components in the III First-Stand synthesis system.

逆转录反应程序:向月经血的总RNA中加入dNTP和oligo(dT),然后65℃处理5min,冰上降温1min;之后加入其它试剂,混匀,置于Mastercycler Nexus PCR仪,50℃50min,85℃5min;之后置于冰上,加入1μL RNase H(浓度为2U/μL),37℃20min。Reverse transcription reaction procedure: add dNTP and oligo(dT) to the total RNA of menstrual blood, then treat at 65°C for 5 minutes, cool down on ice for 1 minute; then add other reagents, mix well, place in a Mastercycler Nexus PCR instrument, 50°C for 50 minutes, 5min at 85°C; then place on ice, add 1 μL RNase H (concentration: 2U/μL), and keep at 37°C for 20min.

加入RNase H的目的为消化未转录的RNA。The purpose of adding RNase H is to digest untranscribed RNA.

3、人工合成用于扩增各个基因的上游引物和下游引物,进行PCR扩增,得到PCR扩增产物;取96孔板,每孔加入PCR扩增产物0.5μL、分子量内标LIZ6000.5μL和Hi-Di甲酰胺9μL,混匀,95℃变性5min,迅速转移至-20℃放置5min,然后用ABI3130xl遗传分析仪进行毛细管电泳检测,得到检测图谱。电泳条件为:进样电压1.2kV,进样时间为30s。之后用GeneMapperID-X软件分析电泳检测结果,检测阈值为50。3. Artificially synthesize the upstream and downstream primers used to amplify each gene, perform PCR amplification, and obtain PCR amplification products; take a 96-well plate, add 0.5 μL of PCR amplification products, molecular weight internal standard LIZ6000.5 μL and 9 μL of Hi-Di formamide was mixed, denatured at 95°C for 5 minutes, quickly transferred to -20°C for 5 minutes, and then detected by capillary electrophoresis with an ABI3130xl genetic analyzer to obtain a detection map. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

PCR反应体系为20μL,由1μL月经血的cDNA、1μL10×PCR Buffer、0.2μLdNTP(浓度为2.5mmol/L)、0.6μL Mg2+(浓度为25mmol/L)、0.1μLHotstarTaq Polymerase(浓度为5U/μL)、1μL上游引物(浓度为10μM)、1μL下游引物(浓度为10μM)和5.1μL去离子水组成。10×PCRBuffer为HotstarTaq Polymerase中的组件。The PCR reaction system was 20 μL, consisting of 1 μL cDNA of menstrual blood, 1 μL 10×PCR Buffer, 0.2 μL dNTP (concentration: 2.5 mmol/L), 0.6 μL Mg 2+ (concentration: 25 mmol/L), 0.1 μL HotstarTaq Polymerase (concentration: 5 U/L) μL), 1 μL upstream primer (10 μM concentration), 1 μL downstream primer (10 μM concentration) and 5.1 μL deionized water. 10×PCRBuffer is a component of HotstarTaq Polymerase.

PCR反应程序为:95℃15min;94℃30s,60℃30s,72℃40s,30个循环;72℃10min,4℃保存。The PCR reaction program was: 95°C for 15 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s; 72°C for 10 min, and stored at 4°C.

通过上述步骤,获得各个基因的特异性引物。Through the above steps, specific primers for each gene were obtained.

4、综合每个基因的扩增条件,选择适宜扩增程序,然后进行复合扩增。复合扩增时,观察各个基因是否均有特异性产物和非特异产物的出现。如果不同基因引物之间产生非特异的杂峰,则需要一一排除,找出这些基因,重新设计并合成引物。此外,不同基因的引物之间还会形成引物二聚体,降低引物的扩增效率,也需要重新设计并合成引物。4. Synthesize the amplification conditions of each gene, select the appropriate amplification program, and then carry out multiple amplification. During multiplex amplification, observe whether each gene has specific products and non-specific products. If there are non-specific miscellaneous peaks between different gene primers, it is necessary to eliminate them one by one, find out these genes, redesign and synthesize primers. In addition, primer dimers will be formed between the primers of different genes, which will reduce the amplification efficiency of the primers, and the primers need to be redesigned and synthesized.

5、反复测试、修改复合扩增体系,用复合扩增体系对月经血的cDNA进行扩增,观察各基因之间是否会有重叠,因为实际过程中会因为扩增产物的序列结构不一致而造成迁移速率偏快或偏慢,导致理论大小与实际大小不符,经常导致基因之间发生重叠或者距离过于接近,影响后续分析。一旦发生此类现象,则需要重新设计该基因引物,再经历步骤3和4,最终获得满足条件的复合扩增体系。5. Repeatedly test and modify the complex amplification system, use the complex amplification system to amplify the cDNA of menstrual blood, and observe whether there will be overlap between the genes, because in the actual process, the sequence structure of the amplification products will be inconsistent. The migration rate is too fast or too slow, resulting in a discrepancy between the theoretical size and the actual size, which often leads to overlapping or too close distances between genes, which affects subsequent analysis. Once such a phenomenon occurs, it is necessary to redesign the gene primers, go through steps 3 and 4, and finally obtain a complex amplification system that meets the conditions.

6、复合扩增体系的调平。具体为:初次复合扩增时,各个引物在反应体系中的浓度均为2μM;根据反应结果调整各个引物对的浓度。6. Leveling of the compound amplification system. Specifically: during the first multiplex amplification, the concentration of each primer in the reaction system is 2 μM; the concentration of each primer pair is adjusted according to the reaction result.

7、优化复合扩增反应参数。用月经血的cDNA对反应体系中的关键组分和环节进行调整,如最优反应体系、扩增循环数、退火温度、延伸时间、HotstarTaq Polymerase用量、引物量、缓冲液浓度等等。7. Optimize the multiplex amplification reaction parameters. Use the cDNA of menstrual blood to adjust the key components and links in the reaction system, such as the optimal reaction system, number of amplification cycles, annealing temperature, extension time, amount of HotstarTaq Polymerase, primer amount, buffer concentration, etc.

经过上述步骤,获得基于6个基因的复合扩增体系。该复合扩增体系由Buffer、dNTP、Mg2+、DNA聚合酶、引物混合物、待测体液的cDNA和水组成;引物混合物由12条引物混合而成;12条引物的名称、核苷酸序列、扩增片段的长度见表1中第1-5列。该复合扩增体系中,dNTP的浓度为0.2mmol/L(即dATP、dTTP、dCTP和dGTP的浓度均为0.2mmol/L),Mg2+的浓度为1.5mmol/L,12条引物的浓度见表1中第6列。其中DNA聚合酶为HotstarTaq Polymerase,在复合扩增体系中的浓度为0.5U/L;Buffer为10×PCR Buffer,10×PCR Buffer在复合扩增体系中的浓度为1×。After the above steps, a multiple amplification system based on 6 genes was obtained. The composite amplification system is composed of Buffer, dNTP, Mg 2+ , DNA polymerase, primer mixture, cDNA of the body fluid to be tested and water; the primer mixture is composed of 12 primers; the names and nucleotide sequences of the 12 primers 1. The length of the amplified fragment is shown in columns 1-5 in Table 1. In this compound amplification system, the concentration of dNTP is 0.2mmol/L (that is, the concentration of dATP, dTTP, dCTP and dGTP is 0.2mmol/L), the concentration of Mg 2 + is 1.5mmol/L, and the concentration of 12 primers See column 6 in Table 1. The DNA polymerase is HotstarTaq Polymerase, the concentration in the multiplex amplification system is 0.5U/L; the Buffer is 10×PCR Buffer, and the concentration of 10×PCR Buffer in the multiplex amplification system is 1×.

表1Table 1

注:FAM表示5’末端进行FAM标记。Note: FAM means that the 5' end is labeled with FAM.

三、单重PCR扩增特异性检测3. Single-plex PCR amplification specific detection

1、HBB基因和HBA基因的特异性检测1. Specific detection of HBB gene and HBA gene

(1)采用miRNeasy mini kit提取外周血的总RNA,然后用Nanodrop 2000c紫外分光光度计进行定量。(1) The total RNA of peripheral blood was extracted by miRNeasy mini kit, and then quantified by Nanodrop 2000c UV spectrophotometer.

(2)完成步骤(1)后,取外周血的总RNA,按照III First-Standsynthesis system说明书的方法进行逆转录,得到外周血的cDNA。(2) After completing step (1), take the total RNA of peripheral blood, according to III First-Standsynthesis system manual method for reverse transcription, to obtain peripheral blood cDNA.

(3)以外周血的cDNA的模板,采用引物对1(由引物1和引物2组成)或引物对2(由引物3和引物4组成)进行PCR扩增,得到PCR扩增产物。(3) Using the template of peripheral blood cDNA, PCR amplification is performed with primer pair 1 (composed of primer 1 and primer 2) or primer pair 2 (composed of primer 3 and primer 4) to obtain PCR amplification products.

(4)取96孔板,每孔加入PCR扩增产物0.5μL、分子量内标LIZ6000.5μL和Hi-Di甲酰胺9μL,混匀,95℃变性5min,迅速转移至-20℃放置5min,然后用ABI3130xl遗传分析仪进行毛细管电泳检测,得到检测图谱。电泳条件为:进样电压1.2kV,进样时间为30s。之后用GeneMapperID-X软件分析电泳检测结果,检测阈值为50。(4) Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of internal molecular weight standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then ABI3130xl Genetic Analyzer was used for capillary electrophoresis detection, and the detection map was obtained. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

2、MMP7基因、MMP10基因、GAPDH基因和ACTB基因的特异性检测2. Specific detection of MMP7 gene, MMP10 gene, GAPDH gene and ACTB gene

(1)采用miRNeasy mini kit提取月经血的总RNA,然后用Nanodrop 2000c紫外分光光度计进行定量。(1) The total RNA of menstrual blood was extracted by miRNeasy mini kit, and then quantified by Nanodrop 2000c UV spectrophotometer.

(2)完成步骤(1)后,取月经血的总RNA,按照III First-Standsynthesis system说明书的方法进行逆转录,得到月经血的cDNA。(2) After completing step (1), take the total RNA of menstrual blood, according to The method of III First-Standsynthesis system instruction manual is carried out reverse transcription, obtains the cDNA of menstrual blood.

(3)以月经血的cDNA的模板,采用引物对3(由引物5和引物6组成)、引物对4(由引物7和引物8组成)、引物对5(由引物9和引物10组成)或引物对6(由引物11和引物12组成)进行PCR扩增,得到PCR扩增产物。(3) With the template of the cDNA of menstrual blood, primer pair 3 (composed of primer 5 and primer 6), primer pair 4 (composed of primer 7 and primer 8), primer pair 5 (composed of primer 9 and primer 10) were used Or perform PCR amplification on primer pair 6 (consisting of primer 11 and primer 12) to obtain a PCR amplification product.

(4)取96孔板,每孔加入PCR扩增产物0.5μL、分子量内标LIZ6000.5μL和Hi-Di甲酰胺9μL,混匀,95℃变性5min,迅速转移至-20℃放置5min,然后用ABI3130xl遗传分析仪进行毛细管电泳检测,得到检测图谱。电泳条件为:进样电压1.2kV,进样时间为30s。之后用GeneMapperID-X软件分析电泳检测结果,检测阈值为50。(4) Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of internal molecular weight standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then ABI3130xl Genetic Analyzer was used for capillary electrophoresis detection, and the detection map was obtained. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

检测结果见图1(a为HBB基因,b为HBA基因,c为MMP7基因,d为MMP10基因,e为GAPDH基因,f为ACTB基因)。结果表明,各个引物对均检测到了特异峰,具有特异性。The detection results are shown in Figure 1 (a is the HBB gene, b is the HBA gene, c is the MMP7 gene, d is the MMP10 gene, e is the GAPDH gene, and f is the ACTB gene). The results showed that each primer pair detected specific peaks and had specificity.

四、多重PCR扩增特异性检测4. Multiplex PCR amplification specific detection

1、外周血样本1. Peripheral blood samples

(1)采用miRNeasy mini kit提取外周血的总RNA,然后用Nanodrop 2000c紫外分光光度计进行定量。(1) The total RNA of peripheral blood was extracted by miRNeasy mini kit, and then quantified by Nanodrop 2000c UV spectrophotometer.

(2)完成步骤(1)后,取外周血的总RNA,按照III First-Standsynthesis system说明书的方法进行逆转录,得到外周血的cDNA。(2) After completing step (1), take the total RNA of peripheral blood, according to III First-Standsynthesis system manual method for reverse transcription, to obtain peripheral blood cDNA.

(3)取9μL实施例1步骤二中3制备的复合扩增体系,加入1μL外周血的cDNA进行PCR扩增,得到PCR扩增产物。(3) Take 9 μL of the composite amplification system prepared in step 2 of Example 1, add 1 μL of peripheral blood cDNA to perform PCR amplification, and obtain PCR amplification products.

(4)取96孔板,每孔加入PCR扩增产物0.5μL、分子量内标LIZ6000.5μL和Hi-Di甲酰胺9μL,混匀,95℃变性5min,迅速转移至-20℃放置5min,然后用ABI3130xl遗传分析仪进行毛细管电泳检测,得到检测图谱。电泳条件为:进样电压1.2kV,进样时间为30s。之后用GeneMapperID-X软件分析电泳检测结果,检测阈值为50。(4) Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of internal molecular weight standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then ABI3130xl Genetic Analyzer was used for capillary electrophoresis detection, and the detection map was obtained. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

检测结果见图2中a。结果表明,外周血中表达HBB基因、HBA基因、GAPDH基因和ACTB基因。The test results are shown in Figure 2a. The results showed that HBB gene, HBA gene, GAPDH gene and ACTB gene were expressed in peripheral blood.

2、月经血样本2. Menstrual blood samples

按照步骤1的方法,将外周血替换为月经血,其它步骤均不变。According to the method of step 1, the peripheral blood was replaced with menstrual blood, and the other steps were unchanged.

检测结果见图2中b。结果表明,月经血中表达HBB基因、HBA基因、MMP7基因、MMP10基因、GAPDH基因和ACTB基因。由于月经血成分复杂,其中混有大量血细胞,因此在月经血样本中检测到HBB基因和HBA基因呈阳性符合预期结果。The test results are shown in Figure 2 b. The results showed that HBB gene, HBA gene, MMP7 gene, MMP10 gene, GAPDH gene and ACTB gene were expressed in menstrual blood. Due to the complex composition of menstrual blood and a large number of blood cells mixed in it, the positive results of HBB gene and HBA gene detected in menstrual blood samples were in line with the expected results.

3、精液样本3. Semen sample

按照步骤1的方法,将外周血替换为精液,其它步骤均不变。According to the method of step 1, the peripheral blood was replaced with semen, and the other steps remained unchanged.

检测结果见图2中c。结果表明,精液中仅表达GAPDH基因和ACTB基因。The test results are shown in c in Figure 2. The results showed that only GAPDH gene and ACTB gene were expressed in semen.

4、阴道分泌液样本4. Vaginal fluid sample

按照步骤1的方法,将外周血替换为阴道分泌液,其它步骤均不变。According to the method of step 1, the peripheral blood was replaced with vaginal secretions, and other steps remained unchanged.

检测结果见图2中d。结果表明,阴道分泌液中仅表达GAPDH基因和ACTB基因。The test results are shown in Figure 2 d. The results showed that only GAPDH gene and ACTB gene were expressed in vaginal fluid.

5、唾液样本5. Saliva sample

按照步骤1的方法,将外周血替换为唾液,其它步骤均不变。According to the method of step 1, the peripheral blood was replaced with saliva, and the other steps were unchanged.

检测结果见图2中e。结果表明,唾液中仅表达GAPDH基因和ACTB基因。The test results are shown in Figure 2 e. The results showed that only GAPDH gene and ACTB gene were expressed in saliva.

实施例2、基于6个基因的复合扩增体系在鉴定待测体液为非血液、月经血还是外周血中的应用Example 2. Application of multiple amplification system based on 6 genes in identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood

1、样本采集1. Sample collection

待测样本为来自河南地区的80个志愿者(年龄在18-35周岁)提供的80份样本。80个志愿者为无关个体,且均知情同意。The samples to be tested were 80 samples provided by 80 volunteers (aged 18-35) from Henan. 80 volunteers were unrelated individuals, and all of them gave informed consent.

样本编号为PB1-PB20的20份样本均为外周血样本。外周血样本为抽取部分志愿者手臂处静脉血获得,-80℃保存。The 20 samples numbered PB1-PB20 are all peripheral blood samples. Peripheral blood samples were obtained from venous blood drawn from the arms of some volunteers and stored at -80°C.

样本编号为MB1-MB20的20份样本均为月经血样本。20份月经血样本为部分志愿者用卫生棉条在月经周期前4天采集,室温晾干1天后-80℃保存。The 20 samples numbered MB1-MB20 are all menstrual blood samples. The 20 menstrual blood samples were collected by some volunteers with tampons 4 days before the menstrual cycle, dried at room temperature for 1 day, and stored at -80°C.

样本编号为SA1-SA20的20份样本均为唾液样本。20份唾液样本为将部分志愿者唾液收集在无菌塑料管中获得(收集前志愿者禁食1h),-80℃保存。The 20 samples numbered SA1-SA20 are all saliva samples. The 20 saliva samples were obtained by collecting saliva from some volunteers in sterile plastic tubes (the volunteers fasted for 1 h before collection), and stored at -80°C.

样本编号为SE1-SE20的20份样本均为精液样本。20份精液样本为将部分志愿者新鲜精液收集在无菌广口塑料杯中获得(收集前志愿者禁欲2天),-80℃保存。The 20 samples with sample numbers SE1-SE20 are all semen samples. 20 semen samples were obtained by collecting fresh semen from some volunteers in sterile wide-mouth plastic cups (the volunteers were abstinent for 2 days before collection), and stored at -80°C.

样本编号为VA1-VA20的20份样本均为阴道分泌液样本。20份阴道分泌液样本为部分志愿者用卫生棉条在在非月经期采集,室温晾干1天后-80℃保存。The 20 samples numbered VA1-VA20 are all vaginal fluid samples. The 20 vaginal fluid samples were collected by some volunteers with tampons during the non-menstrual period, dried at room temperature for 1 day, and stored at -80°C.

2、采用miRNeasy mini kit分别提取待测样本的总RNA,然后用Nanodrop 2000c紫外分光光度计进行定量。2. Use the miRNeasy mini kit to extract the total RNA of the samples to be tested, and then use the Nanodrop 2000c UV spectrophotometer for quantification.

3、分别取待测样本的总RNA,按照III First-Stand synthesissystem说明书的方法进行逆转录,得到待测样本的cDNA。3. Take the total RNA of the sample to be tested respectively, according to III First-Stand synthesissystem manual method for reverse transcription, to obtain the cDNA of the sample to be tested.

4、取9μL实施例1步骤二中3制备的复合扩增体系,加入1μL待测样本的cDNA进行PCR扩增,得到PCR扩增产物。4. Take 9 μL of the composite amplification system prepared in step 2 of Example 1, add 1 μL of the cDNA of the sample to be tested for PCR amplification, and obtain PCR amplification products.

5、取96孔板,每孔加入PCR扩增产物0.5μL、分子量内标LIZ6000.5μL和Hi-Di甲酰胺9μL,混匀,95℃变性5min,迅速转移至-20℃放置5min,然后用ABI3130xl遗传分析仪进行毛细管电泳检测,得到检测图谱。电泳条件为:进样电压1.2kV,进样时间为30s。之后用GeneMapperID-X软件分析电泳检测结果,检测阈值为50。5. Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of molecular weight internal standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then use The ABI3130xl Genetic Analyzer performs capillary electrophoresis detection and obtains a detection map. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

统计结果见表2。结果表明,20份外周血样本中,75%以上的外周血样本可以检测到HBB基因、HBA基因、GAPDH基因和ACTB基因的表达,且任何外周血样本均没有MMP7基因和MMP10基因的表达;20份月经血样本中,75%以上的月经血样本可以检测到HBA基因、HBB基因、MMP7基因和GAPDH基因的表达,50%-75%的月经血样本可以检测到MMP10基因和ACTB基因的表达;20份精液样本中,75%以上的精液样本可以检测到GAPDH基因和ACTB基因的表达,且任何精液样本均没有HBB基因、HBA基因、MMP7基因和MMP10基因的表达;20份唾液样本中,50%-75%的唾液样本可以检测到GAPDH基因和ACTB基因的表达,且任何唾液样本均没有HBB基因、HBA基因、MMP7基因和MMP10基因的表达;20份阴道分泌液样本中,50%以上的阴道分泌液样本可以检测到GAPDH基因和ACTB基因的表达,1份阴道分泌液样本可以检测到MMP10基因的表达,2份阴道分泌液样本可以检测到MMP7基因的表达,这可能是由于部分样本的采集时间处于经期前后,使样本受少量月经血污染所致。管家基因GAPDH、ACTB在不同体液中的检出率存在较大差异。See Table 2 for statistical results. The results showed that among the 20 peripheral blood samples, more than 75% of the peripheral blood samples could detect the expression of HBB gene, HBA gene, GAPDH gene and ACTB gene, and there was no expression of MMP7 gene and MMP10 gene in any peripheral blood sample; 20 Among menstrual blood samples, more than 75% of menstrual blood samples can detect the expression of HBA gene, HBB gene, MMP7 gene and GAPDH gene, and 50%-75% of menstrual blood samples can detect the expression of MMP10 gene and ACTB gene; Among the 20 semen samples, the expression of GAPDH gene and ACTB gene could be detected in more than 75% of the semen samples, and there was no expression of HBB gene, HBA gene, MMP7 gene and MMP10 gene in any semen sample; among the 20 saliva samples, 50 %-75% of saliva samples can detect the expression of GAPDH gene and ACTB gene, and any saliva sample has no expression of HBB gene, HBA gene, MMP7 gene and MMP10 gene; in 20 vaginal fluid samples, more than 50% of The expression of GAPDH gene and ACTB gene can be detected in vaginal fluid samples, the expression of MMP10 gene can be detected in 1 vaginal fluid sample, and the expression of MMP7 gene can be detected in 2 vaginal fluid samples, which may be due to the The collection time was before and after menstruation, which caused the sample to be polluted by a small amount of menstrual blood. The detection rates of housekeeping genes GAPDH and ACTB in different body fluids were quite different.

表2Table 2

注:20为样本个数,“/”前的数字为毛细管电泳检测出峰的样本数。Note: 20 is the number of samples, and the number before "/" is the number of samples with peaks detected by capillary electrophoresis.

上述结果表明,实施例1制备的复合扩增体系可以鉴定样本为血液还是非血液,还可以鉴定待测血液为外周血还是月经血,具有较高的准确性。The above results show that the multiple amplification system prepared in Example 1 can identify whether the sample is blood or non-blood, and can also identify whether the blood to be tested is peripheral blood or menstrual blood, with high accuracy.

<110> 公安部物证鉴定中心<110> Physical Evidence Identification Center of the Ministry of Public Security

<120> 一种鉴定待测体液为非血液、月经血还是外周血的复合扩增体系及其使用的引物组合<120> A complex amplification system for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood and the primer combination used

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Claims (10)

1.引物组合,包括引物组合甲和引物组合乙;1. Primer combinations, including primer combination A and primer combination B; 引物组合甲包括引物1、引物2、引物3和引物4;Primer combination A includes primer 1, primer 2, primer 3 and primer 4; 引物1为序列表中的序列1所示的单链DNA分子;Primer 1 is a single-stranded DNA molecule shown in Sequence 1 in the sequence listing; 引物2为序列表中的序列2所示的单链DNA分子;Primer 2 is a single-stranded DNA molecule shown in Sequence 2 in the sequence listing; 引物3为序列表中的序列3所示的单链DNA分子;Primer 3 is a single-stranded DNA molecule shown in sequence 3 in the sequence listing; 引物4为序列表中的序列4所示的单链DNA分子;Primer 4 is a single-stranded DNA molecule shown in sequence 4 in the sequence listing; 引物组合乙包括引物5、引物6、引物7和引物8;Primer combination B includes primer 5, primer 6, primer 7 and primer 8; 引物5为序列表中的序列5所示的单链DNA分子;Primer 5 is a single-stranded DNA molecule shown in sequence 5 in the sequence listing; 引物6为序列表中的序列6所示的单链DNA分子;Primer 6 is a single-stranded DNA molecule shown in sequence 6 in the sequence listing; 引物7为序列表中的序列7所示的单链DNA分子;Primer 7 is a single-stranded DNA molecule shown in sequence 7 in the sequence listing; 引物8为序列表中的序列8所示的单链DNA分子。Primer 8 is a single-stranded DNA molecule shown in sequence 8 in the sequence listing. 2.权利要求1中所述引物组合甲。2. The primer combination A described in claim 1. 3.权利要求1中所述引物组合乙。3. The primer combination B described in claim 1. 4.如权利要求1所述引物组合、权利要求2所述引物组合甲或权利要求3所述引物组合乙,其特征在于:所述引物组合、所述引物组合甲或所述引物组合乙还包括引物组合丙;4. primer combination as claimed in claim 1, primer combination A as claimed in claim 2 or primer combination second as claimed in claim 3, characterized in that: said primer combination, said primer combination A or said primer combination B also including primer set C; 引物组合丙包括引物9、引物10、引物11和引物12;Primer combination C includes primer 9, primer 10, primer 11 and primer 12; 引物9为序列表中的序列9所示的单链DNA分子;Primer 9 is a single-stranded DNA molecule shown in sequence 9 in the sequence listing; 引物10为序列表中的序列10所示的单链DNA分子;Primer 10 is a single-stranded DNA molecule shown in sequence 10 in the sequence listing; 引物11为序列表中的序列11所示的单链DNA分子;Primer 11 is a single-stranded DNA molecule shown in sequence 11 in the sequence listing; 引物12为序列表中的序列12所示的单链DNA分子。Primer 12 is a single-stranded DNA molecule shown in sequence 12 in the sequence listing. 5.一种复合扩增体系,包括权利要求1或4所述引物组合、或、权利要求2或4所述引物组合甲、或、权利要求3或4所述引物组合乙。5. A multiplex amplification system, comprising the primer combination according to claim 1 or 4, or the primer combination A according to claim 2 or 4, or the primer combination B according to claim 3 or 4. 6.如权利要求5所述复合扩增体系,其特征在于:6. composite amplification system as claimed in claim 5, is characterized in that: 引物1、引物2、引物3和引物4在所述复合扩增体系中的浓度为0.01μM;The concentration of primer 1, primer 2, primer 3 and primer 4 in the multiplex amplification system is 0.01 μM; 引物5和引物6在所述复合扩增体系中的浓度为4.0μM;The concentration of primer 5 and primer 6 in the multiplex amplification system is 4.0 μM; 引物7和引物8在所述复合扩增体系中的浓度为5.0μM;The concentration of primer 7 and primer 8 in the multiplex amplification system is 5.0 μM; 引物9和引物10在所述复合扩增体系中的浓度为0.2μM;The concentration of primer 9 and primer 10 in the multiplex amplification system is 0.2 μM; 引物11和引物12在所述复合扩增体系中的浓度为1.0μM。The concentration of primer 11 and primer 12 in the multiplex amplification system was 1.0 μM. 7.试剂盒甲、试剂盒乙或试剂盒丙;7. Kit A, Kit B or Kit C; 含有权利要求1或4所述引物组合的试剂盒甲;所述试剂盒甲用于鉴定待测体液为非血液、月经血还是外周血;A kit A containing the primer combination according to claim 1 or 4; said kit A is used to identify whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood; 含有权利要求2或4所述引物组合甲的试剂盒乙;所述试剂盒乙用于鉴定待测体液为血液还是非血液;A kit B containing the primer combination A described in claim 2 or 4; said kit B is used to identify whether the body fluid to be tested is blood or non-blood; 含有权利要求3或4所述引物组合乙的试剂盒丙;所述试剂盒丙用于鉴定待测血液为月经血还是外周血。A kit C containing the primer combination B according to claim 3 or 4; said kit C is used to identify whether the blood to be tested is menstrual blood or peripheral blood. 8.X1)或X2)或X3):8. X1) or X2) or X3): X1)权利要求1或4所述引物组合、或、权利要求5或6所述复合扩增体系在鉴定待测体液为非血液、月经血还是外周血中的应用;X1) the primer combination described in claim 1 or 4, or, the application of the composite amplification system described in claim 5 or 6 in identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood; X2)权利要求2或4所述引物组合甲、或、权利要求5或6所述复合扩增体系在鉴定待测体液为血液还是非血液中的应用;X2) the primer combination A described in claim 2 or 4, or, the application of the composite amplification system described in claim 5 or 6 in identifying whether the body fluid to be tested is blood or non-blood; X3)权利要求3或4所述引物组合乙、或、权利要求5或6所述复合扩增体系在鉴定待测血液为月经血还是外周血中的应用。X3) The primer combination B described in claim 3 or 4, or, the application of the multiple amplification system described in claim 5 or 6 in identifying whether the blood to be tested is menstrual blood or peripheral blood. 9.S1)或S2)或S3):9. S1) or S2) or S3): S1)一种鉴定待测体液为非血液、月经血还是外周血的方法,包括如下步骤:以待测体液的cDNA为模板,分别采用引物对1、引物对2、引物对3和引物对4进行PCR扩增,得到PCR扩增产物;引物对1由权利要求1中引物1和引物2组成;引物对2由权利要求1中引物3和引物4组成;引物对3由权利要求1中引物5和引物6组成;引物对4由权利要求1中引物7和引物8组成;然后进行如下判断:S1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, comprising the following steps: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1, primer pair 2, primer pair 3 and primer pair 4 Carry out PCR amplification, obtain PCR amplification product; Primer pair 1 is made up of primer 1 and primer 2 in claim 1; Primer pair 2 is made up of primer 3 and primer 4 in claim 1; Primer pair 3 is made up of primer in claim 1 5 and primer 6 form; Primer pair 4 is made up of primer 7 and primer 8 in claim 1; Carry out following judgment then: 如果采用引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,则该待测体液为或疑似为月经血;If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Contains a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using primer pair 4 contains a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be menstrual blood; 如果采用引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,则该待测体液为或疑似为外周血;If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be peripheral blood; 如果采用引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,采用引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,则该待测体液为或疑似为非血液;If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 If the product does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be non-blood; S2)一种鉴定待测体液为血液还是非血液的方法,包括如下步骤:以待测体液的cDNA为模板,分别采用引物对1和引物对2进行PCR扩增,得到PCR扩增产物;然后进行如下判断:S2) A method for identifying whether the body fluid to be tested is blood or non-blood, comprising the following steps: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1 and primer pair 2 to perform PCR amplification to obtain a PCR amplification product; and then Make the following judgments: 如果采用引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,则待测体液为或疑似为血液;If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243 bp, and the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159 bp, the body fluid to be tested is or is suspected to be blood; 如果采用引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,则该待测体液为或疑似为非血液;If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243 bp, and the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159 bp, then the body fluid to be tested is or is suspected to be non-blood; S3)一种鉴定待测血液为月经血还是外周血的方法,包括如下步骤:以待测血液的cDNA为模板,分别采用引物对3和引物对4进行PCR扩增,得到PCR扩增产物;然后进行如下判断:S3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, comprising the following steps: using the cDNA of the blood to be tested as a template, performing PCR amplification with primer pair 3 and primer pair 4 respectively, to obtain a PCR amplification product; Then make the following judgments: 如果采用引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,则该待测血液为或疑似为月经血;If the PCR amplification product obtained with primer pair 3 contains a DNA fragment with a size of 126 bp, and the PCR amplification product obtained with primer pair 4 contains a DNA fragment with a size of 272 bp, then the blood to be tested is or is suspected to be menstrual blood ; 如果采用引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,则该待测血液为或疑似为外周血。If the PCR amplification product obtained by using primer pair 3 does not contain a DNA fragment with a size of 126 bp, and the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272 bp, then the blood to be tested is or is suspected to be peripheral blood. 10.T1)或T2)或T3):10. T1) or T2) or T3): T1)一种鉴定待测体液为非血液、月经血还是外周血的方法,包括如下步骤:以待测体液的cDNA为模板,分别采用引物对1、引物对2、引物对3、引物对4、引物对5和引物对6进行PCR扩增,得到PCR扩增产物;引物对1由权利要求1中引物1和引物2组成;引物对2由权利要求1中引物3和引物4组成;引物对3由权利要求1中引物5和引物6组成;引物对4由权利要求1中引物7和引物8组成;引物对5由权利要求1中引物9和引物10组成;引物对6由权利要求1中引物11和引物12组成;然后进行如下判断:T1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, comprising the following steps: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1, primer pair 2, primer pair 3, and primer pair 4 1. Primer pair 5 and primer pair 6 carry out PCR amplification, obtain PCR amplification product; Primer pair 1 is made up of primer 1 and primer 2 in claim 1; Primer pair 2 is made up of primer 3 and primer 4 in claim 1; Primer pair Pair 3 is made up of primer 5 and primer 6 in claim 1; Primer pair 4 is made up of primer 7 and primer 8 in claim 1; Primer pair 5 is made up of primer 9 and primer 10 in claim 1; Primer pair 6 is made up of Primer 11 and primer 12 in 1 are composed; then the following judgments are made: 如果采用引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为月经血;If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Contains a DNA fragment with a size of 126bp. The PCR amplification product obtained by using primer pair 4 contains a DNA fragment with a size of 272bp. The PCR amplification product obtained with primer pair 5 contains a DNA fragment with a size of 250bp. Using primer pair 6 If the obtained PCR amplification product contains a DNA fragment with a size of 291bp, the body fluid to be tested is or is suspected to be menstrual blood; 如果采用引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为外周血;If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Does not contain a DNA fragment with a size of 126bp. The PCR amplification product obtained with primer pair 4 does not contain a DNA fragment with a size of 272bp. The PCR amplification product obtained with primer pair 5 contains a DNA fragment with a size of 250bp. If the PCR amplification product obtained in 6 contains a DNA fragment with a size of 291bp, the body fluid to be tested is or is suspected to be peripheral blood; 如果采用引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,采用引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为非血液;If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 The product does not contain a DNA fragment with a size of 126bp. The PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp. The PCR amplification product obtained with primer pair 5 contains a DNA fragment with a size of 250bp. If the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, the body fluid to be tested is or is suspected to be non-blood; T2)一种鉴定待测体液为血液还是非血液的方法,包括如下步骤:以待测体液的cDNA为模板,分别采用引物对1、引物对2、引物对5和引物对6进行PCR扩增,得到PCR扩增产物;然后进行如下判断:T2) A method for identifying whether the body fluid to be tested is blood or non-blood, comprising the steps of: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1, primer pair 2, primer pair 5 and primer pair 6 to carry out PCR amplification , to obtain the PCR amplification product; then carry out the following judgment: 如果采用引物对1得到的PCR扩增产物中含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中含有大小为159bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则待测体液为或疑似为血液;If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 5 Contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the body fluid to be tested is or is suspected to be blood; 如果采用引物对1得到的PCR扩增产物中不含有大小为243bp的DNA片段,采用引物对2得到的PCR扩增产物中不含有大小为159bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则该待测体液为或疑似为非血液;If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 5 If the product contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the body fluid to be tested is or is suspected to be non-blood; T3)一种鉴定待测血液为月经血还是外周血的方法,包括如下步骤:以待测血液的cDNA为模板,分别采用引物对3、引物对4、引物对5和引物对6进行PCR扩增,得到PCR扩增产物;然后进行如下判断:T3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, comprising the steps of: using the cDNA of the blood to be tested as a template, respectively using primer pair 3, primer pair 4, primer pair 5, and primer pair 6 to perform PCR amplification Increase to obtain the PCR amplification product; then make the following judgments: 如果采用引物对3得到的PCR扩增产物中含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中含有大小为272bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则该待测血液为或疑似为月经血;If the PCR amplification product obtained by using primer pair 3 contains a DNA fragment with a size of 126bp, the PCR amplification product obtained by using primer pair 4 contains a DNA fragment with a size of 272bp, and the PCR amplification product obtained by using primer pair 5 Contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the blood to be tested is or is suspected to be menstrual blood; 如果采用引物对3得到的PCR扩增产物中不含有大小为126bp的DNA片段,采用引物对4得到的PCR扩增产物中不含有大小为272bp的DNA片段,采用引物对5得到的PCR扩增产物中含有大小为250bp的DNA片段,采用引物对6得到的PCR扩增产物中含有大小为291bp的DNA片段,则该待测血液为或疑似为外周血。If the PCR amplification product obtained by using primer pair 3 does not contain a DNA fragment with a size of 126bp, the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp, and the PCR amplification product obtained by using primer pair 5 If the product contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the blood to be tested is or is suspected to be peripheral blood.
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