阅读说明：本技术 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 (Composite amplification system for identifying whether body fluid to be detected is non-blood, menstrual blood or peripheral blood and primer combination used by same ) 是由 赵一霞 孙启凡 季安全 胡胜 于 2019-09-02 设计创作，主要内容包括：本发明公开了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。该引物组合由序列表中的序列1至序列12所示的12种DNA分子组成。采用该引物组合构建复合扩增体系,可以鉴定待测体液为非血液、月经血还是外周血,且准确性较高。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。(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.)

1. The primer combination comprises a primer combination A and a primer combination B;

The primer combination A comprises a primer 1, a primer 2, a primer 3 and a primer 4;

the primer 1 is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

The primer 2 is a single-stranded DNA molecule shown as a sequence 2 in a sequence table;

the primer 3 is a single-stranded DNA molecule shown as a sequence 3 in a sequence table;

The primer 4 is a single-stranded DNA molecule shown as a sequence 4 in a sequence table;

The primer combination B comprises a primer 5, a primer 6, a primer 7 and a primer 8;

The primer 5 is a single-stranded DNA molecule shown as a sequence 5 in the sequence table;

the primer 6 is a single-stranded DNA molecule shown as a sequence 6 in a sequence table;

The primer 7 is a single-stranded DNA molecule shown as a sequence 7 in a sequence table;

The primer 8 is a single-stranded DNA molecule shown as a sequence 8 in a sequence table.

2. The primer set A according to claim 1.

3. The primer set B according to claim 1.

4. the primer combination of claim 1, the primer combination A of claim 2, or the primer combination B of claim 3, wherein: the primer combination, the primer combination A or the primer combination B further comprises a primer combination C;

The primer combination C comprises a primer 9, a primer 10, a primer 11 and a primer 12;

The primer 9 is a single-stranded DNA molecule shown as a sequence 9 in a sequence table;

the primer 10 is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;

The primer 11 is a single-stranded DNA molecule shown as a sequence 11 in a sequence table;

the primer 12 is a single-stranded DNA molecule shown as a sequence 12 in a sequence table.

5. A multiplex amplification system comprising the primer combination of claim 1 or 4, or the primer combination A of claim 2 or 4, or the primer combination B of claim 3 or 4.

6. The multiplex amplification system of claim 5, wherein:

The concentration of the primer 1, the primer 2, the primer 3 and the primer 4 in the composite amplification system is 0.01 mu M;

the concentration of the primer 5 and the primer 6 in the composite amplification system is 4.0 mu M;

The concentration of the primer 7 and the primer 8 in the composite amplification system is 5.0 mu M;

the concentration of the primer 9 and the primer 10 in the composite amplification system is 0.2 mu M;

The concentration of primer 11 and primer 12 in the multiplex amplification system was 1.0. mu.M.

7. Kit A, kit B or kit C;

A kit A comprising the primer combination of claim 1 or 4; the kit A is used for identifying whether the body fluid to be detected is non-blood, menstrual blood or peripheral blood;

a kit B comprising the primer combination A according to claim 2 or 4; the kit B is used for identifying whether the body fluid to be detected is blood or non-blood;

A kit C comprising the primer combination B of claim 3 or 4; the kit C is used for identifying whether the blood to be detected is menstrual blood or peripheral blood.

8, X1) or X2) or X3):

X1) the primer combination of claim 1 or 4 or the composite amplification system of claim 5 or 6, in the identification of the body fluid to be tested as non-blood, menstrual blood or peripheral blood;

x2) the primer combination A of claim 2 or 4 or the composite amplification system of claim 5 or 6 is used for identifying whether the body fluid to be detected is blood or non-blood;

x3) the primer combination B of claim 3 or 4 or the composite amplification system of claim 5 or 6, in the identification of the blood to be detected as menstrual blood or peripheral blood.

9, S1) or S2) or S3):

S1) a method for identifying whether a body fluid to be tested is non-blood, menstrual blood or peripheral blood, comprising the steps of: taking cDNA of body fluid to be detected as a template, and respectively adopting a primer pair 1, a primer pair 2, a primer pair 3 and a primer pair 4 to carry out PCR amplification to obtain PCR amplification products; the primer pair 1 consists of a primer 1 and a primer 2 in claim 1; the primer pair 2 consists of a primer 3 and a primer 4 in the claim 1; the primer pair 3 is composed of the primer 5 and the primer 6 in the claim 1; the primer pair 4 is composed of the primer 7 and the primer 8 in the claim 1; then, the following judgment is made:

If the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 3 contains a DNA fragment with the size of 126bp, and the PCR amplification product obtained by adopting the primer pair 4 contains a DNA fragment with the size of 272bp, the body fluid to be detected is or is suspected to be menstrual blood;

If the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 3 does not contain a DNA fragment with the size of 126bp, and the PCR amplification product obtained by adopting the primer pair 4 does not contain a DNA fragment with the size of 272bp, the body fluid to be detected is or is suspected to be peripheral blood;

if the PCR amplification product obtained by the primer pair 1 does not contain a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 does not contain a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, and the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the body fluid to be detected is or is suspected to be non-blood;

S2) a method for identifying whether a body fluid to be tested is blood or non-blood, comprising the steps of: taking cDNA of body fluid to be detected as a template, and respectively adopting a primer pair 1 and a primer pair 2 to carry out PCR amplification to obtain a PCR amplification product; then, the following judgment is made:

if the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, and the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the body fluid to be detected is or is suspected to be blood;

If the PCR amplification product obtained by the primer pair 1 does not contain the DNA fragment with the size of 243bp, and the PCR amplification product obtained by the primer pair 2 does not contain the DNA fragment with the size of 159bp, the body fluid to be detected is or is suspected to be non-blood;

S3) a method for identifying whether blood to be tested is menstrual blood or peripheral blood, comprising the steps of: carrying out PCR amplification by respectively adopting a primer pair 3 and a primer pair 4 by taking cDNA of blood to be detected as a template to obtain a PCR amplification product; then, the following judgment is made:

If the PCR amplification product obtained by adopting the primer pair 3 contains a DNA fragment with the size of 126bp, and the PCR amplification product obtained by adopting the primer pair 4 contains a DNA fragment with the size of 272bp, the blood to be detected is or is suspected to be menstrual blood;

If the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, and the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the blood to be detected is or is suspected to be peripheral blood.

10, T1) or T2) or T3):

t1) a method for identifying whether a body fluid to be tested is non-blood, menstrual blood or peripheral blood, comprising the steps of: taking cDNA of body fluid to be detected as a template, and respectively carrying out PCR amplification by adopting a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5 and a primer pair 6 to obtain PCR amplification products; the primer pair 1 consists of a primer 1 and a primer 2 in claim 1; the primer pair 2 consists of a primer 3 and a primer 4 in the claim 1; the primer pair 3 is composed of the primer 5 and the primer 6 in the claim 1; the primer pair 4 is composed of the primer 7 and the primer 8 in the claim 1; the primer pair 5 is composed of the primer 9 and the primer 10 in claim 1; the primer pair 6 is composed of the primer 11 and the primer 12 in claim 1; then, the following judgment is made:

If the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 3 contains a DNA fragment with the size of 126bp, the PCR amplification product obtained by adopting the primer pair 4 contains a DNA fragment with the size of 272bp, the PCR amplification product obtained by adopting the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by adopting the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be menstrual blood;

If the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 3 does not contain a DNA fragment with the size of 126bp, the PCR amplification product obtained by adopting the primer pair 4 does not contain a DNA fragment with the size of 272bp, the PCR amplification product obtained by adopting the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by adopting the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be peripheral blood;

If the PCR amplification product obtained by the primer pair 1 does not contain a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 does not contain a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be non-blood;

t2) a method for identifying whether a body fluid to be tested is blood or non-blood, comprising the steps of: taking cDNA of body fluid to be detected as a template, and respectively adopting a primer pair 1, a primer pair 2, a primer pair 5 and a primer pair 6 to carry out PCR amplification to obtain PCR amplification products; then, the following judgment is made:

If the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by adopting the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be blood;

If the PCR amplification product obtained by the primer pair 1 does not contain a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 does not contain a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be non-blood;

T3) a method for identifying whether blood to be tested is menstrual blood or peripheral blood, comprising the steps of: taking cDNA of blood to be detected as a template, and respectively carrying out PCR amplification by adopting a primer pair 3, a primer pair 4, a primer pair 5 and a primer pair 6 to obtain PCR amplification products; then, the following judgment is made:

If the PCR amplification product obtained by adopting the primer pair 3 contains a DNA fragment with the size of 126bp, the PCR amplification product obtained by adopting the primer pair 4 contains a DNA fragment with the size of 272bp, the PCR amplification product obtained by adopting the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by adopting the primer pair 6 contains a DNA fragment with the size of 291bp, the blood to be detected is or is suspected to be menstrual blood;

if the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the blood to be detected is or is suspected to be peripheral blood.

Technical Field

The invention belongs to the technical field of forensic science, and particularly relates to 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.

background

By identifying the tissue source of the body fluid spot on the case scene, the tissue source of the sample and the real criminal activity thereof can be deduced, thereby providing important information for case qualification and scene reconstruction and providing important scientific evidence for court science. The common body fluids in case sites include peripheral blood, menstrual blood, saliva, semen, vaginal secretion and the like. Although both peripheral blood and menstrual blood contain a large amount of erythrocytes, menstrual blood contains a larger amount of a mixture of endometrial fragments, cervical mucus, vaginal secretions, and the like than peripheral blood. Whether the on-site blood type test material belongs to peripheral blood or menstrual blood is a very important forensic science problem, and the on-site blood type test material has very important significance for case qualification, case site reconstruction and case evidence provision; particularly for sexual assault, female missing and certain injuries, where a female victim is said to have hematuria resulting from a violent attack, it is necessary to determine whether there is menstrual blood contamination of the source of blood cells in the urine. Therefore, the differentiation between menstrual blood and peripheral blood is still the key point and difficulty for forensic body fluid identification. The traditional blood mark identification method mainly comprises biochemical analysis and immunological analysis, namely, hemoglobin and derivatives thereof in blood are used for identification, but the method is time-consuming and labor-consuming, consumes test materials and is easy to generate false positive or false negative results. The trace amount of detection material obtained from the crime scene needs to be identified by a more sensitive and accurate identification method.

A large number of researches show that mRNA marker detection based on molecular biology level is expected to become an effective method for identifying the body fluid speckle tissue attribute source. The method relies on the unique expression profiles of mRNA in different body fluids to identify the body fluids, has the advantages of strong specificity, high sensitivity, only need of trace detection materials and the like, and is compatible with the current DNA/RNA co-extraction technology, namely, DNA and mRNA are simultaneously extracted from biological detection materials, wherein the DNA is used for polymorphic typing, and the mRNA is used for tissue source identification, so that the working efficiency can be improved, and the trace detection materials are fully utilized. The research of European DNA typing group shows that the blood stain can be successfully identified by mRNA analysis method in the laboratory without RNA detection experience, different body fluids are mixed and then RT-PCR composite amplification is carried out, the mRNA gene locus detection result of the tissue specificity of each body fluid is successfully obtained, and the source of the body fluid is accurately judged.

Disclosure of Invention

The purpose of the present invention is to identify whether a body fluid to be measured is non-blood, menstrual blood or peripheral blood.

The invention firstly protects a primer combination, which can comprise a primer combination A and a primer combination B.

the primer combination A can comprise a primer 1, a primer 2, a primer 3 and a primer 4;

The primer 1 can be a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

The primer 2 can be a single-stranded DNA molecule shown as a sequence 2 in a sequence table;

The primer 3 can be a single-stranded DNA molecule shown as a sequence 3 in a sequence table;

The primer 4 can be a single-stranded DNA molecule shown as a sequence 4 in a sequence table.

The primer combination B can comprise a primer 5, a primer 6, a primer 7 and a primer 8;

The primer 5 can be a single-stranded DNA molecule shown as a sequence 5 in a sequence table;

the primer 6 can be a single-stranded DNA molecule shown as a sequence 6 in a sequence table;

The primer 7 can be a single-stranded DNA molecule shown as a sequence 7 in a sequence table;

The primer 8 can be a single-stranded DNA molecule shown as a sequence 8 in a sequence table.

the primer 1, the primer 3, the primer 5 and the primer 7 are all labeled with fluorescence.

The 5' ends of the primer 1, the primer 3, the primer 5 and the primer 7 are all labeled with fluorescence.

The fluorescent label may specifically be a FAM label.

The primer combination A can specifically consist of the primer 1, the primer 2, the primer 3 and the primer 4.

The primer combination B can specifically consist of the primer 5, the primer 6, the primer 7 and the primer 8.

The primer combination can specifically consist of the primer combination A and the primer combination B.

The invention also protects the primer combination A.

The invention also protects the primer combination B.

Any one of the primer combinations can also comprise a primer combination C; the primer combination C can comprise a primer 9, a primer 10, a primer 11 and a primer 12;

the primer 9 can be a single-stranded DNA molecule shown as a sequence 9 in a sequence table;

The primer 10 can be a single-stranded DNA molecule shown as a sequence 10 in a sequence table;

the primer 11 can be a single-stranded DNA molecule shown as a sequence 11 in a sequence table;

the primer 12 can be a single-stranded DNA molecule shown as a sequence 12 in a sequence table.

the primer combination C can specifically consist of the primer 9, the primer 10, the primer 11 and the primer 12.

The primer combination can specifically consist of the primer combination A, the primer combination B and the primer combination C.

Any one of the primer combination A can also comprise the primer combination C.

the primer combination A can specifically consist of the primer 1, the primer 2, the primer 3, the primer 4 and the primer combination C.

Any one of the primer combinations B can also comprise the primer combination C.

the primer combination B can specifically consist of the primer 5, the primer 6, the primer 7, the primer 8 and the primer combination C.

Hereinbefore, 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 may 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.

The invention also provides a composite amplification system which comprises any one of the primer combinations, or any one of the primer combinations A, or any one of the primer combinations B.

The composite amplification system can specifically consist of any one of the primer combinations.

the composite amplification system can specifically comprise any one of the primer combinations A.

the composite amplification system can specifically comprise any one of the primer combinations B.

In the multiplex amplification system, the concentration of the primer 1, the primer 2, the primer 3 and the primer 4 in the multiplex amplification system is 0.01. mu.M. The concentration of the primer 5 and the primer 6 in the composite amplification system is 4.0 mu M. The concentration of the primer 7 and the primer 8 in the composite amplification system is 5.0 mu M. The concentration of the primer 9 and the primer 10 in the composite amplification system is 0.2 mu M. The concentration of the primer 11 and the primer 12 in the composite amplification system is 1.0. mu.M.

in the above composite amplification system, the composite amplification system may further comprise reagents required for performing a PCR amplification reaction; the "reagents required for performing a PCR amplification reaction" does not include primers required for a PCR amplification reaction.

The "reagents required for performing a PCR amplification reaction" may include at least one of DNA polymerase, dNTP, and Mg2 +. The concentration of the DNA polymerase in the multiplex amplification system may be 0.5U/. mu.L. The concentration of the dNTPs in the multiplex amplification system can be 0.2mmol/L (i.e., the concentrations of dATP, dTTP, dCTP, and dGTP are all 0.2 mmol/L). The concentration of the Mg2+ in the multiplex amplification system can be 1.5 mmol/L.

The composite amplification system can specifically comprise any one of the primer combinations and reagents required for carrying out PCR amplification reaction.

the composite amplification system can specifically comprise any one of the primer combinations A and reagents required for PCR amplification reaction.

the composite amplification system can specifically comprise any one of the primer combinations B and reagents required for PCR amplification reaction.

The invention also protects a kit A containing any one of the primer combinations; the kit A is used for identifying whether the body fluid to be detected is non-blood, menstrual blood or peripheral blood.

The invention also protects a kit B containing any one of the primer combinations A; the kit B is used for identifying whether the body fluid to be detected is blood or non-blood.

The invention also protects a kit C containing any one of the primer combinations B; the kit C is used for identifying whether the blood to be detected is menstrual blood or peripheral blood.

The preparation method of any one of the composite amplification system, the kit A, the kit B or the kit C also belongs to the protection scope of the invention.

the method for preparing any one of the composite amplification system, the kit A, the kit B or the kit C can comprise the step of packaging each primer in any one of the primer combinations, any one of the primer combinations A or any one of the primer combinations B independently.

the following X1) or X2) or X3) also belong to the scope of protection of the present invention.

x1) or the multiplex amplification system (especially the multiplex amplification system comprising the primer combination) in identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood.

X2) any one of the primer combination A or the multiplex amplification system (especially the multiplex amplification system comprising any one of the primer combination A) in the identification of the body fluid to be detected as blood or non-blood.

X3) the primer combination B or the multiplex amplification system (especially the multiplex amplification system comprising the primer combination B) in the identification of the blood to be detected as menstrual blood or peripheral blood.

the invention also protects S1) or S2) or S3).

S1) a method for identifying whether a body fluid to be tested is non-blood, menstrual blood or peripheral blood, which comprises the steps of: taking cDNA of body fluid to be detected as a template, and respectively adopting a primer pair 1, a primer pair 2, a primer pair 3 and a primer pair 4 to carry out PCR amplification to obtain PCR amplification products; the primer pair 1 consists of the primer 1 and the primer 2; the primer pair 2 consists of the primer 3 and the primer 4; the primer pair 3 consists of the primer 5 and the primer 6; the primer pair 4 consists of the primer 7 and the primer 8; then, the following judgment is made:

if the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 3 contains a DNA fragment with the size of 126bp, and the PCR amplification product obtained by adopting the primer pair 4 contains a DNA fragment with the size of 272bp, the body fluid to be detected is or is suspected to be menstrual blood;

if the PCR amplification product obtained by the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, and the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the body fluid to be detected is or is suspected to be peripheral blood;

if the PCR amplification product obtained by adopting the primer pair 1 does not contain a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 does not contain a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 3 does not contain a DNA fragment with the size of 126bp, and the PCR amplification product obtained by adopting the primer pair 4 does not contain a DNA fragment with the size of 272bp, the body fluid to be detected is or is suspected to be non-blood.

s2) a method for identifying whether a body fluid to be tested is blood or non-blood, comprising the steps of: carrying out PCR amplification by respectively adopting the primer pair 1 and the primer pair 2 by taking cDNA of body fluid to be detected as a template to obtain a PCR amplification product; then, the following judgment is made:

If the PCR amplification product obtained by adopting the primer pair 1 contains a DNA fragment with the size of 243bp, and the PCR amplification product obtained by adopting the primer pair 2 contains a DNA fragment with the size of 159bp, the body fluid to be detected is or is suspected to be blood;

If the PCR amplification product obtained by the primer pair 1 does not contain the DNA fragment with the size of 243bp, and the PCR amplification product obtained by the primer pair 2 does not contain the DNA fragment with the size of 159bp, the body fluid to be detected is or is suspected to be non-blood.

S3) a method for identifying whether blood to be tested is menstrual blood or peripheral blood, comprising the steps of: carrying out PCR amplification by respectively adopting the primer pair 3 and the primer pair 4 by taking cDNA of blood to be detected as a template to obtain a PCR amplification product; then, the following judgment is made:

if the PCR amplification product obtained by the primer pair 3 contains a DNA fragment with the size of 126bp, and the PCR amplification product obtained by the primer pair 4 contains a DNA fragment with the size of 272bp, the blood to be detected is or is suspected to be menstrual blood;

If the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, and the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the blood to be detected is or is suspected to be peripheral blood.

the invention also protects T1) or T2) or T3).

T1) a method for identifying whether a body fluid to be tested is non-blood, menstrual blood or peripheral blood, which comprises the steps of: taking cDNA of body fluid to be detected as a template, and respectively carrying out PCR amplification by adopting the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5 and the primer pair 6 to obtain PCR amplification products; then, the following judgment is made:

If the PCR amplification product obtained by the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 3 contains a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 contains a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be menstrual blood;

If the PCR amplification product obtained by the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be peripheral blood;

If the PCR amplification product obtained by the primer pair 1 does not contain a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 does not contain a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be non-blood.

T2) a method for identifying whether a body fluid to be tested is blood or non-blood, comprising the steps of: taking cDNA of body fluid to be detected as a template, and respectively carrying out PCR amplification by adopting the primer pair 1, the primer pair 2, the primer pair 5 and the primer pair 6 to obtain PCR amplification products; then, the following judgment is made:

if the PCR amplification product obtained by the primer pair 1 contains a DNA fragment with the size of 243bp, the PCR amplification product obtained by the primer pair 2 contains a DNA fragment with the size of 159bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be blood;

If the PCR amplification product obtained by adopting the primer pair 1 does not contain a DNA fragment with the size of 243bp, the PCR amplification product obtained by adopting the primer pair 2 does not contain a DNA fragment with the size of 159bp, the PCR amplification product obtained by adopting the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by adopting the primer pair 6 contains a DNA fragment with the size of 291bp, the body fluid to be detected is or is suspected to be non-blood.

t3) a method for identifying whether blood to be tested is menstrual blood or peripheral blood, which comprises the steps of: carrying out PCR amplification by using cDNA of blood to be detected as a template and adopting the primer pair 3, the primer pair 4, the primer pair 5 and the primer pair 6 respectively to obtain a PCR amplification product; then, the following judgment is made:

if the PCR amplification product obtained by the primer pair 3 contains a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 contains a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the blood to be detected is or is suspected to be menstrual blood;

If the PCR amplification product obtained by the primer pair 3 does not contain a DNA fragment with the size of 126bp, the PCR amplification product obtained by the primer pair 4 does not contain a DNA fragment with the size of 272bp, the PCR amplification product obtained by the primer pair 5 contains a DNA fragment with the size of 250bp, and the PCR amplification product obtained by the primer pair 6 contains a DNA fragment with the size of 291bp, the blood to be detected is or is suspected to be peripheral blood.

any of the above body fluids to be tested may be peripheral blood, menstrual blood, saliva, semen or vaginal secretion.

Any one of the above blood to be tested may be peripheral blood or menstrual blood.

Any of the above blood may be peripheral blood or menstrual blood.

Any of the above non-blood may be saliva, semen or vaginal fluid.

Any of the above-described PCR amplification products can be detected by capillary electrophoresis.

In one embodiment of the invention, 20 peripheral blood samples, 20 saliva samples, 20 semen samples, 20 menstrual blood samples and 20 vaginal secretion samples were obtained from 80 volunteers (aged 18-35 years) in the south of Henan. The composite amplification system provided by the invention is adopted to identify 80 samples, and the results are as follows: among 20 peripheral blood samples, more than 75% of the peripheral blood samples can detect the expression of the HBB gene, the HBA gene, the GAPDH gene and the ACTB gene, and any peripheral blood sample has no expression of the MMP7 gene and the MMP10 gene; in 20 menstrual blood samples, more than 75% of the menstrual blood samples can detect the expression of the HBA gene, the HBB gene, the MMP7 gene and the GAPDH gene, and 50% -75% of the menstrual blood samples can detect the expression of the MMP10 gene and the ACTB gene; more than 75% of 20 semen samples can detect the expression of GAPDH gene and ACTB gene, and any semen sample has no expression of HBB gene, HBA gene, MMP7 gene and MMP10 gene; of the 20 saliva samples, 50% -75% of the 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; of 20 vaginal secretion samples, more than 50% of the vaginal secretion samples can detect the expression of GAPDH gene and ACTB gene, 1 vaginal secretion sample can detect the expression of MMP10 gene, and 2 vaginal secretion samples can detect the expression of MMP7 gene. The result shows that the composite amplification system provided by the invention can identify whether the body fluid to be detected is blood, peripheral blood or menstrual blood, and can also identify whether the blood to be detected is peripheral blood or menstrual blood, and the accuracy is higher. The invention provides accurate scientific basis for determining case property, conviction and sentencing, and has important application value.

Drawings

FIG. 1 shows the detection of single PCR amplification specificity.

FIG. 2 shows the detection of the specificity of multiplex PCR amplification.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

the experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

the quantitative tests in the following examples, all set up three replicates and the results averaged.

in the following examples, all primers were synthesized by sangon. miRNeasy Mini Kit is a product of Qiagen, Germany. III First-Strand Synthesis System is a product of Invitrogen corporation, USA. HotstarTaq Polymerase is a product of Qiagen, Germany. Hi-Di formamide, molecular weight internal standard LIZ600 and 3500XL genetic analyzers are all products of Applied Biosystems, USA. The Mastercycler Nexus PCR instrument is a product of Eppendorf, Germany. Nanodrop 2000c UV Spectrophotometer is a product of Thermo Scientific, USA.