CN116287317A

CN116287317A - Composite amplification system, primer and kit for identifying mixed body fluid

Info

Publication number: CN116287317A
Application number: CN202310361860.7A
Authority: CN
Inventors: 高知枭; 梁伟波; 肖园园; 屈胜秋; 陈德智; 刘桂宏; 郑亚子; 吴秋硕; 于在亮; 李迎澳; 陈初光
Original assignee: Suzhou Microread Gene Technology Co ltd
Current assignee: Suzhou Microread Gene Technology Co ltd
Priority date: 2023-04-06
Filing date: 2023-04-06
Publication date: 2023-06-23

Abstract

The invention discloses a composite amplification system, a primer and a kit for identifying mixed body fluid. The multiplex amplification system comprises primer sequences shown as SEQ ID NO. 1-48, and can amplify the following STR sites simultaneously: HBB, HBA, PPBP, HBD, ALAS2, AMI, PRM1, MSMB, KLK3, SEMG1, KLK2, PRM2, HTN3, STATH, MUC7, KRT13, KRT4, SPRR2A, MYOZ1, HBD1, CYP2B7P, β2-MG, UBC, and PGK. The invention can amplify a plurality of specific markers of four human body fluid spots (blood, semen, saliva and vaginal secretion) and housekeeping gene mRNA simultaneously, can rapidly and accurately analyze the human characteristic markers, further identify the types of common body fluid spots of forensics and help correctly identify the tissue sources of on-site biological detection materials of cases.

Description

Composite amplification system, primer and kit for identifying mixed body fluid

Technical Field

The invention belongs to the technical field of forensic detection and identification, and particularly relates to a composite amplification system, a primer and a kit for identifying mixed body fluid.

Background

In recent decades, DNA analysis technology in the forensic field has been helpful to find relevant evidence between criminals and crime sites, reducing the scope of suspects, and although DNA analysis methods have been widely accepted as important aids for identifying criminal cases, due to the diversity of case forms, inspection methods by DNA alone have failed to meet the needs of all criminal cases. The accurate identification of tissue sources of on-site biological detection materials is helpful for deducing the occurrence process and on-site reconstruction of cases, and the STR typing detection technology of DNA can judge individual sources of common biological detection materials such as saliva, blood, semen and the like, but the existing DNA detection kit cannot distinguish different body fluid types from the same individual due to the identity of the DNA.

Different tissues and fluids each contain their unique protein or enzyme molecules. Traditional forensic physics and physics experiments have mainly detected these specific protein molecules through some enzymatic reactions or immunological tests to screen and confirm various body fluids and tissues. The traditional identification methods are generally simple, convenient and quick, have higher specificity and accuracy on small body fluid tissues such as blood marks, sperm spots and the like, but have certain limitations. Firstly, cross reaction exists between part of body fluid and tissues, so that the specificity of the body fluid is reduced; secondly, the traditional identification method can damage the detection material, and the identification methods of different tissues are difficult to be compatible, so that additional detection material is consumed, which is extremely unfavorable for biological detection materials with small quantity; again, the enzyme protein is not as stable as DNA, making detection of old or spoiled degradation detection materials difficult. In addition, the result judgment of the traditional method mostly depends on the level and experience of operators, and the risk of misjudgment caused by human factors is increased.

Currently, forensic science identifies tissue sources with widely used molecular markers mainly including three types of messenger RNA (mRNA), microRNA (MicroRNA, miRNA), and DNA methylation; in addition, the microbial flora may also be used for identification of sources of body fluids. Thus, there are mainly the following methods for detecting the type of body fluid spot:

1. MicroRNA analysis

Micrornas (microRNAs, miRNAs) are a class of endogenous non-coding small molecule RNAs ranging in length from 16-29nt, with an average length of 22nt, found widely in a variety of eukaryotic cells. miRNAs are known as "modulators" of gene expression by either directly cleaving target mRNA or inhibiting translation of target mRNA by fully/incompletely complementarily binding to the 3' untranslated region of target mRNAs, thereby participating in the regulation of cell growth, differentiation, apoptosis, senescence, disease and tumor development. It has been demonstrated that there are differences in the expression of miRNAs between different body fluids and tissues, rendering miRNAs a novel molecular tool for forensic tissue source identification.

Studies have shown that miRNAs are resistant to rnase degradation, and their content and molecular structure are not significantly altered when strong acids, strong bases or temperature extremes are added, and that miRNAs are small in size, so that the biggest advantage of miRNAs detection is greater sensitivity and stability than tissue/body fluid identification using mRNA. However, it should be noted that the expression of miRNAs is relative, and that detection thereof relies on RT-PCR, and that normalization is difficult.

2. Tissue differential methylation assay

Numerous studies have demonstrated that there are numerous regions of tissue-specific differential methylation (tissuespecific differentially methylated regions, tdrs) between different mammalian tissues, thereby conferring DNA methylation profiles to various cells and tissues, which lay a theoretical basis for forensic body fluids and tissue identification using DNA methylation markers. Fan Guangyao and the like, and the tDMRs of the promoter region of the DDX4 gene are shown to be specific to semen and have high methylation level, so that semen and non-semen can be effectively identified. Lee et al used a bisulphite sequencing method to select the tDMRs of 5 genes for methylation analysis, found that the tDMRs of both DACT1 and USP49 genes showed semen-specific hypomethylation, and used MSP technique to initially identify the presence of sperm plaques. The research adopts the technical methods including methylation sensitive restriction enzyme digestion, specific target site complex amplification, capillary electrophoresis and automatic fluorescence detection technology, is completely compatible with the existing DNA typing technology, so that no extra sample is consumed, and the sensitivity comparable with DNA typing is shown, so that the tissue specific DNA methylation is expected to become a novel genetic marker with good application prospect in tissue identification. Currently, the major detection techniques for DNA methylation are pyrosequencing and second generation sequencing, which are incompatible with conventional STR detection; DNA methylation detection relies primarily on bisulfite conversion, but this method results in degradation of DNA and is therefore unsuitable for degradation/trace amounts of materials. Furthermore, DNA methylation is affected by a variety of factors, which may have a certain impact on forensic applications of tissue-specific differential methylation.

3. Microbiological method

The bacteria can only specifically survive in specific body fluid tissue or body fluid tissue by utilizing different flora, and is suitable for old examination materials, and the bacterial concentration in the body fluid of a human body is 10 ⁸ ～10 ⁹ Around cfu/mL, this concentration ensures the possibility of microbiological testing of body fluid patches. However, the detected microorganism species of the sample will be changed due to the growth or degradation of the microorganism itself; and along with the degradation of human tissue components, the growth of microorganisms in the environment and other reasons, the micro-organisms are generatedThe biological species may also vary.

4. Tissue/body fluid specific mRNA assay

The protein expression profile varies from tissue to tissue due to physiological function differences, which is the theoretical basis for body fluid/tissue identification using mRNA. Numerous studies have demonstrated that different body fluids or tissues have their specifically expressed mRNA genes. For example, blood-specific genes SPTB, PBGD, glycoA, HBA, HBB and ALAS2, etc.; saliva specific genes stat h and HTN3; semen specific genes PRM1 and KLK2, SEMG1, and the like; menstrual blood specific genes MMP7, 10 and 11; skin tissue gene CDSN, LOR, KRT, vaginal secretion HBD1, MUC4, and the like. The identification of tissue/body fluid sources by tissue-specific mRNA has the advantage of not only higher specificity and sensitivity, but also sharing an analytical platform with current DNA typing.

The method has the main advantages of high sensitivity and high specificity, can detect the specific markers of various body fluids in one composite reaction, is compatible with the current nucleic acid extraction and analysis flow, saves the experiment cost and has practical value; mRNA is also the earliest marker used for identifying body fluids, and can be said to be the most deeply studied molecular marker for identifying body fluids, and has great potential in identifying mixed spots. The system for identifying the body fluid spots on the market is limited to a scientific research stage, a mature kit aiming at the mRNA sites and capable of identifying common body fluid spot types does not appear yet, and the development of the kit for detecting the body fluid types by utilizing the mRNA sites is a new development direction.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a compound amplification system, a primer and a kit for identifying mixed body fluid, and the system simultaneously amplifies a plurality of specific markers of four human body fluid spots (blood, semen, saliva and vaginal secretion) and housekeeping gene mRNA, so that human characteristic marker analysis can be rapidly and accurately carried out, thereby identifying common body fluid spot types of forensics and helping to accurately identify tissue sources of on-site biological detection materials of cases.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a composite amplification system for identifying mixed body fluid, which comprises primer sequences shown in SEQ ID NO. 1-48, and can simultaneously amplify the following STR sites:

HBB, HBA, PPBP, HBD, ALAS2, AMI, PRM1, MSMB, KLK3, SEMG1, KLK2, PRM2, HTN3, STATH, MUC7, KRT13, KRT4, SPRR2A, MYOZ1, HBD1, CYP2B7P, β2-MG, UBC, and PGK; specific site and amplification primer information is shown in table 1:

TABLE 1 sites and amplification primers

According to the invention, through expression trend and specificity research of the characteristic marker, the 24 selected sites comprise 6 sites AMI, PPBP, ALAS, HBD, HBB and HBA of blood; semen 6 sites PRM1, PRM2, MSMB, KLK3, SEMG1 and KLK2; saliva 6 sites HTN3, STATH, MUC7, KRT4, KRT13 and SPRR2A; 3 sites of vaginal secretion MYOZ1, HBD1, CYP2B7P; 3 positions beta 2-MG, UBC, PGK of housekeeping genes. These body fluid spots have good site specificity, and housekeeping genes are stably expressed in various types of body fluid spots.

Further, the 5' end of the primer pair was fluorescently labeled, as FAM, HEX, TAMRA and ROX, respectively.

Further, the 24 sites are divided into four groups, wherein the first group is CYP2B7P, HBD1, MYOZ1, beta 2-MG, UBC and PGK, and the fluorescent label of the primer pair for amplifying the CYP2B7P, HBD1, MYOZ1, beta 2-MG, UBC and PGK sites is FAM;

the second group is HTN3, stat h, MUC7, KRT4, KRT13, and SPRR2A, the fluorescent label of the primer pair amplifying HTN3, stat h, MUC7, KRT4, KRT13, and SPRR2A sites is HEX;

the third group is PRM1, PRM2, MSMB, KLK3, SEMG1 and KLK2, and the fluorescent label of the primer pair for amplifying the sites of PRM1, PRM2, MSMB, KLK3, SEMG1 and KLK2 is TAMRA;

the fourth group is AMI, PPBP, ALAS, HBD, HBB and HBA, and the fluorescent label of the primer pair amplifying AMI, PPBP, ALAS, HBD, HBB and HBA sites is ROX.

Further, the mixed body fluid includes at least one of blood, saliva, semen, and vaginal secretions.

A primer combination for amplifying the composite amplification system comprises sequences shown as SEQ ID NO. 1-48.

The invention adopts a four-color fluorescent marking system to group and perform fluorescent marking on the 24 loci: FAM markers MYOZ1, HBD1, CYP2B7P, β2-MG, UBC and PGK; HEX markers HTN3, STATH, MUC7, KRT4, KRT13 and SPRR2A; TAMRA marks PRM1, PRM2, MSMB, KLK3, SEMG1 and KLK2; R0X marks AMI, PPBP, ALAS, HBD, HBB and HBA.

Meanwhile, in designing mRNA primers, it is required that one of the primers spans at least one exon-exon junction to ensure that the primer does not bind to DNA and that the specificity of mRNA product is ensured. Constructing a composite system according to the length of the products of the screening sites, and requiring the sites with different fluorescent modifications to be mutually spaced, so that the sites cannot be mutually influenced. And mixing all the primers for a multiplex amplification experiment to determine that no non-specific amplification phenomenon exists. Meanwhile, the standard substance in the detection component adopts Orange mark, so that the size of the amplified product of each site can be clearly distinguished by the mark.

A kit for identifying a mixed body fluid, the kit comprising the primer combination of claim 5.

Further, buffer, template cDNA and Taq DNA polymerase are also included.

Further, the PCR buffer composition includes: l0mM ammonium sulfate, 10mM potassium chloride, 50mM Tris-HCl pH8.3, 2mM magnesium ions and 0.2mM dNTPs, taq DNA polymerase was used in an amount of 2U.

Further, the reaction conditions for the amplification of the amplification system are:

step 1: denaturation at 95 ℃ for 5 min, step 2: denaturation at 94℃for 20 seconds, step 3 annealing at 59℃for 90 seconds, step 4: extending at 60 ℃ for 60 minutes, repeating the steps from 2 to 3 for 28 times, and finally extending at 15 ℃.

A method for analyzing and identifying STR locus in mixed body fluid is characterized by that said multiplex amplification system, primer combination or kit is used to detect DNA.

The use of the above multiplex amplification system, primer combination or kit for individual identification, paternity testing, population genetics analysis and/or construction of a human DNA database and body fluid identification.

The invention has the beneficial effects that:

1. containing 24 mRNAs, common specific markers and housekeeping genes covering four body fluid plaques. The information quantity is large, and the compatibility is good.

2. The template has wide application range, and is suitable for forensic cDNA analysis in material evidence cases involving four body fluid spots (blood, semen, saliva and vaginal secretion) cells.

3. The system has good specificity and stability, and no non-specific amplification product is generated after repeated verification for many times, so that the signal intensity is stable.

4. The sensitivity is high, the minimum detection limit can reach 0.1ng, and the type of the body fluid spot can be accurately judged through the cDNA template quantity of 0.1ng.

Drawings

FIG. 1 is a diagram of amplification of cDNA extracted from blood;

FIG. 2 is a diagram of amplification of extract cDNA of fine spots;

FIG. 3 is a saliva extracted cDNA amplification map;

FIG. 4 is a cDNA amplification map of vaginal secretion extraction;

FIG. 5 is an amplification of cDNA extracted from a 1:1 mixture of saliva and vaginal secretions;

FIG. 6 is an amplification plot of cDNA extracted from a 1:1 mixture of blood and semen;

FIG. 7 is an amplification plot of cDNA extracted from a 1:25 mixed sample of blood and saliva;

FIG. 8 is an amplification of cDNA extracted from a 1:25 mixed sample of blood and vaginal secretions;

FIG. 9 is an amplification plot of cDNA extracted from a 1:25 mix of semen and saliva;

FIG. 10 is a schematic of cDNA amplification from semen and vaginal secretion 1:25 samples;

FIG. 11 is a reverse transcription amplification plot of 0.1ng RNA extracted from a blood sample;

FIG. 12 is a reverse transcription amplification plot of 0.1ng RNA extracted from semen samples;

FIG. 13 is a reverse transcription amplification plot of 50ng RNA extracted from saliva samples;

FIG. 14 is a reverse transcription amplification plot of 10ng RNA extracted from vaginal secretion samples;

FIG. 15 is a drawing of cDNA amplification extracted from a real case sample;

FIG. 16 is a drawing showing cDNA amplification by extracting a sample of a real case.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

Example 1STR site screening

The protein expression profile varies from tissue to tissue due to physiological function differences, which is the theoretical basis for body fluid/tissue identification using mRNA. Numerous studies have now demonstrated that different body fluids or tissues have their specifically expressed mRNA genes. For example, blood-specific genes SPTB, PBGD, glycoA, HBA, HBB and ALAS2, etc.; saliva specific genes stat h and HTN3; semen specific genes PRM1 and KLK3, SEMG1, and the like; menstrual blood specific genes MMP7, 10 and 11; skin tissue gene CDSN, LOR, KRT, HBD1, MUC4, MUC7, etc. of vaginal secretion.

Finally, a large number of literature queries and experimental tests confirm that the characteristic marker with high expression quantity and good specificity and housekeeping genes with stable expression in various body fluid types are selected as sites of a composite amplification system, wherein the sites comprise 6 sites AMI, PPBP, ALAS, HBD, HBB and HBA of blood; semen 6 sites PRM1, PRM2, MSMB, KLK3, SEMG1 and KLK2; saliva 6 sites HTN3, STATH, MUC7, KRT4, KRT13 and SPRR2A; 3 sites of vaginal secretion MYOZ1, HBD1 and CYP2B7P; the 3 sites of the housekeeping gene are beta 2-MG, UBC and PGK.

Thus, the combination forms 24 specific marker loci amplified in one PCR reaction system. The sites used were AMI, HBB, HBA, PPBP, HBD, ALAS, PRM1, MSMB, KLK3, SEMG1, KLK2, PRM2, HTN3, STATH, MUC7, KRT13, KRT4, SPRR2A, MYOZ1, HBD1, CYP2B7P, beta 2-MG, UBC and PGK.

Example 2 primer design

The primers are designed by adopting software such as Primer5, NCBI Blast and the like, and the Tm value of each Primer is ensured to be within the range of (60+/-3) DEG C as much as possible when the primers are designed, the amplification efficiency is similar, and the amplified products of each pair of primers are ensured to be more than 10bp in size. After the design is completed, the interaction between primer dimer and different primers is analyzed by software such as AutoDimer, if the interaction can generate nonspecific products or the dimer needs to be redesigned, until the primer sequence meeting the requirement is obtained.

The PCR system and the amplification conditions are adjusted according to the electrophoresis result to obtain the amplification conditions common to 24 pairs of primers. It is finally desirable that all primer pairs appear as bright and more single bands of interest under the same system and amplification conditions. The sequence of the designed primer is shown as SEQ ID NO. 1-48.

Example 3 separate detection of different types of body fluid plaque samples

1. Four body fluid spot detection alone (four samples are from the department of public security material evidence identification center)

2. RNA extraction and cDNA Synthesis

Four kinds of body fluid spot total RNA are extracted by TRIzol method, after the RNA extraction is completed, the total RNA is subjected to reverse transcription by using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) reverse transcription kit to synthesize cDNA.

3. The reaction system:

the reaction reagents (Buffer, primer Mix, dNTP, etc.) are mixed in an oscillating way, and then mixed according to the volume ratio of the table (except the templates), and the mixture is prepared into a PCR reaction mixture, and 9 mu L of the mixture is packaged in a PCR reaction tube, and finally 1 mu L of the template is added into each reaction tube, and the mixture is centrifuged and then enters the next step. The composition of the reaction system is shown in Table 2.

TABLE 2 Standard amplification reaction System

Component name	Volume (mu L)
		Sterile deionized water	3
2.5 Xreaction buffer	4
		Primer mixture	2
Template cDNA	1
		Total volume of	10μL

4. PCR reaction procedure:

the PCR reaction tube was placed on an amplicon apparatus, and the following procedure was designed and run:

step 1: denaturation at 95 ℃ for 5 min, step 2: denaturation at 94℃for 20 seconds, step 3 annealing at 59℃for 90 seconds, step 4: extending at 60 ℃ for 60 minutes, repeating the steps from 2 to 3 for 28 times, and finally extending at 15 ℃. After the operation is finished, the product is stored in a refrigerator at 4 ℃.

5. Capillary electrophoresis detection

QD550 internal standard and formamide were proportioned to 2.5:100, 9. Mu.L of the mixture was added to a 96-well plate, 1. Mu.L of the amplification product sample was added thereto, and the mixture was allowed to stand for several minutes, centrifuged, and then placed on a genetic analyzer 3130, and ready for detection.

6. Data analysis

The original data is imported, a File menu of the main page is selected Add sample to Project, a sample File is found, a folder is selected, an add to list is clicked, an add is clicked, and the sample File is displayed in a Project window: analysis parameters are selected. Analysis methods, panels, and size standards are defined. Browsing the original data of sample electrophoresis, optionally selecting a sample file name, and selecting 'raw data' under a 'sample' menu. Moving the tracking line to stop the cursor at the right side of the primer peak (before the first red internal standard peak), and taking the value displayed on the X axis at the lower left corner of the window at the moment as a starting point in analysis method analysis parameters; clicking the green analysis button, and displaying a save project dialog box, naming and saving, and the software starts to process the data, and the lower left corner after analysis is completed is displayed analysis completed. The resulting data were analyzed using GeneMapper software and a map was generated.

7. Experimental results

FIG. 1 shows the amplification of a blood sample, where 6 markers AMI, PPBP, ALAS, HBD, HBB, HBA of blood and housekeeping gene β2-MG can be detected; FIG. 2 shows saliva sample amplification, 6 markers HTN3, STATH, MUC7, KRT4, KRT13, SPRR2A and housekeeping gene β2-MG of saliva can be detected; FIG. 3 shows the amplification of semen samples, where 6 markers PRM1, PRM2, MSMB, KLK3, SEMG1, KLK2 and housekeeping genes β2-MG, UBC, PGK can be detected; FIG. 4 shows vaginal discharge sample amplification, and the 2-characteristic markers HBD1, CYP2B7P and housekeeping gene beta 2-MG, UBC, PGK of vaginal discharge can be detected.

As can be seen from the detection results of FIGS. 1 to 4, the primer pair designed in the application can well amplify sample cDNA derived from four types of body fluid spots alone, and the corresponding feature markers and housekeeping genes can be detected. The primer designed by the application has good specificity and accuracy.

Example 4 detection of Mixed body fluid spotting

1. The mixed body fluid spot sample is obtained by mixing body fluid spot samples of a material evidence identification center of the public security department

2. Preparation of body fluid spot mixed sample

The body fluid spot sample of the physical evidence identification center of the public security department is mixed according to a specific proportion to obtain different types of body fluid spot mixed samples, which comprises the following specific steps: saliva and vaginal secretion 1:1 mixed sample, blood and semen 1:1 mixed sample, blood and saliva 1:25 mixed sample, blood and vaginal secretion 1:25 mixed sample, semen and saliva 1:25 mixed sample, semen and vaginal secretion 1:25 sample.

3. The reaction system:

4. PCR reaction procedure:

5. Capillary electrophoresis detection

6. Data analysis

7. Experimental results

FIG. 5 shows a 1:1 mixed sample of saliva and vaginal secretions, with detection of the vaginal secretion signatures HBD1, MYOZ1, CYP2B7P, saliva signatures KRT4, STATH, KRT13, SPRR2A, HTN3, housekeeping genes β2-MG, UBC, PGK.

FIG. 6 shows the amplification of a 1:1 mix of blood and semen, the detection of the semen signatures PRM1, PRM2, MSMB, KLK3, SEMG1, KLK2, the blood signatures AMI, PPBP, ALAS, HBD, HBB, HBA, housekeeping genes β2-MG, UBC, PGK.

FIG. 7 shows amplification of a 1:25 mixed sample of blood and saliva, the blood signatures AMI, PPBP, ALAS, HBD, HBB, HBA, saliva signatures KRT4, STATH, KRT13, SPRR2A, MUC7, HTN3, housekeeping genes β2-MG, UBC, PGK were detected.

FIG. 8 shows a 1:25 mixed sample of blood and vaginal secretion, with blood signatures AMI, PPBP, ALAS, HBD, HBB, HBA detected, vaginal secretion signatures HBD1, MYOZ1, CYP2B7P, housekeeping genes β2-MG, UBC, PGK.

FIG. 9 shows a 1:25 mix of semen and saliva, with the markers PRM1, PRM2, MSMB, KLK3, SEMG1, KLK2, saliva markers KRT4, STATH, KRT13, SPRR2A, HTN3, housekeeping genes β2-MG, UBC, PGK detected.

FIG. 10 shows a 1:25 sample of semen and vaginal secretions, with the detection of the semen characteristic markers PRM1, PRM2, MSMB, KLK3, SEMG1,

KLK2, characteristic markers of vaginal secretion HBD1, MYOZ1, CYP2B7P, housekeeping genes beta 2-MG, UBC, PGK.

According to the detection results shown in fig. 5 to 10, the multiplex amplification system is shown to be applicable to detection of mixed samples, and the type of mixing can be confirmed according to the characteristic markers corresponding to the body fluid spots shown by the results.

Example 5 sensitivity detection

1. RNA extraction, dilution and cDNA synthesis

Four kinds of body fluid spot total RNA are extracted by adopting a TRIzol method, after the RNA extraction is finished, the RNA extracting solution is diluted into different RNA template amounts (50, 25, 10, 5, 1, 0.5 and 0.1 ng), and the RNA with different template amounts is subjected to reverse transcription by using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) reverse transcription kit to synthesize cDNA.

2. Reaction system

The reaction reagents (Buffer, primer Mix, dNTP, etc.) are mixed in an oscillating way, and then mixed according to the volume ratio of the table (except the templates), and the mixture is prepared into a PCR reaction mixture, and 9 mu L of the mixture is packaged in a PCR reaction tube, and finally 1 mu L of the template is added into each reaction tube, and the mixture is centrifuged and then enters the next step. The composition of the reaction system is shown in Table 3.

TABLE 3 reaction system

3. PCR reaction procedure

4. Capillary electrophoresis detection

5. Data analysis

FIG. 11 is a reverse transcription amplification plot of 0.1ng RNA extracted from a blood sample; FIG. 12 is a reverse transcription amplification plot of 0.1ng RNA extracted from semen samples; FIG. 13 is a reverse transcription amplification plot of 50ng RNA extracted from saliva samples; FIG. 14 is a reverse transcription amplification plot of 10ng RNA extracted from vaginal secretion samples. As is clear from the detection results shown in FIGS. 11 to 14, the minimum detection limit of the detection system constructed based on the designed detection primer in the present application can be as low as 0.1ng.

Example 5 true unknown sample detection

1. Case sample detection (case sample is from the department of public security material evidence identification center).

2. RNA extraction and cDNA Synthesis

Extracting total RNA of the mixed body fluid spots by adopting a TRIzol method, and carrying out reverse transcription on the total RNA by using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) reverse transcription kit to synthesize cDNA after the RNA extraction is finished.

3. The reaction system:

4. PCR reaction procedure:

5. Capillary electrophoresis detection

6. Data analysis

7. Experimental results

Fig. 15 shows that the characteristic markers HBD1 of vaginal secretion, and the characteristic markers PPBP, ALAS2, HBD, HBB, HBA of blood, housekeeping gene β2-MG, UBC, PGK, were detected, and the sample was judged to be a mixture of vaginal secretion and blood. FIG. 16 detection of saliva signature markers KRT4, KRT13, SPRR2A, housekeeping gene β2-MG, judged that the sample was saliva.

According to the detection results shown in fig. 15 and 16, it is shown that the amplification system designed by the application can be used for detecting specific compositions in unknown body fluid, can be used for detecting real case samples, and can confirm sample types according to the body fluid spot corresponding feature markers displayed by the results.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.

Claims

1. A composite amplification system for identifying mixed body fluids, characterized in that the composite amplification system comprises primer sequences shown as SEQ ID NO. 1-48, and can simultaneously amplify the following STR sites:

HBB, HBA, PPBP, HBD, ALAS2, AMI, PRM1, MSMB, KLK3, SEMG1, KLK2, PRM2, HTN3, STATH, MUC7, KRT13, KRT4, SPRR2A, MYOZ1, HBD1, CYP2B7P, β2-MG, UBC, and PGK.

2. The composite amplification system of claim 1, wherein the 5' ends of the primer pairs are fluorescently labeled, with fluorescent labels FAM, HEX, TAMRA and ROX, respectively.

3. The multiplex amplification system of claim 2, wherein the fluorescent label of the primer pair that amplifies CYP2B7P, HBD1, MYOZ1, β2-MG, UBC and PGK sites is FAM;

the fluorescent label of the primer pair amplifying HTN3, STATH, MUC7, KRT4, KRT13 and SPRR2A sites is HEX;

the fluorescent labels of the primer pairs for amplifying the PRM1, PRM2, MSMB, KLK3, SEMG1 and KLK2 sites are TAMRA;

the fluorescent label of the primer pair amplifying AMI, PPBP, ALAS, HBD, HBB and HBA sites was ROX.

4. The composite amplification system of any one of claims 1-3, wherein the mixed body fluid comprises at least one of blood, saliva, semen, and vaginal secretions.

5. A primer combination for amplifying the multiplex amplification system according to any one of claims 1 to 4, wherein the primer combination comprises a sequence shown in SEQ ID NO.1 to 48.

6. A kit for identifying mixed body fluids, comprising the primer combination of claim 5.

7. The kit of claim 6, further comprising a buffer, a template cDNA and Taq DNA polymerase.

8. A method for the analytical identification of STR sites in a mixed body fluid, characterized in that DNA is detected using the multiplex amplification system according to any one of claims 1 to 4, or the primer combination according to claim 5, or the kit according to claim 6.

9. Use of the multiplex amplification system of any one of claims 1 to 4, the primer combination of claim 5 or the kit of claim 6 for individual identification, paternity testing, population genetics analysis and/or construction of human DNA databases and body fluid identification.