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

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

A compound amplification method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood The system and the primer combinations used

technical field

The invention belongs to the field of forensic technology, and in particular relates to a compound amplification system for identifying whether a body fluid to be tested is non-blood, menstrual blood or peripheral blood and a primer combination used therefor.

Background technique

By identifying the tissue source of body fluid stains at the crime scene, the source of sample tissue and its real criminal activities can be inferred, thereby providing important information for case characterization and scene reconstruction, and important scientific evidence for forensic science. Common bodily fluids at the scene of the case include peripheral blood, menstrual blood, saliva, semen, and vaginal secretions. Among them, although both peripheral blood and menstrual blood contain a large number of red blood cells, compared with peripheral blood, menstrual blood also contains a large number of mixtures such as endometrial fragments, cervical mucus, and vaginal secretions. Whether the on-site blood samples belong to peripheral blood or menstrual blood is a very important forensic scientific issue, which is often of great significance to the characterization of the case, reconstruction of the scene of the case, and provision of court evidence; especially for rape cases, cases of missing women and In some injury cases, female victims claim to have hematuria due to violent assault, in which case it is necessary to determine whether the source of blood cells in the urine is contaminated with menstrual blood. Therefore, the distinction between menstrual blood and peripheral blood, two blood-like body fluids, is still the focus and difficulty of forensic body fluid identification. Traditional bloodstain identification methods are mainly biochemical and immunological analysis methods, that is, using hemoglobin and its derivatives in blood for identification, but such methods are not only time-consuming and laborious, but also consume a lot of sample materials, and are prone to false positive or false negative results. A more sensitive and accurate identification method is urgently needed for the identification of trace samples obtained from crime scenes.

A large number of studies have shown that the detection of mRNA markers based on molecular biology is expected to become an effective method for the identification of the source of tissue properties of body fluid stains. This method relies on the unique expression profiles of mRNA in different body fluids to identify body fluids. It has the advantages of strong specificity, high sensitivity, and requires only a small amount of sample material. It is also compatible with the current DNA/RNA co-extraction technology, that is, simultaneous extraction from biological samples Extract DNA and mRNA, in which DNA is used for polymorphic typing, and mRNA is used for tissue source identification, which can not only improve work efficiency, but also make full use of trace samples. Research by the European DNA Typing Group has shown that in laboratories with no experience in RNA detection, mRNA analysis can still be used to successfully identify blood stains. After mixing different body fluids, RT-PCR multiplex amplification is performed, and the specificity of each body fluid tissue is successfully obtained. mRNA gene locus detection results, and accurate judgment on the source of body fluids.

Contents of the invention

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

The present invention first protects the primer combination, which may include primer combination A and primer combination B.

The primer combination A may include primer 1, primer 2, primer 3 and primer 4;

The primer 1 can be a single-stranded DNA molecule shown in sequence 1 in the sequence listing;

The primer 2 can be a single-stranded DNA molecule shown in sequence 2 in the sequence listing;

The primer 3 can be a single-stranded DNA molecule shown in sequence 3 in the sequence listing;

The primer 4 can be a single-stranded DNA molecule shown in sequence 4 in the sequence listing.

The primer combination B may include primer 5, primer 6, primer 7 and primer 8;

The primer 5 can be a single-stranded DNA molecule shown in sequence 5 in the sequence listing;

The primer 6 can be a single-stranded DNA molecule shown in sequence 6 in the sequence listing;

The primer 7 can be a single-stranded DNA molecule shown in sequence 7 in the sequence listing;

The primer 8 can be a single-stranded DNA molecule shown in sequence 8 in the sequence listing.

The primer 1, the primer 3, the primer 5 and the primer 7 are all labeled with fluorescent light.

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

The fluorescent label can specifically be a FAM label.

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

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

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

The present invention also protects the primer combination A.

The present invention also protects the primer combination B.

Any of the primer combinations described above may also include primer combination C; the primer combination C may include primer 9, primer 10, primer 11 and primer 12;

The primer 9 can be a single-stranded DNA molecule shown in sequence 9 in the sequence listing;

The primer 10 can be a single-stranded DNA molecule shown in sequence 10 in the sequence listing;

The primer 11 can be a single-stranded DNA molecule shown in sequence 11 in the sequence listing;

The primer 12 can be a single-stranded DNA molecule shown in sequence 12 in the sequence listing.

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

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

Any of the above-mentioned primer combination A may also include the primer combination C.

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

Any of the above-mentioned primer combinations B may also include the primer combination C.

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

Above, the molar ratio of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11 and primer 12 can be 0.01:0.01:0.01:0.01 :4.0:4.0:5.0:5.0:0.2:0.2:1.0:1.0.

The present invention also protects a composite amplification system, comprising any of the above-mentioned primer combinations, or, any of the above-mentioned primer combination A, or, any of the above-mentioned primer combination B.

The multiplex amplification system can be specifically composed of any one of the primer combinations described above.

The multiplex amplification system can be specifically composed of any one of the above-mentioned primer sets A.

The multiplex amplification system can be specifically composed of any one of the above-mentioned primer combinations B.

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

In the above composite amplification system, the composite amplification system may also include reagents required for PCR amplification reaction; the "reagents required for PCR amplification reaction" does not include primers required for PCR amplification reaction.

The "reagents required for PCR amplification reaction" may include at least one of DNA polymerase, dNTP and Mg ²⁺ . The concentration of the DNA polymerase in the multiplex amplification system may be 0.5U/μL. The concentration of the dNTP in the multiplex amplification system may be 0.2mmol/L (that is, the concentrations of dATP, dTTP, dCTP and dGTP are all 0.2mmol/L). The concentration of the Mg ²⁺ in the multiple amplification system may be 1.5mmol/L.

The multiplex amplification system may be specifically composed of any one of the primer combinations described above and reagents required for PCR amplification reactions.

The multiplex amplification system can be specifically composed of any one of the above-mentioned primer sets A and reagents required for PCR amplification reaction.

The multiplex amplification system can be specifically composed of any one of the above-mentioned primer combinations B and reagents required for PCR amplification reaction.

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

The present invention also protects the kit B containing any one of the above-mentioned primer combinations A; the kit B is used to identify whether the body fluid to be tested is blood or non-blood.

The present invention also protects the kit C containing any one of the above-mentioned primer combinations B; the kit C is used to identify whether the blood to be tested is menstrual blood or peripheral blood.

The preparation method of any one of the above multiplex amplification systems, kit A, kit B or kit C also belongs to the scope of protection of the present invention.

The preparation method of any of the above-mentioned composite amplification systems, kit A, kit B or kit C may include combining any of the above-mentioned primer combinations, any of the above-mentioned primer combination A or any of the above-mentioned The step of individually packaging each primer in primer combination B.

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

X1) Any of the above-mentioned primer combinations, or, any of the above-mentioned composite amplification systems (especially referring to the composite amplification system including any of the above-mentioned primer combinations) is useful in identifying whether the body fluid to be tested is non-blood, menstrual blood or Application in peripheral blood.

X2) Any of the above-mentioned primer combination A, or, any of the above-mentioned composite amplification systems (especially referring to the above-mentioned composite amplification system including any of the above-mentioned primer combination A) in identifying whether the body fluid to be tested is blood or non-blood in the application.

X3) Any of the above-mentioned primer combinations B, or, any of the above-mentioned composite amplification systems (especially referring to the composite amplification system including any of the above-mentioned primer combinations B) is useful in identifying whether the blood to be tested is menstrual blood or peripheral application in blood.

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

S1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, using primer pair 1, primer pair 2, primer pair 3 and primer pair respectively 4. Perform PCR amplification to obtain a PCR amplification product; the primer pair 1 is composed of the primer 1 and the primer 2; the primer pair 2 is composed of the primer 3 and the primer 4; the primer pair 3 is composed of the primer 5 and the primer 6; the primer pair 4 is composed of the primer 7 and the primer 8; and then judged as follows:

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

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

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

S2) A method for identifying whether the body fluid to be tested is blood or non-blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, respectively using the primer pair 1 and the primer pair 2 to perform PCR amplification to obtain PCR Amplify the product; then judge as follows:

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

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

S3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the blood to be tested as a template, respectively using the primer pair 3 and the primer pair 4 to perform PCR amplification to obtain PCR amplification product; then judge as follows:

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

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

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

T1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, using the primer pair 1, the primer pair 2, and the The primer pair 3, the primer pair 4, the primer pair 5 and the primer pair 6 carry out PCR amplification to obtain a PCR amplification product; then judge as follows:

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

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

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

T2) A method for identifying whether the body fluid to be tested is blood or non-blood, which may include the following steps: using the cDNA of the body fluid to be tested as a template, using the primer pair 1, the primer pair 2, the primer pair 5 and the primer pair respectively. Described primer is carried out PCR amplification to 6, obtains PCR amplification product; Then carry out following judgment:

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

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

T3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, which may include the following steps: using the cDNA of the blood to be tested as a template, using the primer pair 3, the primer pair 4, and the primer pair 5 respectively Carry out PCR amplification with described primer pair 6, obtain PCR amplification product; Then carry out following judgment:

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

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

Any of the aforementioned body fluids to be tested can be peripheral blood, menstrual blood, saliva, semen or vaginal secretions.

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

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

Any of the aforementioned non-blood can be saliva, semen or vaginal fluid.

Any of the aforementioned PCR amplification products can be detected by capillary electrophoresis.

In one embodiment of the present invention, 20 peripheral blood samples, 20 saliva samples, 20 semen samples, 20 menstrual blood samples and 20 A sample of vaginal fluid. The multiple amplification system provided by the present invention was used to identify 80 samples, and the results were as follows: among the 20 peripheral blood samples, the expression of HBB gene, HBA gene, GAPDH gene and ACTB gene could be detected in more than 75% of the peripheral blood samples, And there is no expression of MMP7 gene and MMP10 gene in any peripheral blood sample; in 20 menstrual blood samples, more than 75% of menstrual blood samples can detect the expression of HBA gene, HBB gene, MMP7 gene and GAPDH gene, 50%-75 The expression of MMP10 gene and ACTB gene can be detected in % of menstrual blood samples; the expression of GAPDH gene and ACTB gene can be detected in more than 75% of the 20 semen samples, and there is no HBB gene, HBA gene in any semen sample , MMP7 gene and MMP10 gene expression; in 20 saliva samples, GAPDH gene and ACTB gene expression could be detected in 50%-75% of the saliva samples, and any saliva sample had no HBB gene, HBA gene, MMP7 gene and MMP10 gene Gene expression; in 20 vaginal fluid samples, the expression of GAPDH gene and ACTB gene can be detected in more than 50% of vaginal fluid samples, the expression of MMP10 gene can be detected in 1 vaginal fluid sample, and the expression of MMP10 gene can be detected in 2 vaginal fluid samples The sample can detect the expression of MMP7 gene. The results show that the complex amplification system provided by the invention can identify whether the body fluid to be tested is blood, peripheral blood or menstrual blood, and can also identify whether the blood to be tested is peripheral blood or menstrual blood, all of which have high accuracy. The invention provides accurate scientific basis for clarifying the nature of the case, conviction and sentencing, etc., and has important application value.

Description of drawings

Figure 1 is a single-plex PCR amplification specific detection.

Figure 2 is the multiplex PCR amplification specific detection.

Detailed ways

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

The experimental methods in the following examples are conventional methods unless otherwise specified.

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

Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

In the following examples, all primers were synthesized by Sangon Company. miRNeasy Mini Kit is a product of Qiagen, Germany. III First-Strand Synthesis System is a product of Invitrogen Corporation of the United States. HotstarTaq Polymerase is a product of Qiagen, Germany. Hi-Di formamide, molecular weight internal standard LIZ600 and 3500XL genetic analyzer are all products of Applied Biosystems, USA. The MastercyclerNexus PCR instrument is a product of Eppendorf, Germany. Nanodrop 2000c ultraviolet spectrophotometer is a product of American ThermoScientific Company.

Embodiment 1, the preparation of the compound amplification system based on 6 genes

1. Screening of specifically expressed genes

The inventors of the present invention obtained a large number of candidate genes specifically expressed in peripheral blood, menstrual blood, saliva, semen, and vaginal secretions by consulting the literature, comprehensively considered the sequence structure of each gene, and finally screened and obtained 4 specifically expressed genes (respectively) HBA gene, HBB gene, MMP7 gene, MMP10 gene) and two housekeeping genes (GAPDH gene and ACTB gene respectively).

2. Preparation of multiple amplification system based on 6 genes

According to the nucleotide sequence of each gene and the length range of the amplified fragment (between 100bp-300bp), different amplified fragment lengths were reasonably selected to distinguish each gene, and then a multiple amplification system based on 6 genes was prepared. Specific steps are as follows:

1. The total RNA of menstrual blood was extracted with miRNeasy mini kit, and then quantified with a Nanodrop 2000c UV spectrophotometer. The same sample was repeated three times to calculate the average value.

2. After completing step 1, take the total RNA of menstrual blood and follow the The method of III First-Standsynthesissystem instruction manual is carried out reverse transcription, obtains the cDNA of menstrual blood.

The reverse transcription reaction system was 20 μL, consisting of 200ng total RNA of menstrual blood, 1 μL dNTP (concentration: 2.5 mmol/L), 1 μL oligo( ^dT ) (concentration: 25mmol/L), 2μL DTT (concentration: 0.1mol/L), 1μL SuperScriptTMⅢRT (concentration: 200U/μL), 1μL RNaseOUTTM (concentration: 40U/μL) and nuclease-free water. oligo(dT), 10×RT Buffer, SuperScriptTMⅢRT and RNaseOUTTM are all Components in the III First-Stand synthesis system.

Reverse transcription reaction procedure: add dNTP and oligo(dT) to the total RNA of menstrual blood, then treat at 65°C for 5 minutes, cool down on ice for 1 minute; then add other reagents, mix well, place in a Mastercycler Nexus PCR instrument, 50°C for 50 minutes, 5min at 85°C; then place on ice, add 1 μL RNase H (concentration: 2U/μL), and keep at 37°C for 20min.

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

3. Artificially synthesize the upstream and downstream primers used to amplify each gene, perform PCR amplification, and obtain PCR amplification products; take a 96-well plate, add 0.5 μL of PCR amplification products, molecular weight internal standard LIZ6000.5 μL and 9 μL of Hi-Di formamide was mixed, denatured at 95°C for 5 minutes, quickly transferred to -20°C for 5 minutes, and then detected by capillary electrophoresis with an ABI3130xl genetic analyzer to obtain a detection map. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

The PCR reaction system was 20 μL, consisting of 1 μL cDNA of menstrual blood, 1 μL 10×PCR Buffer, 0.2 μL dNTP (concentration: 2.5 mmol/L), 0.6 μL Mg ²⁺ (concentration: 25 mmol/L), 0.1 μL HotstarTaq Polymerase (concentration: 5 U/L) μL), 1 μL upstream primer (10 μM concentration), 1 μL downstream primer (10 μM concentration) and 5.1 μL deionized water. 10×PCRBuffer is a component of HotstarTaq Polymerase.

The PCR reaction program was: 95°C for 15 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s; 72°C for 10 min, and stored at 4°C.

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

4. Synthesize the amplification conditions of each gene, select the appropriate amplification program, and then carry out multiple amplification. During multiplex amplification, observe whether each gene has specific products and non-specific products. If there are non-specific miscellaneous peaks between different gene primers, it is necessary to eliminate them one by one, find out these genes, redesign and synthesize primers. In addition, primer dimers will be formed between the primers of different genes, which will reduce the amplification efficiency of the primers, and the primers need to be redesigned and synthesized.

5. Repeatedly test and modify the complex amplification system, use the complex amplification system to amplify the cDNA of menstrual blood, and observe whether there will be overlap between the genes, because in the actual process, the sequence structure of the amplification products will be inconsistent. The migration rate is too fast or too slow, resulting in a discrepancy between the theoretical size and the actual size, which often leads to overlapping or too close distances between genes, which affects subsequent analysis. Once such a phenomenon occurs, it is necessary to redesign the gene primers, go through steps 3 and 4, and finally obtain a complex amplification system that meets the conditions.

6. Leveling of the compound amplification system. Specifically: during the first multiplex amplification, the concentration of each primer in the reaction system is 2 μM; the concentration of each primer pair is adjusted according to the reaction result.

7. Optimize the multiplex amplification reaction parameters. Use the cDNA of menstrual blood to adjust the key components and links in the reaction system, such as the optimal reaction system, number of amplification cycles, annealing temperature, extension time, amount of HotstarTaq Polymerase, primer amount, buffer concentration, etc.

After the above steps, a multiple amplification system based on 6 genes was obtained. The composite amplification system is composed of Buffer, dNTP, Mg ²⁺ , DNA polymerase, primer mixture, cDNA of the body fluid to be tested and water; the primer mixture is composed of 12 primers; the names and nucleotide sequences of the 12 primers 1. The length of the amplified fragment is shown in columns 1-5 in Table 1. In this compound amplification system, the concentration of dNTP is 0.2mmol/L (that is, the concentration of dATP, dTTP, dCTP and dGTP is 0.2mmol/L), the concentration of Mg ² + is 1.5mmol/L, and the concentration of 12 primers See column 6 in Table 1. The DNA polymerase is HotstarTaq Polymerase, the concentration in the multiplex amplification system is 0.5U/L; the Buffer is 10×PCR Buffer, and the concentration of 10×PCR Buffer in the multiplex amplification system is 1×.

Table 1

Note: FAM means that the 5' end is labeled with FAM.

3. Single-plex PCR amplification specific detection

1. Specific detection of HBB gene and HBA gene

(1) The total RNA of peripheral blood was extracted by miRNeasy mini kit, and then quantified by Nanodrop 2000c UV spectrophotometer.

(2) After completing step (1), take the total RNA of peripheral blood, according to III First-Standsynthesis system manual method for reverse transcription, to obtain peripheral blood cDNA.

(3) Using the template of peripheral blood cDNA, PCR amplification is performed with primer pair 1 (composed of primer 1 and primer 2) or primer pair 2 (composed of primer 3 and primer 4) to obtain PCR amplification products.

(4) Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of internal molecular weight standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then ABI3130xl Genetic Analyzer was used for capillary electrophoresis detection, and the detection map was obtained. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

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

(1) The total RNA of menstrual blood was extracted by miRNeasy mini kit, and then quantified by Nanodrop 2000c UV spectrophotometer.

(2) After completing step (1), take the total RNA of menstrual blood, according to The method of III First-Standsynthesis system instruction manual is carried out reverse transcription, obtains the cDNA of menstrual blood.

(3) With the template of the cDNA of menstrual blood, primer pair 3 (composed of primer 5 and primer 6), primer pair 4 (composed of primer 7 and primer 8), primer pair 5 (composed of primer 9 and primer 10) were used Or perform PCR amplification on primer pair 6 (consisting of primer 11 and primer 12) to obtain a PCR amplification product.

(4) Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of internal molecular weight standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then ABI3130xl Genetic Analyzer was used for capillary electrophoresis detection, and the detection map was obtained. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

The detection results are shown in Figure 1 (a is the HBB gene, b is the HBA gene, c is the MMP7 gene, d is the MMP10 gene, e is the GAPDH gene, and f is the ACTB gene). The results showed that each primer pair detected specific peaks and had specificity.

4. Multiplex PCR amplification specific detection

1. Peripheral blood samples

(1) The total RNA of peripheral blood was extracted by miRNeasy mini kit, and then quantified by Nanodrop 2000c UV spectrophotometer.

(2) After completing step (1), take the total RNA of peripheral blood, according to III First-Standsynthesis system manual method for reverse transcription, to obtain peripheral blood cDNA.

(3) Take 9 μL of the composite amplification system prepared in step 2 of Example 1, add 1 μL of peripheral blood cDNA to perform PCR amplification, and obtain PCR amplification products.

(4) Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of internal molecular weight standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then ABI3130xl Genetic Analyzer was used for capillary electrophoresis detection, and the detection map was obtained. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

The test results are shown in Figure 2a. The results showed that HBB gene, HBA gene, GAPDH gene and ACTB gene were expressed in peripheral blood.

2. Menstrual blood samples

According to the method of step 1, the peripheral blood was replaced with menstrual blood, and the other steps were unchanged.

The test results are shown in Figure 2 b. The results showed that HBB gene, HBA gene, MMP7 gene, MMP10 gene, GAPDH gene and ACTB gene were expressed in menstrual blood. Due to the complex composition of menstrual blood and a large number of blood cells mixed in it, the positive results of HBB gene and HBA gene detected in menstrual blood samples were in line with the expected results.

3. Semen sample

According to the method of step 1, the peripheral blood was replaced with semen, and the other steps remained unchanged.

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

4. Vaginal fluid sample

According to the method of step 1, the peripheral blood was replaced with vaginal secretions, and other steps remained unchanged.

The test results are shown in Figure 2 d. The results showed that only GAPDH gene and ACTB gene were expressed in vaginal fluid.

5. Saliva sample

According to the method of step 1, the peripheral blood was replaced with saliva, and the other steps were unchanged.

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

Example 2. Application of multiple amplification system based on 6 genes in identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood

1. Sample collection

The samples to be tested were 80 samples provided by 80 volunteers (aged 18-35) from Henan. 80 volunteers were unrelated individuals, and all of them gave informed consent.

The 20 samples numbered PB1-PB20 are all peripheral blood samples. Peripheral blood samples were obtained from venous blood drawn from the arms of some volunteers and stored at -80°C.

The 20 samples numbered MB1-MB20 are all menstrual blood samples. The 20 menstrual blood samples were collected by some volunteers with tampons 4 days before the menstrual cycle, dried at room temperature for 1 day, and stored at -80°C.

The 20 samples numbered SA1-SA20 are all saliva samples. The 20 saliva samples were obtained by collecting saliva from some volunteers in sterile plastic tubes (the volunteers fasted for 1 h before collection), and stored at -80°C.

The 20 samples with sample numbers SE1-SE20 are all semen samples. 20 semen samples were obtained by collecting fresh semen from some volunteers in sterile wide-mouth plastic cups (the volunteers were abstinent for 2 days before collection), and stored at -80°C.

The 20 samples numbered VA1-VA20 are all vaginal fluid samples. The 20 vaginal fluid samples were collected by some volunteers with tampons during the non-menstrual period, dried at room temperature for 1 day, and stored at -80°C.

2. Use the miRNeasy mini kit to extract the total RNA of the samples to be tested, and then use the Nanodrop 2000c UV spectrophotometer for quantification.

3. Take the total RNA of the sample to be tested respectively, according to III First-Stand synthesissystem manual method for reverse transcription, to obtain the cDNA of the sample to be tested.

4. Take 9 μL of the composite amplification system prepared in step 2 of Example 1, add 1 μL of the cDNA of the sample to be tested for PCR amplification, and obtain PCR amplification products.

5. Take a 96-well plate, add 0.5 μL of PCR amplification product, 0.5 μL of molecular weight internal standard LIZ6000 and 9 μL of Hi-Di formamide to each well, mix well, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then use The ABI3130xl Genetic Analyzer performs capillary electrophoresis detection and obtains a detection map. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 30s. Afterwards, the electrophoresis detection results were analyzed with GeneMapperID-X software, and the detection threshold was 50.

See Table 2 for statistical results. The results showed that among the 20 peripheral blood samples, more than 75% of the peripheral blood samples could detect the expression of HBB gene, HBA gene, GAPDH gene and ACTB gene, and there was no expression of MMP7 gene and MMP10 gene in any peripheral blood sample; 20 Among menstrual blood samples, more than 75% of menstrual blood samples can detect the expression of HBA gene, HBB gene, MMP7 gene and GAPDH gene, and 50%-75% of menstrual blood samples can detect the expression of MMP10 gene and ACTB gene; Among the 20 semen samples, the expression of GAPDH gene and ACTB gene could be detected in more than 75% of the semen samples, and there was no expression of HBB gene, HBA gene, MMP7 gene and MMP10 gene in any semen sample; among the 20 saliva samples, 50 %-75% of saliva samples can detect the expression of GAPDH gene and ACTB gene, and any saliva sample has no expression of HBB gene, HBA gene, MMP7 gene and MMP10 gene; in 20 vaginal fluid samples, more than 50% of The expression of GAPDH gene and ACTB gene can be detected in vaginal fluid samples, the expression of MMP10 gene can be detected in 1 vaginal fluid sample, and the expression of MMP7 gene can be detected in 2 vaginal fluid samples, which may be due to the The collection time was before and after menstruation, which caused the sample to be polluted by a small amount of menstrual blood. The detection rates of housekeeping genes GAPDH and ACTB in different body fluids were quite different.

Table 2

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

The above results show that the multiple amplification system prepared in Example 1 can identify whether the sample is blood or non-blood, and can also identify whether the blood to be tested is peripheral blood or menstrual blood, with high accuracy.

<110> Physical Evidence Identification Center of the Ministry of Public Security

<120> A complex amplification system for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood and the primer combination used

<160> 12

<170> PatentIn version 3.5

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Claims (10)

1. Primer combinations, including primer combination A and primer combination B; Primer combination A includes primer 1, primer 2, primer 3 and primer 4; Primer 1 is a single-stranded DNA molecule shown in Sequence 1 in the sequence listing; Primer 2 is a single-stranded DNA molecule shown in Sequence 2 in the sequence listing; Primer 3 is a single-stranded DNA molecule shown in sequence 3 in the sequence listing; Primer 4 is a single-stranded DNA molecule shown in sequence 4 in the sequence listing; Primer combination B includes primer 5, primer 6, primer 7 and primer 8; Primer 5 is a single-stranded DNA molecule shown in sequence 5 in the sequence listing; Primer 6 is a single-stranded DNA molecule shown in sequence 6 in the sequence listing; Primer 7 is a single-stranded DNA molecule shown in sequence 7 in the sequence listing; Primer 8 is a single-stranded DNA molecule shown in sequence 8 in the sequence listing. 2. The primer combination A described in claim 1. 3. The primer combination B described in claim 1. 4. primer combination as claimed in claim 1, primer combination A as claimed in claim 2 or primer combination second as claimed in claim 3, characterized in that: said primer combination, said primer combination A or said primer combination B also including primer set C; Primer combination C includes primer 9, primer 10, primer 11 and primer 12; Primer 9 is a single-stranded DNA molecule shown in sequence 9 in the sequence listing; Primer 10 is a single-stranded DNA molecule shown in sequence 10 in the sequence listing; Primer 11 is a single-stranded DNA molecule shown in sequence 11 in the sequence listing; Primer 12 is a single-stranded DNA molecule shown in sequence 12 in the sequence listing. 5. A multiplex amplification system, comprising the primer combination according to claim 1 or 4, or the primer combination A according to claim 2 or 4, or the primer combination B according to claim 3 or 4. 6. composite amplification system as claimed in claim 5, is characterized in that: The concentration of primer 1, primer 2, primer 3 and primer 4 in the multiplex amplification system is 0.01 μM; The concentration of primer 5 and primer 6 in the multiplex amplification system is 4.0 μM; The concentration of primer 7 and primer 8 in the multiplex amplification system is 5.0 μM; The concentration of primer 9 and primer 10 in the multiplex amplification system is 0.2 μM; The concentration of primer 11 and primer 12 in the multiplex amplification system was 1.0 μM. 7. Kit A, Kit B or Kit C; A kit A containing the primer combination according to claim 1 or 4; said kit A is used to identify whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood; A kit B containing the primer combination A described in claim 2 or 4; said kit B is used to identify whether the body fluid to be tested is blood or non-blood; A kit C containing the primer combination B according to claim 3 or 4; said kit C is used to identify whether the blood to be tested is menstrual blood or peripheral blood. 8. X1) or X2) or X3): X1) the primer combination described in claim 1 or 4, or, the application of the composite amplification system described in claim 5 or 6 in identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood; X2) the primer combination A described in claim 2 or 4, or, the application of the composite amplification system described in claim 5 or 6 in identifying whether the body fluid to be tested is blood or non-blood; X3) The primer combination B described in claim 3 or 4, or, the application of the multiple amplification system described in claim 5 or 6 in identifying whether the blood to be tested is menstrual blood or peripheral blood. 9. S1) or S2) or S3): S1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, comprising the following steps: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1, primer pair 2, primer pair 3 and primer pair 4 Carry out PCR amplification, obtain PCR amplification product; Primer pair 1 is made up of primer 1 and primer 2 in claim 1; Primer pair 2 is made up of primer 3 and primer 4 in claim 1; Primer pair 3 is made up of primer in claim 1 5 and primer 6 form; Primer pair 4 is made up of primer 7 and primer 8 in claim 1; Carry out following judgment then: If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Contains a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using primer pair 4 contains a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be menstrual blood; If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be peripheral blood; If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 If the product does not contain a DNA fragment with a size of 126bp, and the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp, then the body fluid to be tested is or is suspected to be non-blood; S2) A method for identifying whether the body fluid to be tested is blood or non-blood, comprising the following steps: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1 and primer pair 2 to perform PCR amplification to obtain a PCR amplification product; and then Make the following judgments: If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243 bp, and the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159 bp, the body fluid to be tested is or is suspected to be blood; If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243 bp, and the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159 bp, then the body fluid to be tested is or is suspected to be non-blood; S3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, comprising the following steps: using the cDNA of the blood to be tested as a template, performing PCR amplification with primer pair 3 and primer pair 4 respectively, to obtain a PCR amplification product; Then make the following judgments: If the PCR amplification product obtained with primer pair 3 contains a DNA fragment with a size of 126 bp, and the PCR amplification product obtained with primer pair 4 contains a DNA fragment with a size of 272 bp, then the blood to be tested is or is suspected to be menstrual blood ; If the PCR amplification product obtained by using primer pair 3 does not contain a DNA fragment with a size of 126 bp, and the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272 bp, then the blood to be tested is or is suspected to be peripheral blood. 10. T1) or T2) or T3): T1) A method for identifying whether the body fluid to be tested is non-blood, menstrual blood or peripheral blood, comprising the following steps: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1, primer pair 2, primer pair 3, and primer pair 4 1. Primer pair 5 and primer pair 6 carry out PCR amplification, obtain PCR amplification product; Primer pair 1 is made up of primer 1 and primer 2 in claim 1; Primer pair 2 is made up of primer 3 and primer 4 in claim 1; Primer pair Pair 3 is made up of primer 5 and primer 6 in claim 1; Primer pair 4 is made up of primer 7 and primer 8 in claim 1; Primer pair 5 is made up of primer 9 and primer 10 in claim 1; Primer pair 6 is made up of Primer 11 and primer 12 in 1 are composed; then the following judgments are made: If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Contains a DNA fragment with a size of 126bp. The PCR amplification product obtained by using primer pair 4 contains a DNA fragment with a size of 272bp. The PCR amplification product obtained with primer pair 5 contains a DNA fragment with a size of 250bp. Using primer pair 6 If the obtained PCR amplification product contains a DNA fragment with a size of 291bp, the body fluid to be tested is or is suspected to be menstrual blood; If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 Does not contain a DNA fragment with a size of 126bp. The PCR amplification product obtained with primer pair 4 does not contain a DNA fragment with a size of 272bp. The PCR amplification product obtained with primer pair 5 contains a DNA fragment with a size of 250bp. If the PCR amplification product obtained in 6 contains a DNA fragment with a size of 291bp, the body fluid to be tested is or is suspected to be peripheral blood; If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 3 The product does not contain a DNA fragment with a size of 126bp. The PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp. The PCR amplification product obtained with primer pair 5 contains a DNA fragment with a size of 250bp. If the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, the body fluid to be tested is or is suspected to be non-blood; T2) A method for identifying whether the body fluid to be tested is blood or non-blood, comprising the steps of: using the cDNA of the body fluid to be tested as a template, respectively using primer pair 1, primer pair 2, primer pair 5 and primer pair 6 to carry out PCR amplification , to obtain the PCR amplification product; then carry out the following judgment: If the PCR amplification product obtained by using primer pair 1 contains a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 contains a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 5 Contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the body fluid to be tested is or is suspected to be blood; If the PCR amplification product obtained by using primer pair 1 does not contain a DNA fragment with a size of 243bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA fragment with a size of 159bp, and the PCR amplification product obtained by using primer pair 5 If the product contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the body fluid to be tested is or is suspected to be non-blood; T3) A method for identifying whether the blood to be tested is menstrual blood or peripheral blood, comprising the steps of: using the cDNA of the blood to be tested as a template, respectively using primer pair 3, primer pair 4, primer pair 5, and primer pair 6 to perform PCR amplification Increase to obtain the PCR amplification product; then make the following judgments: If the PCR amplification product obtained by using primer pair 3 contains a DNA fragment with a size of 126bp, the PCR amplification product obtained by using primer pair 4 contains a DNA fragment with a size of 272bp, and the PCR amplification product obtained by using primer pair 5 Contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the blood to be tested is or is suspected to be menstrual blood; If the PCR amplification product obtained by using primer pair 3 does not contain a DNA fragment with a size of 126bp, the PCR amplification product obtained by using primer pair 4 does not contain a DNA fragment with a size of 272bp, and the PCR amplification product obtained by using primer pair 5 If the product contains a DNA fragment with a size of 250bp, and the PCR amplification product obtained by using primer pair 6 contains a DNA fragment with a size of 291bp, then the blood to be tested is or is suspected to be peripheral blood.