CN110540988A

CN110540988A - 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

Info

Publication number: CN110540988A
Application number: CN201910822152.2A
Authority: CN
Inventors: 赵一霞; 孙启凡; 季安全; 胡胜
Original assignee: Institute of Forensic Science Ministry of Public Security PRC
Current assignee: Institute of Forensic Science Ministry of Public Security PRC
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2019-12-06

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

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

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.

Example 1 preparation of 6 Gene-based multiplex amplification System

Screening of specific expression genes

the inventor of the invention obtains a large number of candidate genes specifically expressed in peripheral blood, menstrual blood, saliva, semen and vaginal secretion by consulting the literature, finally screens and obtains 4 specifically expressed genes (HBA gene, HBB gene, MMP7 gene and MMP10 gene) and two housekeeping genes (GAPDH gene and ACTB gene respectively) by comprehensively considering the sequence structure of each gene.

Preparation of composite amplification system based on 6 genes

Reasonably selecting different amplified fragment lengths to distinguish each gene according to the nucleotide sequence of each gene and the amplified fragment length range (between 100bp and 300 bp), and then preparing a composite amplification system based on 6 genes. The method comprises the following specific steps:

1. total RNA of menstrual blood was extracted using miRNeasy mini kit, and then quantified using a Nanodrop 2000c ultraviolet spectrophotometer, and the average value was calculated by repeating the same sample three times.

2. after the step 1 is completed, the total RNA of menstrual blood is taken and reverse transcription is carried out according to the method of the III First-Stand synthesis instruction to obtain the cDNA of menstrual blood.

The reverse transcription reaction system was 20. mu.L, consisting of 200ng of total menstrual blood RNA, 1. mu.L of dNTP (concentration of 2.5mmol/L), 1. mu.L of oligo (dT) (concentration of 50. mu.M), 2. mu.L of 10 XT Buffer, 4. mu.L of Mg2+ (concentration of 25mmol/L), 2. mu.L of DTT (concentration of 0.1mol/L), 1. mu.L of SuperScript III RT (concentration of 200U/. mu.L), 1. mu.L of NaseOUTTM (concentration of 40U/. mu.L), and water for enucleation. oligo (dT), 10 × RT Buffer, SuperScriptTM III RT and RNaseOUTTM are all components of the III First-stage synthesis system.

Reverse transcription reaction procedure: adding dNTP and oligo (dT) into total RNA of menstrual blood, treating at 65 ℃ for 5min, and cooling on ice for 1 min; then adding other reagents, mixing uniformly, placing in a Mastercycler Nexus PCR instrument, 50min at 50 ℃, 5min at 85 ℃; then, the mixture was placed on ice, and 1. mu.L of RNase H (concentration: 2U/. mu.L) was added thereto at 37 ℃ for 20 min.

the purpose of adding RNase H is to digest the untranscribed RNA.

3. Artificially synthesizing an upstream primer and a downstream primer for amplifying each gene, and carrying out PCR amplification to obtain a PCR amplification product; taking a 96-well plate, adding 0.5 mu L of PCR amplification product, 6000.5 mu L of molecular weight internal standard LIZ and 9 mu L of Hi-Di formamide into each well, uniformly mixing, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ for placing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a detection map. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 30 s. The results of the electrophoretic tests were then analyzed by GeneMapperID-X software, with a detection threshold of 50.

The PCR reaction system was 20. mu.L, and consisted of 1. mu.L of menstrual blood cDNA, 1. mu.L of 10 XPCR Buffer, 0.2. mu.L of dNTP (concentration of 2.5mmol/L), 0.6. mu.L of Mg2+ (concentration of 25mmol/L), 0.1. mu.L of LHottstar Taq Polymerase (concentration of 5U/. mu.L), 1. mu.L of forward primer (concentration of 10. mu.M), 1. mu.L of reverse primer (concentration of 10. mu.M), and 5.1. mu.L of deionized water. 10 XPCR Buffer is a module in HotstarTaq Polymerase.

the PCR reaction program is: 15min at 95 ℃; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 40 s; preserving at 72 deg.C for 10min and 4 deg.C.

Through the above steps, specific primers for each gene were obtained.

4. Combining the amplification conditions of each gene, selecting a proper amplification program, and then carrying out composite amplification. During multiplex amplification, whether specific products and non-specific products appear in each gene was observed. If non-specific hybrid peaks are generated among different gene primers, the genes need to be eliminated one by one, and the primers need to be redesigned and synthesized. In addition, primer dimers are formed between primers of different genes, which reduces the amplification efficiency of the primers and requires redesign and synthesis of primers.

5. and (2) repeatedly testing and modifying the composite amplification system, amplifying cDNA of menstrual blood by using the composite amplification system, and observing whether the genes are overlapped, wherein the migration rate is faster or slower due to inconsistent sequence structures of amplification products in the actual process, so that the theoretical size is not consistent with the actual size, and the genes are overlapped or too close to each other, thereby influencing the subsequent analysis. Once the phenomenon occurs, the gene primer needs to be redesigned and then goes through steps 3 and 4, and finally, a composite amplification system meeting the conditions is obtained.

6. Leveling of the multiplex amplification system. The method specifically comprises the following steps: during primary composite amplification, the concentration of each primer in a reaction system is 2 mu M; the concentration of each primer pair is adjusted according to the reaction result.

7. And optimizing the parameters of the composite amplification reaction. The cDNA of menstrual blood is used to adjust key components and links in the reaction system, such as optimal reaction system, number of amplification cycles, annealing temperature, extension time, amount of HotstarTaq Polymerase, amount of primers, concentration of buffer, and the like.

Through the steps, a composite amplification system based on 6 genes is obtained. The composite amplification system consists of Buffer, dNTP, Mg2+, DNA polymerase, primer mixture, cDNA of body fluid to be detected and water; the primer mixture is formed by mixing 12 primers; the names, nucleotide sequences, and lengths of the amplified fragments of the 12 primers are shown in columns 1-5 of Table 1. In the multiplex amplification system, the concentration of dNTP was 0.2mmol/L (i.e., the concentrations of dATP, dTTP, dCTP and dGTP were all 0.2mmol/L), the concentration of Mg2+ was 1.5mmol/L, and the concentrations of 12 primers are shown in column 6 of Table 1. Wherein the DNA Polymerase is HotstarTaq Polymerase, and the concentration of the DNA Polymerase in the composite amplification system is 0.5U/L; the Buffer is 10 XPCR Buffer, and the concentration of the 10 XPCR Buffer in the composite amplification system is 1X.

TABLE 1

Note: FAM indicates FAM labeling at the 5' end.

triple, single PCR amplification specificity detection

1. Specific detection of HBB Gene and HBA Gene

(1) Total RNA from peripheral blood was extracted using miRNeasy mini kit and then quantified using a Nanodrop 2000c UV spectrophotometer.

(2) After the step (1) is completed, the total RNA of peripheral blood is taken and reverse transcription is carried out according to the method of the III First-Stand synthesis system instruction, and cDNA of the peripheral blood is obtained.

(3) And carrying out PCR amplification by using a cDNA template of peripheral blood and adopting a primer pair 1 (consisting of a primer 1 and a primer 2) or a primer pair 2 (consisting of a primer 3 and a primer 4) to obtain a PCR amplification product.

(4) Taking a 96-well plate, adding 0.5 mu L of PCR amplification product, 6000.5 mu L of molecular weight internal standard LIZ and 9 mu L of Hi-Di formamide into each well, uniformly mixing, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ for placing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a detection map. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 30 s. The results of the electrophoretic tests were then analyzed by GeneMapperID-X software, with a detection threshold of 50.

2. specific detection of MMP7 gene, MMP10 gene, GAPDH gene, and ACTB gene

(1) Total RNA from menstrual blood was extracted using miRNeasy mini kit and then quantified using a Nanodrop 2000c ultraviolet spectrophotometer.

(2) After the step (1) is completed, the total RNA of menstrual blood is taken and reverse transcription is carried out according to the method of the III First-Stand synthesis system instruction, and then the cDNA of menstrual blood is obtained.

(3) Using a cDNA template of menstrual blood, and carrying out PCR amplification by using a primer pair 3 (consisting of a primer 5 and a primer 6), a primer pair 4 (consisting of a primer 7 and a primer 8), a primer pair 5 (consisting of a primer 9 and a primer 10) or a primer pair 6 (consisting of a primer 11 and a primer 12) to obtain a PCR amplification product.

The results are shown in FIG. 1(a is HBB gene, b is HBA gene, c is MMP7 gene, d is MMP10 gene, e is GAPDH gene, and f is ACTB gene). The result shows that each primer pair detects a specific peak and has specificity.

Fourth, multiple PCR amplification specificity detection

1. Peripheral blood sample

(3) and adding 1 mu L of cDNA of peripheral blood into 9 mu L of the composite amplification system prepared in the second step 3 of the example 1 for PCR amplification to obtain a PCR amplification product.

The detection result is shown as a in FIG. 2. The results showed that HBB gene, HBA gene, GAPDH gene and ACTB gene were expressed in peripheral blood.

2. menstrual blood sample

Peripheral blood was replaced with menstrual blood according to the method of step 1, and the other steps were not changed.

The detection result is shown in b in FIG. 2. The results showed that the HBB gene, HBA gene, MMP7 gene, MMP10 gene, GAPDH gene and ACTB gene were expressed in menstrual blood. Since menstrual blood has a complex composition in which a large number of blood cells are mixed, detection of positive HBB gene and HBA gene in a menstrual blood sample matches the expected result.

3. semen sample

Replacing peripheral blood with semen according to the method of step 1, and keeping other steps unchanged.

The results are shown in fig. 2 c. The results showed that only GAPDH gene and ACTB gene were expressed in semen.

4. Vaginal secretion sample

Peripheral blood was replaced with vaginal secretions according to the procedure of step 1, all other steps remaining unchanged.

the results are shown in fig. 2 d. The results showed that only GAPDH gene and ACTB gene were expressed in vaginal secretions.

5. Saliva sample

Peripheral blood was replaced with saliva according to the method of step 1, with no other steps.

The results are shown in FIG. 2, e. The results showed that only GAPDH gene and ACTB gene were expressed in saliva.

Example 2 application of 6-gene-based multiplex amplification system to identification of body fluid to be tested as non-blood, menstrual blood or peripheral blood

1. sample collection

The samples to be tested were 80 samples from 80 volunteers (aged 18-35 years) from the Henan region. 80 volunteers were unrelated and all gave informed consent.

20 samples with sample numbers PB1-PB20 were peripheral blood samples. Peripheral blood samples were obtained from venous blood drawn from the arms of a portion of volunteers and stored at-80 ℃.

20 samples with sample numbers MB1-MB20 were menstrual blood samples. 20 menstrual blood samples were collected 4 days before the menstrual cycle with a tampon from some volunteers, air dried at room temperature for 1 day, and stored at-80 deg.C.

The 20 samples with sample numbers SA1-SA20 were all saliva samples. 20 saliva samples were obtained by collecting a portion of the volunteer saliva in sterile plastic tubes (volunteer fasted for 1h before collection), -stored at 80 ℃.

the 20 samples with sample numbers SE1-SE20 were semen samples. 20 semen samples were obtained by collecting a portion of fresh semen from volunteers in sterile wide-mouth plastic cups (volunteers were prohibited for 2 days before collection), and stored at-80 ℃.

the 20 samples with sample numbers VA1-VA20 were vaginal fluid samples. 20 vaginal secretion samples were collected by a tampon from some volunteers during non-menstrual periods, air dried at room temperature for 1 day and stored at-80 ℃.

2. total RNA of samples to be detected is respectively extracted by using miRNeasy mini kit, and then quantification is carried out by using a Nanodrop 2000c ultraviolet spectrophotometer.

3. respectively taking the total RNA of the sample to be detected, and carrying out reverse transcription according to the method of the III First-stage synthesis system instruction so as to obtain the cDNA of the sample to be detected.

4. and (3) adding 1 mu L of cDNA of a sample to be detected into 9 mu L of the composite amplification system prepared in the step two in the embodiment 1 for PCR amplification to obtain a PCR amplification product.

5. Taking a 96-well plate, adding 0.5 mu L of PCR amplification product, 6000.5 mu L of molecular weight internal standard LIZ and 9 mu L of Hi-Di formamide into each well, uniformly mixing, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ for placing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a detection map. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 30 s. The results of the electrophoretic tests were then analyzed by GeneMapperID-X software, with a detection threshold of 50.

the statistical results are shown in Table 2. The results show that more than 75% of 20 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, which may be caused by that the samples are polluted by a small amount of menstrual blood when the collection time of part of the samples is around the menstrual period. The detection rates of housekeeping genes GAPDH and ACTB in different body fluids are greatly different.

TABLE 2

Note: 20 is the number of samples, and the number before "/" is the number of samples in which peaks were detected by capillary electrophoresis.

the results show that the composite amplification system prepared in example 1 can identify whether the sample is blood or non-blood, can also identify whether the blood to be detected is peripheral blood or menstrual blood, and has higher accuracy.

<110> material evidence identification center of public security department

<120> a multiplex amplification system for identifying whether a body fluid to be measured is non-blood, menstrual blood or peripheral blood, and a primer combination used in the same

<160> 12

<170> PatentIn version 3.5

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Claims

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 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 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;

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;