KR101210657B1 - Kit and method for identifying body fluid - Google Patents

Abstract

PURPOSE: A body fluid-specific marker composition and a method for detecting body fluid using the same are provided to effectively identify body fluid from a biological sample. CONSTITUTION: A composition for detecting body fluid contains: a primer set containing sequence numbers 1 and 2 for identifying PPBP in blood; a primer set containing sequence numbers 13 and 14 for identifying FDCSP in spittle; a primer set containing sequence numbers 23 and 24 for identifying MSMB in semen; and primer set containing sequence numbers 31 and 32 for identifying MSLN in virginal discharge.

Description

Kit for identifying body fluids and a method for identifying body fluids using the same

The present invention relates to a body fluid identification kit comprising a fluid-specific marker composition and a body fluid identification method using the same.

Forensic identification of the tissue origin of a bodily fluid sample is very important for crimes such as sexual assault. Until now, immunological detection of specific protein markers has been the main method of identifying fluids in forensics. PSA for semen and hemoglobin for blood were used as protein markers. However, immunological tests can often cross-react with other species or tissues, resulting in less accuracy. In addition, most immunological tests rely on simple color change observation, which is difficult to quantify, and sometimes fails to recognize itself, especially when there are very few forensic samples. The use of RNA for forensic bodily fluid identification has been discussed, as it is possible to obtain RNA with reliable tissue specificity and genomic DNA (gDNA) extraction. Genomic DNA can be used for conventional STR analysis, but RNA can be used to identify tissue-specific information that DNA does not have. In addition, multiplex PCR techniques can be used to simultaneously analyze a large number of tissue-specific genes that can identify multiple tissues, thus saving the limited amount of forensic sample used in the assay. RNA assays can now be used for standard endpoint PCR or quantitative PCR analysis widely used in forensic laboratories.

The biggest obstacle to forensic RNA analysis is the instability of the RNA molecule itself, which can be easily degraded by RNases present everywhere. Recent studies, however, have found that mRNA samples obtained from crime scenes are stable enough for forensic use [1, 2]. RNA has been successfully isolated from forensic samples using commercially available RNA extraction kits and DNA / RNA co-extraction methods [3, 4, 5, 6]. These findings show that fluid types can be distinguished by measuring the expression level of tissue specific mRNA markers using forensic RNA samples.

Various tissue specific mRNA markers have been found for the identification of body fluids, including frequently encountered blood, saliva, semen, vaginal secretions and menstrual blood [2, 7, 8, 9, 10, 11, 12]. Many humor-specific mRNA markers have been reported in previous studies through single gene-based assays. For example, SPTB, PBGD and HBA1 have been reported as blood specific mRNA markers, MMP7 and MMP11 have been reported as menstrual blood specific mRNA markers, STATH and HTN3 as saliva specific mRNA markers, and semen specific mRNA markers. PRM1, PRM2, and KLK3 were reported, and HBD1 and MUC4 were reported as vaginal fluid specific mRNA markers [11, 13, 14, 15, 16, 17]. mRNA expression was measured using reverse transcription followed by standard end-point PCR (RT-PCR) or reverse transcription followed by quantitative PCR (qRT-PCR) or capillary electrophoresis [2, 7, 8]. , 9, 10, 11, 12, 18, 19]. Multiprex qRT-PCR techniques have also been developed to detect various mRNA markers at once [15, 20, 21]. These methods have the advantage of being sensitive to the analysis of mRNA expression, but only a few genes could be tested. In order to understand the gene expression characteristics of forensic body fluid samples and further investigate the possibility of new mRNA markers, a systematic approach was needed to analyze the entire transcript.

DNA microarrays are useful tools for analyzing whole transcripts and discovering new biological markers. Many researchers have used DNA microarray techniques to search for tissue specific genes [22, 23, 24]. In the forensic field, specific markers of mRNA and microRNA for the identification of blood and saliva have been found using DNA microarray techniques [25, 26]. However, the investigation of specific mRNA markers for four body fluids (blood, saliva, semen, vaginal secretion) by whole genome assay has not been performed yet.

One embodiment is to provide a bodily fluid specific marker composition.

Another embodiment is to provide a kit for identifying a body fluid comprising a body fluid specific marker composition.

Yet another embodiment is to provide a method of identifying a body fluid in a biological sample using a body fluid identification kit.

One aspect includes a primer set for identifying PPBP in blood consisting of a primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 1 and a primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 2;

A primer set for identifying NKG7 in blood consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 3 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 4;

A set of primers for identifying CCL5 in blood consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 5 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 6;

A primer set for identifying NRGN in blood consisting of a primer comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 7 and a primer comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 8;

A primer set for identifying GZMH in blood consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 9 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 10;

A primer set for identifying PRF1 in blood consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 11 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 12;

A set of primers for identifying FDCSP in saliva consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 13 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 14;

A set of primers for identifying MUC7 in saliva consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 15 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 16;

A set of primers for identifying KLK4 in saliva consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 17 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 18;

A set of primers for identifying HTN1 in saliva consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 19 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 20;

A set of primers for identifying HTN3 in saliva consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 21 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 22;

A primer set for identifying MSMB in semen consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 23 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 24;

A primer set for identifying NKX3-1 in semen consisting of a primer comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 25 and a primer comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 26;

A primer set for identifying SEMG1 in semen consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 27 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 28;

A primer set for identifying PRM2 in semen consisting of a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 29 and a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 30;

A set of primers for identifying MSLN in vaginal secretions consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 31 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 32;

A primer set for identifying SERPINB3 in a vaginal secretion consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 33 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 34; And

A group consisting of a set of primers for identifying MMP7 in vaginal secretions consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 35 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 36 A bodily fluid specific marker composition comprising two or more primer sets selected from is provided.

Another aspect includes a primer set for identifying PPBP in blood consisting of a primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 47 and a primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 48, and A probe comprising 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 37;

Primer set for identifying NKG7 in blood consisting of a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 49 and a primer comprising at least 15 consecutive nucleotides of SEQ ID NO: 50 and SEQ ID NO: 38 A probe comprising at least 15 consecutive nucleotides of a nucleotide sequence of;

A probe set comprising a primer set consisting of SEQ ID NO: 51 and SEQ ID NO: 52 for identifying NRGN in blood and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 39;

A probe set comprising a primer set consisting of SEQ ID NO: 53 and SEQ ID NO: 54 for identifying FDCSP in saliva and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 40;

A probe set comprising a primer set consisting of SEQ ID NO: 55 and SEQ ID NO: 56 for identifying MUC7 in saliva and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 41;

A probe set comprising a primer set consisting of SEQ ID NO: 57 and SEQ ID NO: 58 for identifying KLK4 in saliva and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 42;

A probe set comprising a primer set consisting of SEQ ID NO: 59 and SEQ ID NO: 60 for identifying MSMB in semen and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 43;

A probe set comprising a primer set consisting of SEQ ID NO: 61 and SEQ ID NO: 62 for identifying NKX3-1 in semen and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 44;

A probe set comprising a primer set consisting of SEQ ID NO: 63 and SEQ ID NO: 64 for identifying MSLN in vaginal secretions and at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 45; And

At least two polynucleotide combinations selected from the group consisting of a primer set consisting of SEQ ID NO: 65 and SEQ ID NO: 66 and a probe comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 46 for identifying MMP7 in the vaginal secretion It provides a body fluid specific marker composition.

The term primer is a single stranded oligonucleotide that can serve as an initiation of template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. Means. Suitable lengths of primers are typically 15 to 30 nucleotides, although varying depending on various factors, such as temperature and the use of the primer. Short primers may generally require lower temperatures to form a hybridization complex that is sufficiently stable with the template. The terms "forward primer" and "reverse primer" refer to primers that bind to the 3 'end and the 5' end, respectively, of a portion of the template that is amplified by a polymerase chain reaction. The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer set according to one embodiment does not need to have a sequence that is perfectly complementary to the nucleotide sequence that is a template, and it is interpreted that it is sufficient to have sufficient complementarity within a range capable of hybridizing to the sequence and acting as a primer. The design of such primers can be easily carried out by those skilled in the art by referring to the nucleotide sequence of the polynucleotide to be a template, for example, using a primer design program (for example, PRIMER 3, VectorNTI program). have.

On the other hand, the primer according to one embodiment is hybridized or annealed to one site of the template to form a double chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described in Joseph Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al.,Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, D.C. (1985). For example, the primer may comprise at least 10 or at least 15 contiguous nucleotides in any one of SEQ ID NO: 1 to SEQ ID NO: 36, SEQ ID NO: 47 to SEQ ID NO: 66, wherein the primer is It may be an oligonucleotide having a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 36, SEQ ID NO: 47 to SEQ ID NO: 66.

The term "probe" refers to a linear oligomer with natural or modified monomers or bonds, including deoxyribonucleotides and / or ribonucleotides that can hybridize to a specific polynucleotide sequence. For example, the probe may be single stranded for maximum efficiency in hybridization.

The probe according to one embodiment may be a sequence that is perfectly complementary to the polynucleotide sequence that is a template, but may be a sequence that is substantially complementary to the extent that it does not prevent specific hybridization. Conditions suitable for hybridization are as described above. The term “substantially complementary sequence” means a sequence that can hybridize with a polynucleotide that is a template under stringent conditions known in the art. The term "stringent conditions" refers to Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985), stringent conditions can be determined by controlling temperature, ionic strength (buffer concentration) and the presence of compounds such as organic solvents, and the like, depending on the sequence being hybridized. Can be. For example, stringent conditions may be a) washed with 50 ° C. temperature and 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate, or b) hybridization buffer (50% formamide, 2 × SSC and 10%). Hybridization to 55 ° C. in dextran sulfate), followed by washing at 55 ° C. with EDTA-containing 0.1 × SSC. According to one embodiment, the probe may be a TaqMan probe.

According to one embodiment, the probe may further comprise a detectable label attached to the end. The term "detectable label" refers to an atom or molecule that specifically enables the detection of a molecule comprising a label among the same kind of molecules without a label. The detectable label is for example Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy2, Cy3.18, Cy3.5, Cy3, Cy5.18, Cy5.5, Cy5, Cy7, Oregon Green, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green 500, Oregon Green 514, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59 , SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, SYBR Green, YO-PRO- 1, YO-PRO-3, YOYO-1, YOYO-3, and thiazole orange (thiazo le orange), but is not limited thereto.

According to one embodiment, the composition comprises a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 for identifying PPBP in the blood;

A primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4 for identifying NKG7 in blood;

A primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8 for identifying NRGN in blood;

A primer set consisting of SEQ ID NO: 13 and SEQ ID NO: 14 for identifying FDCSP in saliva;

A primer set consisting of SEQ ID NO: 15 and SEQ ID NO: 16 for identifying MUC7 in saliva;

A primer set consisting of SEQ ID NO: 17 and SEQ ID NO: 18 for identifying KLK4 in saliva;

A primer set consisting of SEQ ID NO: 23 and SEQ ID NO: 24 for identifying MSMB in semen;

A primer set consisting of SEQ ID NO: 25 and SEQ ID NO: 26 for identifying NKX3-1 in semen;

A primer set consisting of SEQ ID NO: 33 and SEQ ID NO: 34 for identifying MSLN in vaginal secretions; And

It may comprise one or more primer sets selected from the group consisting of a primer set consisting of SEQ ID NO: 37 and SEQ ID NO: 38 for identifying MMP7 in the vaginal secretion.

According to another embodiment, the composition comprises a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and a probe consisting of SEQ ID NO: 37 for identifying PPBP in the blood;

A primer set consisting of SEQ ID NO: 13 and SEQ ID NO: 14 and a probe consisting of SEQ ID NO: 40 for identifying FDCSP in saliva;

A primer set consisting of SEQ ID NO: 23 and SEQ ID NO: 24 and a probe consisting of SEQ ID NO: 43 for identifying MSMB in semen; And

It may comprise a combination of two or more polynucleotides selected from the group consisting of a primer set consisting of SEQ ID NO: 33 and SEQ ID NO: 34 and a probe consisting of SEQ ID NO: 45 for identifying MSLN in the vaginal secretion.

Another aspect provides a kit for identifying a bodily fluid comprising the bodily fluid specific marker composition. According to one embodiment, the kit may be to identify blood, saliva, semen or vaginal fluid.

According to one embodiment, the kit may further comprise a reaction buffer, DNA polymerase, dNTP (mixture comprising dATP, dCTP, dGTP and dTTP). The DNA polymerase is for example Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus furiosus Heat stable DNA polymerase obtained from (Pfu). In addition, reaction buffers are compounds that are added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction, which buffer solutions are well known in the art, For example, it may be, but is not limited to, Tris, Tricine, MOPS, or HEPES. In addition, the kit may further include a DNA polymerase joiner, if necessary.

Another aspect includes contacting a biological sample with the body fluid identification kit; And

It provides a method for identifying a body fluid from a biological sample comprising the step of identifying one or more body fluids selected from the group consisting of blood, saliva, semen and vaginal fluid from the biological sample.

According to one embodiment, the biological sample may be a human tissue or cell itself, or a nucleic acid extracted from the tissue or cell. For example, the nucleic acid may be extracted from a part of a human cell such as human hair or body fluid found at a crime scene. Extraction of nucleic acids from human cells can be performed using nucleic acid extraction kits well known in the art. As described above, a method of separately extracting nucleic acids from the biological sample may be performed, but since the cell membranes of human cells may be destroyed by the high temperature generated during the PCR process, nucleic acids in the cells may be exposed to the outside, thereby The sample itself can also be used directly for a PCR reaction.

According to one embodiment, the polymerase chain reaction may be RT-PCR, or qRT-PCR, may be a multiplex polymerase chain reaction (multiplex PCR). That is, two or more primer sets included in the kit can be simultaneously put into a PCR reaction solution, and various body fluid specific genes can be identified from one PCR reaction. According to one embodiment, the humor-specific gene is blood specific genes PPBP, NKG7, CCL5, NRGN, GZMH, PRF1, saliva-specific genes FDCSP, MUC7, KLK4, HTN1, HTN3, semen-specific gene MSMB , NKX3-1, SEMG1, PRM2, vaginal secretion-specific genes SERPINB3, MMP7 and MSLN may be one or more genes selected from the group, and most preferably the method is PPBP, FDCSP, It may be to check whether the presence of the MSMB and MSLN at the same time.

According to the kit for identifying a body fluid according to one embodiment, it is possible to effectively identify the body fluid from a biological sample.

1 shows selected humoral specific candidate genes. DNA microarray experiments were performed with a total of 24 Korean body fluid samples of 6 samples per body fluid using the Illumina BeadChip Array platform. Based on the Q value of each humor (Q <= 1.8) 137 humoral specific candidate genes were selected. Unsupervised hierarchical clustering was performed with 137 candidate genes (red: blood, blue: saliva, green: semen, yellow: vaginal secret).
FIG. 2 shows unsupervised hierarchical clustering (red: blood, blue: saliva, green: semen, yellow: vaginal secretion) of 137 candidate genes to assess their value as general fluid specific properties. Indicates. Expression patterns of 137 candidate genes were examined using DNA microarray data from 113 non-Affymetrix U133 Plus 2 (GPL570) platforms obtained from the GEO database, revealing general humor-specific features.
3 shows validation using RT-PCR of selected humoral specific mRNA markers. RT-PCR analysis was performed with two total RNA samples per body fluid. Humoral specific expression patterns of 18 representative genes were verified.
4 shows verification using qRT-PCR of selected humoral specific mRNA markers. qRT-PCR analysis was performed with 7 or 8 total RNA samples per body fluid. Humoral specific expression patterns of four mRNA markers (blood: PPBP, saliva: FDCSP, semen: MSLN, vaginal secretion: MSMB) were verified by qRT-PCR (bl: blood, sa: saliva, se: semen, va: vaginal secretion). (A) blood, (B) saliva, (C) semen, (D) vaginal secretions.
5 shows validation using multiplex qRT-PCR probes of four selected body fluid specific mRNA markers. Multiplex qRT-PCR was performed with probes with five different dyes attached. Expression of the GAPDH gene was measured together for use as an endogenous control (bl: blood, sa: saliva, se: semen, va: vaginal secretion). (A) blood, (B) saliva, (C) semen, (D) vaginal secretions.
6 shows DNA / RNA co-extraction followed by multiplex qRT-PCR and traditional STR analysis. After DNA / RNA co-extraction, RNA was reverse transcribed and used for multiplex qRT-PCR. (A) blood, (B) saliva, (C) semen, (D) vaginal secretions.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

1. Experimental Method

1.1. Sample Collection and RNA Preparation

Four body fluid samples of blood, saliva, semen and vaginal secretions were collected from healthy Korean volunteers and anonymized according to local ethical regulations. To prepare intact RNA for use in microarrays, whole blood, saliva and part of semen were transferred to a centrifuge tube, and the vaginal fluid sample was immediately collected by dipping a swab with vaginal secretion into a lysis buffer. It was. Total RNA for microarrays was isolated using the manufacturer's protocol using Qiagen's RNeasy Mini kit. For saliva, some RNA samples were isolated using Qiagen's RNeasy Protect Saliva Mini kit. Qualitative and quantitative analysis of the extracted total RNA was confirmed using Bio-Rad's Experion ^™ RNA StdSens. Qiagen's AllPrep DNA / RNA Mini kit was used to prepare genomic DNA and total RNA simultaneously.

1.2. DNA microarray experiment

Total RNA was amplified using Ambion's Illumina TotalPrep ^™ RNA Amplification Kit. RNA amplification consists of cRNA synthesis, biotin adhesion, and in vitro transcription. Through these processes, the RNA was transformed into a biotinylated cRNA, which was then conjugated with the Illumina BeadChip. After conjugation, the BeadChip was scanned with an Illumina BeadArray Reader ^™ laser scanner. The scanned images were converted to intensity values using the Illumina BeadStudio ^™ 3.4 program.

1.3. DNA Microarray Data Analysis

The intensity values obtained from the Illumina BeadStudio ^™ 3.4 program were totally normalized by the displacement value method [27]. Shannon entropy (H) was used to measure the overall specificity of the genes [28, 29]. Given the degree of expression of the g gene ( g = 1, 2,…, G) in N tissues ( t = 1, 2,…, N), the relative expression of the g gene in t tissues is given by Is defined as:

General formula I

In the general formula, w _{g, t} means the expression level of the g gene in t tissue. In addition, the entropy of gene expression distribution is computed like the following general formula II.

Formula II

Low entropy values indicate that the gene expression pattern is specific. Although Shannon entropy represents one metric for assessing the overall gene expression profile, it does not provide information about which gene is specifically expressed in which tissue. Therefore, in order to measure the specificity of gene expression in each tissue, a statistical value Q _{g | t} was introduced, which can be represented by the following general formula III.

General formula Ⅲ

Low Q _{g | t} indicates that the expression pattern of specific genes in that tissue is specific [28, 29]. Candidate genes were selected based on a Q value lower than 1.8. Then, genes that are expressed together in more than two body fluids were manually removed. Unsupervised hierarchical clustering and visualization were performed with the MEV 4.0 program (http://www.tm4.org/). DNA microarray data of the Affymetrix U133 Plus 2 (GPL570) platform for four body fluids was collected from the GEO database (http://www.ncbi.nlm.nih.gov/geo/) provided by NCBI. The collected GEO accession numbers are GSE6872, GSE7307, GSE7451, GSE8764, GSE11622, GSE12446, GSE13494, GSE14245, GSE14642, GSE17340, GSE20266, GSE22331, GSE25518. Collected microarray data was totally normalized by MAS5 method using affy package [30]. Two DNA microarray data sets for four body fluids were visualized and provided on a web server at http://medicalgenome.kribb.re.kr/forensic. All initial data was stored in GBI of NCBI as accession number of GSE34844.

1.4. RT-PCR and qRT-PCR

Reverse transcription was performed with Bio-Rad's iScript ^TM cDNA Synthesis kit using total RNA as a template. RT-PCR primers were designed using Primer3 software. RT-PCR was performed using Novelzyme ^™ Taq Plus Premix. Β-actin gene (SEQ ID NO: 70 and SEQ ID NO: 71) was used as a housekeeping control for standardization. The base sequences of the nucleic acid primers are shown in Table 1 below.

SEQ ID NO: Primer name Base sequence One PPBP-F1 TTTGGAAGTGATCGGGAAAG 2 PPBP-R1 TTGATTCTGGGAGCATCTGG 3 NKG7-F1 GTGAGCTTCCTGGTCCTGTC 4 NKG7-R1 CCAGGAGAAGAAGGTCTGGA 5 CCL5-F1 CGCTGTCATCCTCATTGCTA 6 CCL5-R1 GAGCACTTGCCACTGGTGTA 7 NRGN-F1 GGACTGCTGCACCGAGAAC 8 NRGN-R1 GCGCTCTCCGCTCTTTATCT 9 GZMH-F1 CAGAAGGACTGCCAGTGTGA 10 GZMH-R1 ACTCCTGGAGGTGTCCCTTT 11 PRF1-F1 GCTGGACGTGACTCCTAAGC 12 PRF1-R1 GATGAAGTGGGTGCCGTAGT 13 C4ORF7-F1 CAAGGCCACAGTGAAACTCA 14 C4ORF7-R1 GGCCTTCTTCTTTGCTTCCT 15 MUC7-F1 AAAACTCTGCCGCTGTTTGT 16 MUC7-R1 CAGCGTTTGTGCAGACATTT 17 KLK4-F1 AAAACTCTGCCGCTGTTTGT 18 KLK4-R1 AATGAACGGCTTCTGGTGAG 19 HTN1-F1 TTTTTGTCTTTGCTTTAGTCTTGG 20 HTN1-R1 AATTTGATCCATAGTCCCCATAAA 21 HTN3-F1 TCTTGGCTCTCATGCTTTCC 22 HTN3-R1 GCCTCGATGTGAATGATGC 23 MSMB-F1 GTGATCTTTGCCACCTTCGT 24 MSMB-R1 CGTAGCAAGTGCATGTCTCA 25 NKX3-1-F1 CACGAGCAGCCAGAGACAG 26 NKX3-1-R1 ATGGCTGAACTTCCTCTCCA 27 SEMG1-F1 TTTCCCTGCTCCTCATCTTG 28 SEMG1-R1 GCCTTTGGATTCAGTTTGTTG 29 PRM2-F1 CTATAGGCGCAGACACTGCT 30 PRM2-R1 TGCCTTCTGCATGTTCTCTT 31 MSLN-F1 ATTTGAAGGCGCTCAGTCAG 32 MSLN-R1 CTGCCGTAGGATCCAGTCC 33 SERPINB3-F1 ATTGTGTTGCTGCCAAATGA 34 SERPINB3-R1 GGCCTTGTGTAGGACTCCAG 35 MMP7-F1 TGCTCACTTCGATGAGGATG 36 MMP7-R1 TGGGGATCTCCATTTCCATA

A dual fluorescence attached probe sold by Metabion was used for qRT-PCR. The qRT-PCR reaction was performed on a CFX96 ^™ Real-Time PCR machine (C1000 ^™ Thermal Cycler, Bio-Rad) using THUNDERBIRD ^™ Probe qRT-PCR Mix under the following conditions: initial denaturation, Then, 45 cycles of denaturation, annealing and extension processes at 94 ° C. for 15 seconds, and final annealing / elongation at 56 ° C. for 1 minute. GAPDH gene was used as a housekeeping control for normalization. The base sequences of the nucleic acid probes and primers used for qRT-PCR are shown in Table 2 below.

SEQ ID NO: Primer / Probe Name Base sequence 37 PPBP-TM 5'-Yakima Yellow-CTG CTG CTT CTG TCA TTG CTG CTG-BHQ-1-3 ' 38 NKG7-TM 5'-Yakima Yellow-TGA CGC AGA CCT TCA GCA TTA TGG C-BHQ-1-3 ' 39 NRGN-TM 5'-Fam-CCC CGA AAA CTC GCC TGG ATT TTG-BHQ-1-3 ' 40 FDCSP-TM 5'-Fam-AAC CAA CAG CCA CTG CCA AGA TG-BHQ-1-3 ' 41 MUC7-TM 5'-Yakima Yellow-CAC AGA AGG CAT CAT CAC CAA TCA CC-BHQ-1-3 ' 42 KLK4-TM 5'-Fam-TGT AGA CAC CTG GCA CGC CA-BHQ-1-3 ' 43 MSMB-TM 5'-Yakima Yellow-AGT TGT CAG TCT GCC ACT CCG A-BHQ-1-3 ' 44 NKX3-1-TM 5'-Fam-TGA ACT TCC TCT CCA ACT CGA TCA CCT G-BHQ-1-3 ' 45 MSLN-TM 5'-Yakima Yellow-CCA CAG TCA ACG GCA GCA CC-BHQ-1-3 ' 46 MMP7-TM 5'-Fam-TAT GCG ACT CAC CGT GCT GTG T-BHQ-1-3 ' 47 PPBP-F2 5'-AGA CCA CTT CAT GCC TTG-3 ' 48 PPBP-R2 5'-CAG CGG AGT TCA GCA TAC-3 ' 49 NKG7-F2 5'-GGA CAT CAT ATC AGG CTA CAT C-3 ' 50 NKG7-R2 5'-CAG GAC AGG ACC AGG AAG-3 ' 51 NRGN-F2 5'-CGG ACG ACG ACA TTC TAG-3 ' 52 NRGN-R2 5'-GCT CTC CGC TCT TTA TCT TC-3 ' 53 FDCSP-F2 5'-ACA GCG TCA GAG AGA AAG-3 ' 54 FDCSP-R2 5'-CGT TCC TGG TCT TGA GAG-3 ' 55 MUC7-F2 5'-CGT TCA GTG AAG GTC GAG-3 ' 56 MUC7-R2 5'-GCA GAC ATT TAT AGG ACT TTC TAA-3 ' 57 KLK4-F2 5'-GTG TCT TTC GGA AAA GCC-3 ' 58 KLK4-R2 5'-CGG TTT TCT CTA TCC ACT CA-3 ' 59 MSMB-F2 5'-GGA GTT CCA GGA GAT TCA-3 ' 60 MSMB-R2 5'-GTA GCA AGT GCA TGT CTC-3 ' 61 NKX3-1-F2 5'-GTT GGA CTC TGA AAA CAC TTC-3 ' 62 NKX3-1-R2 5'-GCC GAC AGG TAC TTC TGA-3 ' 63 MSLN-F2 5'-CTC AGT CAG CAG AAT GTG A-3 ' 64 MSLN-R2 5'-GGG TCC CAG AAG TTT CTG-3 ' 65 MMP7-F2 5'-CAA CCA TAG GTC CAA GAA CA-3 ' 66 MMP7-R2 5'-GTT CCC ACT GTA GCT CAC-3 ' 67 GAPDH-F2 5'-AAG GCT GAG AAC GGG AAG-3 ' 68 GAPDH-R2 5'-GGA CTC CAC GAC GTA CTC-3 ' 69 GAPDH-TM 5'-CAL Fluor Red 610-CCA TCA CCA TCT TCC AGG AGC GAG A-BHQ-2-3 '

The degree of expression was quantified using the ΔC _t method. Bio-Rad's iQ ^™ Multiplex Powermix was used for the multiplex qRT-PCR. The qRT-PCR reaction was performed on a CFX96 ^™ Real-Time PCR machine (C1000 ^™ Thermal Cycler, Bio-Rad) under the following conditions: initial denaturation at 95 ° C. for 1 minute, then 45 cycles at 95 ° C. for 15 seconds. Denaturation, annealing and extension process, and final annealing / elongation for 1 minute at 56 ° C. GAPDH gene was used as a housekeeping control for normalization. The probe for multiplex qRT-PCR was used by attaching the following five fluorescent dyes to the same base sequence (probe sequence of PPBP, FDCSP, MSMB, MSLN) used in Table 2 above: FAM, Yakima Yellow, IRD700 , Cy5 and CAL Fluor Red 610. The fluorescence intensity was measured using five filters: FAM, HEX, Quasar705, Cy5 and CAL Fluor Red 610.

Meanwhile, probes and primers for multiplex qRT-PCR were added at the following concentrations: PPBP probe, 0.33 μM, FDCSP probe, 0.67 μM, MSMB probe, 0.33 μM, MSLN probe, 0.33 μM, GAPDH probe, 0.33 μM , PPBP forward primer, 0.33 μM, PPBP reverse primer, 0.33 μM, FDCSP forward primer, 0.67 μM, FDCSP reverse primer, 0.67 μM, MSMB forward primer, 0.33 μM, MSMB reverse primer, 0.33 μM, MSLN forward primer, 0.67 μM, MSLN reverse primer, 0.67 μM, GAPDH forward primer, 0.33 μM, GAPDH reverse primer, 0.33 μM.

1.5. STR  analysis

Genomic DNA (gDNA) was extracted simultaneously with total RNA with Qiagen's AllPrep DNA / RNA Mini kit. gDNA was quantified using Applied Biosystems' Quantifiler ^™ Human DNA Quantification Kit in the 7500 Real-Time PCR System according to the manufacturer's protocol. Autosomal STR amplification was performed by Applied Biosystems' AmpFlSTR  Identifiler  The PCR Amplification Kit was used according to the manufacturer's protocol. PCR products were analyzed and detected using Applied Biosystems '3730 DNA Analyzer capillary electrophoresis system, and STR profiles were analyzed using Applied Biosystems' GeneMapper ID program.

2. Experimental results

2.1. Gene Expression Profiling

Gene expression profiling experiments were performed to find mRNA markers specific to four types of body fluids (blood, saliva, semen and vaginal secretions). Total RNA was extracted from each bodily fluid sample using a commercially available RNA preparation kit, and the quality of total RNA was confirmed using Experion ^™ . However, most of the total RNA samples (particularly saliva and vaginal secretions) showed poor quality due to severe degradation. Therefore, it was first confirmed whether DNA microarray experiments could be performed even with such poor RNA samples. One of the widely used DNA microarray platforms, Illumina BeadChip Array ^™, is the first step in cRNA synthesis, biotin attachment, and RNA amplification through in vitro transcription. The success of the Illumina BeadChip Array ^™ experiment depends largely on RNA amplification. RNA amplification was performed and the resulting cRNA was confirmed to be properly synthesized for use in the next procedure. As a result, the quality of the total RNA was poor, but most of the samples were amplified enough to be conjugated to the Illumina BeadChip. Therefore, the 24 cRNA samples (6 samples per body fluid) were selected and conjugated with Illumina BeadChip and read with a laser scanner. The overall intensities of 24 cRNA conjugated images were identified during the scanning process, and six of the 24 images were found to have low intensity values. These samples were still useful for selecting humoral specific genes. These results confirm that the Illumina BeadChip DNA microarray platform is suitable for forensic fluid research.

2.2. Gene Expression Data Analysis

Shannon Entropy (H) and Q-statistics (Q) were used to select each humoral specific gene. The standardized intensity values of 24 samples were used to calculate the H and Q values of all genes in the DNA microarray. Then, candidate genes with a Q value of less than 1.8 were selected for at least one type of body fluid based on these Q values. First 158 genes were selected, but some of them were not specific to only one type of body fluid. In particular, 21 genes were expressed together in saliva and vaginal secretions. Thus, the 21 genes were removed and then 137 potential humoral specific genes were obtained. These candidate genes are composed of 40 types of blood specific, 80 types of semen specific, 4 types of saliva, and 13 types of vaginal fluid specific genes. When unsupervised hierarchical clustering was performed with the 137 candidate genes, it was found that four kinds of body fluids can be completely identified by these genes (FIG. 1). The results indicate that 137 candidate genes were successfully selected to show humoral specificity in 24 Korean samples when using the Illumina BeadChip Array ^™ platform.

2.3. Analysis of Public Expression Datasets

Publicly available gene expression data sets were obtained from GEO (http://www.ncbi.nlm.nih.gov/geo/) to demonstrate the reliability of 137 candidate genes in other ethnic groups. . Data were collected using the Affymetrix U133 Plus2 platform from four types of body fluid samples. A total of 113 samples were collected, including 40 for blood, 37 for saliva, 13 for semen and 23 for vaginal fluid. After standardizing the intensity values by the MAS5 algorithm, unsupervised hierarchical clustering was performed by extracting the expression values of 137 candidate genes. These 137 genes were clearly distinguished according to four body fluids in 113 samples (FIG. 2). Therefore, it was confirmed that these 137 humor-specific genes could be applied as general humor-specific characteristics in various ethnic groups and microarray platforms.

2.4. 40 additional selections from 137 candidate genes

From a practical point of view, investigating all 137 candidate genes for bodily fluid identification is expensive and time consuming, so we sought to select representative genes from 137 candidates. Three criteria were used to select representative candidate genes: 1) rank of Q values in each body fluid, 2) specificity in both datasets, and 3) absolute value of expression. According to these three criteria, 22 representative blood genes, 4 saliva-specific and 7 semen-specific representative candidate genes were selected. Vaginal secretion samples did not obtain adequate specific genes that met the three conditions. Therefore, for vaginal secretions, candidate genes were selected using more relaxed Q value conditions (Table 3). Several well-known humoral specific markers, including PPBP (blood), HBA1 (blood), HTN1 (saliva), HTN3 (saliva), PRM1 (sperm), PRM2 (sperm), have also been successfully selected from the data set of this experiment. (Table 3), suggesting that the approach of this example to identify specific markers for the four body fluids was efficient.

Gene symbol Full name Entropy Q_blood Q_saliva Q_semen Q_vagina blood




















C21orf7 chromosome 21 open reading frame 7 1.07 1.41 5.13 4.70 4.93 CCL5 chemokine (C-C motif) ligand 5 0.69 0.87 5.74 5.78 4.85 CD247 CD247 molecule 1.11 1.49 5.23 5.43 4.16 CD3D CD3d molecule, delta (CD3-TCR complex) 1.19 1.60 4.93 4.94 4.56 CD52 CD52 molecule 1.15 1.55 4.19 5.47 5.02 CLC Charcot-Leyden crystal protein 0.77 0.98 5.15 5.37 5.33 CX3CR1 chemokine (C-X3-C motif) receptor 1 1.01 1.32 5.06 5.16 4.73 ETS1 v-ets erythroblastosis virus E26 oncogene
homolog 1 (avian) 1.08 1.43 5.62 4.49 4.74 FGFBP2 FGFBP2 fibroblast growth factor binding
protein 2 1.16 1.55 4.90 4.94 4.68 GNLY granulysin 1.10 1.46 5.00 5.00 4.68 GZMA granzyme A (granzyme 1, cytotoxic Tlymphocyte-
associated serine esterase 3) 1.13 1.51 5.23 5.32 4.19 HBA1 hemoglobin, alpha 1 0.61 0.76 5.56 5.75 5.39 HBB hemoglobin, beta 0.34 0.40 6.67 6.81 5.85 KLRB1 killer cell lectin-like receptor subfamily
B, member 1 1.01 1.32 5.22 5.24 4.54 LYZ lysozyme 1.04 1.41 3.70 6.20 5.68 MNDA myeloid cell nuclear differentiation
antigen 0.97 1.28 4.09 6.16 5.26 NKG7 natural killer cell group 7 sequence 0.64 0.80 5.80 5.82 4.99 NRGN neurogranin (protein kinase C substrate,
RC3) 1.01 1.31 4.95 5.00 5.00 PPBP pro-platelet basic protein (chemokine (CX-
C motif) ligand 7) 0.52 0.63 5.80 5.98 5.62 PRF1 perforin 1 (pore forming protein) 1.13 1.51 4.90 5.09 4.60 RUNX3 runt-related transcription factor 3 1.23 1.72 5.29 5.72 3.71 TUBB1 tubulin, beta 1 class VI 1.18 1.58 4.86 4.84 4.76 saliva


FDCSP follicular dendritic cell secreted protein 0.77 5.47 0.98 5.23 5.15 HTN1 histatin 1 0.83 5.23 1.06 5.37 4.99 HTN3 histatin 3 0.37 6.29 0.45 6.20 6.24 MUC7 mucin 7, secreted 1.26 4.74 1.71 4.79 4.73 semen





MSMB microseminoprotein, beta- 0.34 6.69 6.27 0.41 6.19 NEFH neurofilament, heavy polypeptide 0.69 5.73 5.01 0.86 5.58 NKX3-1 NK3 homeobox 1 0.38 6.44 5.76 0.46 6.54 PRM1 protamine 1 0.97 5.10 5.05 1.26 4.91 PRM2 protamine 2 0.66 5.55 5.39 0.83 5.46 SEMG1 semenogelin I 0.48 5.87 5.98 0.59 5.82 SEMG2 semenogelin II 1.21 4.89 4.70 1.62 4.80 vaginal swab





PLAU plasminogen activator, urokinase 0.84 6.28 3.90 6.23 1.10 CFB complement factor B 0.86 5.55 4.71 5.28 1.10 IGFBP3 insulin-like growth factor binding protein
3 1.35 4.68 4.44 4.96 1.86 SERPINB3 serpin peptidase inhibitor, clade B
(ovalbumin), member 3 1.38 4.84 4.39 4.78 1.91 S100A7 S100 calcium binding protein A7 1.46 4.74 4.32 4.78 2.07 MMP7 matrix metallopeptidase 7 1.51 4.68 4.50 4.55 2.16 MSLN mesothelin 1.52 4.74 4.37 4.60 2.18

2.5. RT - PCR and qRT - PCR  Fluid specific using technique Markers  Verification

28 genes were selected for validation using RT-PCR and qRT-PCR techniques. First, RT-PCR analysis was performed using two samples for each body fluid. As a result, it was confirmed that 18 genes of 28 genes are specifically expressed in humor (Figure 3). Successfully validated mRNA markers include six blood specific genes (PPBP, NKG7, CCL5, NRGN, GZMH, PRF1), five saliva specific genes (FDCSP, MUC7, KLK4, HTN1, HTN3), and four semen specific Genes (MSMB, NKX3-1, SEMG1, PRM2) and three vaginal secretion specific genes (SERPINB3, MMP7, MSLN).

Ten mRNA markers among the 18 genes showing humor-specific expression patterns were again confirmed by qRT-PCR using a TaqMan® probe. First, qRT-PCR analysis was performed using two samples per body fluid. As a result, most of the mRNA markers except KLK4 showed the same fluid-specific expression pattern as the RT-PCR result. Of these, four markers, one per body fluid, were selected (blood: PPBP, saliva: FDCSP, semen: MSMB, vaginal fluid: MSLN), and the expression pattern of the markers was determined by qRT-PCR using a larger number of samples. Further verification. 4 shows the humoral specific expression of the four representative genes (PPBP, FDSCP, MSMB, MSLN).

Multiplexed qRT-PCR probes for the four mRNA markers were designed for practical application in forensic body fluid identification. Multiplex qRT-PCR was performed using five different dyes attached to the probe all at once. As a result, it was confirmed that the four body fluids were successfully distinguished from the multiplex qRT-PCR analysis using five dyes (Fig. 5).

2.6. DNA Of RNA  Joint extraction and simultaneous fluid identification STR  analysis

In most forensic samples, it is essential to purify the gDNA to perform STR amplification and fragment analysis for human identification. In experiments, extracting DNA and RNA simultaneously is useful because it saves the consumption of the sample and the gDNA fraction can be maintained even if the RNA is severely degraded. A commercial DNA / RNA co-extraction kit (AllPrep DNA / RNA Mini Kit, Qiagen) was tested for separate forensic body fluid samples (blood, semen, saliva, vaginal fluid). Multiplex qRT-PCR was performed using RNA fractions, while gDNA fractions were used to perform STR amplification of autosomes according to existing protocols.

In all humoral samples subjected to DNA / RNA co-extraction, 15 identifiers of the autosomal STR locus were successfully analyzed simultaneously with the Amelogenin locus. In addition, RNA obtained simultaneously with gDNA was tested by the multiplex qRT-PCR technique for body fluid identification. The multiplex qRT-PCR system of the present invention using four newly selected mRNA markers was able to successfully distinguish the type of humor while showing specific expression of the humoral marker (FIG. 6). These results indicate that the obtained RNA is also suitable for RT-PCR analysis, just as gDNA obtained by DNA / RNA co-extraction has a good enough quality for STR analysis.

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Attach an electronic file to a sequence list

Claims (10)

delete delete A primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 for identifying PPBP in blood;
A primer set consisting of SEQ ID NO: 13 and SEQ ID NO: 14 for identifying FDCSP in saliva;
A primer set consisting of SEQ ID NO: 23 and SEQ ID NO: 24 for identifying MSMB in semen; And
Composition for detecting body fluids comprising a primer set consisting of SEQ ID NO: 31 and SEQ ID NO: 32 for identifying MSLN in vaginal secretion. A primer set consisting of SEQ ID NO: 47 and SEQ ID NO: 48 and a probe consisting of SEQ ID NO: 37 for identifying PPBP in blood;
A primer set consisting of SEQ ID NO: 53 and SEQ ID NO: 54 and a probe consisting of SEQ ID NO: 40 for identifying FDCSP in saliva;
A primer set consisting of SEQ ID NO: 59 and SEQ ID NO: 60 and a probe consisting of SEQ ID NO: 43 for identifying MSMB in semen; And
Composition for detecting body fluids comprising a primer set consisting of SEQ ID NO: 63 and SEQ ID NO: 64 for identifying MSLN in the vaginal secretion and a probe consisting of SEQ ID NO: 45. delete Kit for identifying a body fluid comprising the composition of claim 3 or 4. delete The kit of claim 6, wherein the kit identifies blood, saliva, semen or vaginal fluid. delete Contacting the biological sample with the kit for identifying body fluids of claim 6; And
Identifying one or more body fluids selected from the group consisting of blood, saliva, semen and vaginal fluid from the biological sample.

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