MRNA (messenger ribonucleic acid) marker combination and product and application thereof as well as forensic human menstrual blood or human menstrual blood spot detection method

Abstract

The invention belongs to the technical field of forensic medicine, and particularly relates to an mRNA marker combination, a product and application thereof and a forensic human menstrual blood or human menstrual blood spot detection method. The mRNA marker combination comprises a reference gene, a human blood specific mRNA marker and a human menstrual blood specific mRNA marker; the human blood specific mRNA marker is composed of HBA, HBB, ALAS2 and GYPA; the human menstrual blood specific mRNA marker is composed of MMP7, MMP10, SFRP4 and STC1. Standardized identification suggestions are given according to mRNA marker combination expression conditions, the problems of insufficient specificity, flow disorder and the like in the prior art are solved, human menstrual blood or human menstrual blood spots can be accurately distinguished from peripheral blood and other body fluids, cross-platform compatible standardized detection is realized, evidence support is provided for missing cases, and the detection method is suitable for clinical application. The method has important practical significance for improving the forensic physical evidence identification efficiency.

Description

Technical Field

[0001] This invention belongs to the field of forensic medicine technology, specifically relating to a combination of mRNA biomarkers and their products, applications, and a method for detecting forensic human menstrual blood or human menstrual blood spots. Background Technology

[0002] In forensic science, the accurate identification of bodily fluid stains and their tissue origin is a core aspect of crime scene reconstruction and case investigation. Blood, as the most common biological sample at crime scenes, plays a crucial role in constructing a complete chain of evidence and determining the nature of the case, especially in sexual assault cases, missing women cases, and injury cases involving female victims. This identification directly affects the direction of the investigation and the determination of legal facts.

[0003] Human menstrual blood is a special bodily fluid formed by the shed endometrial tissue, cervical mucus, vaginal secretions, and peripheral blood, resulting from cyclical changes in the ovaries. The dried stains it forms contain not only red blood cells and hemoglobin but also unique components such as endometrial cells and cervical mucus proteins. Traditional methods for identifying human menstrual blood (stains), such as cell morphology observation and specific biomolecular detection, suffer from low specificity and insufficient sensitivity, particularly in old, trace, or mixed samples. For example, microscopic observation of endometrial cells relies on sample integrity and is easily interfered with by bleeding from the reproductive organs; while plasmin activity detection can indirectly reflect the characteristics of human menstrual blood, it cannot exclude interference from other plasmin-rich bodily fluids. With the development of molecular biology techniques, RNA-based detection methods offer a new direction for the identification of human menstrual blood (stains). Messenger RNA (mRNA), as a single-stranded ribonucleic acid that carries genetic information and guides protein synthesis, has tissue-specific expression characteristics that make it an ideal biomarker for identifying fluid-derived sources. Compared with traditional identification methods, mRNA-based detection technology has higher sensitivity and specificity, and can obtain effective information from trace or degraded samples.

[0004] Patent CN104419753B provides a method and system for identifying the tissue origin of bodily fluids in Chinese individuals at the gene level. The method includes detecting the expression levels of seven genes in the bodily fluids: GYPA, SPTB, MMP-7, STATH, MUC4, PRM2, and TGM4. This method can effectively determine the tissue origin of bodily fluids in Chinese individuals at the gene level, greatly simplifying the steps for identifying the tissue origin of bodily fluids or stains at criminal investigation scenes. It can provide accurate scientific evidence for clarifying the nature of a case, identifying suspects, and determining guilt and sentencing. However, this method relies on the fluorescence signal of a genetic analyzer to determine the amplification products. If the sample contains PCR inhibitors (such as humic acid), it may inhibit amplification or produce non-specific fluorescence, affecting the accuracy of the results.

[0005] However, current mRNA-based human menstrual blood (spot) identification technology still faces challenges in standardization: significant differences exist among laboratories in biomarker selection (such as the choice between single biomarkers and combinations of multiple biomarkers), detection platforms (real-time quantitative PCR, capillary electrophoresis, and targeted high-throughput sequencing), and result interpretation standards (threshold setting, internal reference gene selection), leading to a lack of comparability of cross-laboratory data and limiting the technology's universality. Given these technical challenges, developing more efficient and accurate human menstrual blood (spot) identification methods is of great significance for improving the forensic science and technology system and promoting fair trial of cases. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a combination of mRNA biomarkers and their products, applications, and a method for detecting forensic human menstrual blood or human menstrual blood spots.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a combination of mRNA biomarkers, which includes: an internal reference gene, a human blood-specific mRNA biomarker, and a human menstrual blood-specific mRNA biomarker; the human blood-specific mRNA biomarker is composed of HBA, HBB, ALAS2, and GYPA; the human menstrual blood-specific mRNA biomarker is composed of MMP7, MMP10, SFRP4, and STC1.

[0008] Preferably, the internal reference genes include 18S rRNA and ACTB.

[0009] On the other hand, the present invention provides a primer composition for amplifying the mRNA marker combination described in any of the above claims.

[0010] Specifically, the nucleotide sequence of the primer composition is shown in SEQ ID NO.1-20.

[0011] Preferably, the nucleotide sequence of the primer for the 18S rRNA is shown in SEQ ID NO.1-2.

[0012] More preferably, the nucleotide sequence of the forward primer of the 18S rRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2.

[0013] Preferably, the nucleotide sequence of the primer for ACTB is shown in SEQ ID NO.3-4.

[0014] More preferably, the nucleotide sequence of the forward primer of the ACTB is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0015] Preferably, the nucleotide sequence of the primer for HBA is shown in SEQ ID NO.5-6.

[0016] More preferably, the nucleotide sequence of the forward primer of the HBA is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6.

[0017] Preferably, the nucleotide sequence of the primer for HBB is shown in SEQ ID NO.7-8.

[0018] More preferably, the nucleotide sequence of the forward primer of HBB is shown in SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.8.

[0019] Preferably, the nucleotide sequence of the primer for ALAS2 is shown in SEQ ID NO.9-10.

[0020] More preferably, the nucleotide sequence of the forward primer of ALAS2 is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10.

[0021] Preferably, the nucleotide sequence of the primer for GYPA is shown in SEQ ID NO.11-12.

[0022] More preferably, the nucleotide sequence of the forward primer of GYPA is shown in SEQ ID NO.11, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.12.

[0023] Preferably, the nucleotide sequence of the primer for MMP7 is shown in SEQ ID NO.13-14.

[0024] More preferably, the nucleotide sequence of the forward primer of MMP7 is shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14.

[0025] Preferably, the nucleotide sequence of the primer for MMP10 is shown in SEQ ID NO.15-16.

[0026] More preferably, the nucleotide sequence of the forward primer of MMP10 is shown in SEQ ID NO.15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.16.

[0027] Preferably, the nucleotide sequence of the primer for SFRP4 is shown in SEQ ID NO.17-18.

[0028] More preferably, the nucleotide sequence of the forward primer of SFRP4 is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.18.

[0029] Preferably, the nucleotide sequence of the primer for STC1 is shown in SEQ ID NO.19-20.

[0030] More preferably, the nucleotide sequence of the forward primer of STC1 is shown in SEQ ID NO.19, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.20.

[0031] Specifically, in the primer composition, the final concentration of each primer used to amplify the mRNA marker combination is 0.06-0.2 µM.

[0032] Preferably, the final concentration of the primers used to amplify 18S rRNA is 0.06 µM; Preferably, the final concentration of the primers used to amplify ACTB is 0.2 µM; Preferably, the final concentration of the primers used to amplify HBA or HBB is 0.075 µM; Preferably, the final concentration of the primers used to amplify ALAS2, GYPA, MMP7, MMP10, SFRP4, or STC1 is 0.2 µM.

[0033] In another aspect, the present invention provides the use of the mRNA biomarker combination or primer composition described in any of the above claims in the preparation of a forensic human menstrual blood or human menstrual blood spot detection kit.

[0034] In another aspect, the present invention provides a kit for detecting forensic human menstrual blood or human menstrual blood spots, the kit comprising any of the above-described mRNA marker combinations or primer compositions.

[0035] Specifically, the kit also includes one or more of the following: PCR buffer, PCR polymerase, DNA extraction reagent, sequencing library construction reagent, nucleic acid purification reagent, positive control, and negative control.

[0036] In another aspect, the present invention provides a method for detecting forensic human menstrual blood or human menstrual blood spots based on a combination of mRNA biomarkers, the method comprising using any of the above-described mRNA biomarker combinations, primer compositions or kits.

[0037] Specifically, the method includes the following steps: S1. Extract genomic RNA from the sample and reverse transcribe the RNA into cDNA; S2. Using cDNA as a template, a primer combination is used to specifically amplify the combination of mRNA markers; S3. Sequencing is performed using qPCR, capillary electrophoresis, or targeted high-throughput sequencing platforms: S4. Based on the test results, provide an assessment opinion.

[0038] Specifically, the samples include one or more of the following: blood, blood spots, oral cells, semen, sperm stains, bones, hair, saliva, saliva stains, sweat, and amniotic fluid containing fetal cells.

[0039] Preferably, the blood includes: human peripheral blood and / or human menstrual blood.

[0040] Preferably, the blood spots include: human peripheral blood spots and / or human menstrual blood spots.

[0041] Specifically, the aforementioned expert opinions include: ① tending to believe that the tested sample does not contain human blood or human blood spots; ② tending to believe that the tested sample contains human blood or human blood spots, but does not contain human menstrual blood or human menstrual blood spots; ③ tending to believe that the tested sample contains human blood or human blood spots, but cannot give a preference opinion on whether it contains human menstrual blood or human menstrual blood spots; ④ tending to believe that the tested sample contains human menstrual blood or human menstrual blood spots.

[0042] Preferably, the criterion for the identification opinion ① that the tested sample does not contain human blood or human blood spots is: ≤2 human blood-specific mRNA markers are expressed in the tested sample.

[0043] Preferably, the identification opinion ② tends to consider that the tested sample contains human blood or human blood spots, but does not contain human menstrual blood or human menstrual blood spots. The criteria for this judgment are: in the tested sample, at least 3 human blood-specific mRNA markers are expressed, and no human menstrual blood-specific mRNA markers are expressed.

[0044] Preferably, the identification opinion ③ tends to believe that the tested sample contains human blood or human blood spots. The criterion for determining whether the sample contains human menstrual blood or human menstrual blood spots is: at least 3 human blood-specific mRNA markers are expressed in the tested sample, and only 1 human menstrual blood-specific mRNA marker is expressed or it cannot be determined whether human menstrual blood-specific mRNA markers are expressed.

[0045] Preferably, the identification opinion ④ tends to consider the following criteria for determining whether the tested sample contains human menstrual blood or human menstrual blood spots: at least 3 human blood-specific mRNA markers are expressed in the tested sample, and at least 2 human menstrual blood-specific mRNA markers are expressed.

[0046] The beneficial effects of this invention are as follows: 1. This invention provides an mRNA biomarker combination with a specific primer composition designed and qPCR amplification concentration optimized. It can accurately distinguish between human menstrual blood (spots) and other bodily fluids (spots) such as peripheral blood and semen, avoiding misjudgment caused by insufficient specificity in traditional methods.

[0047] 2. The mRNA biomarker set of this invention is compatible with three mainstream platforms: qPCR, capillary electrophoresis, and targeted high-throughput sequencing. By unifying primer concentrations and result interpretation rules, it solves the industry problem of incomparable data from different laboratories and promotes the standardization of the detection process.

[0048] 3. The mRNA biomarker combination and detection method of the present invention provides a standardized solution for forensic science. Through specific primer combinations and a strict positive / negative control verification system, four types of identification opinions can be quickly generated, which directly assists in determining the direction of case investigation and constructing the legal evidence chain, and has important practical significance in forensic medicine. Attached Figure Description

[0049] Figure 1 The figures show the specific melting curves of mRNA markers in the forensic menstrual blood (spot) detection system using qPCR. In the figure, A represents the specific melting curve of 18S rRNA and ACTB using qPCR; B represents the specific melting curve of HBA, HBB, ALAS2, and GYPA using qPCR; C represents the specific melting curve of MMP7, MMP10, and STC1 using qPCR; and D represents the specific melting curve of SFRP4 using qPCR. Detailed Implementation

[0050] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of protection of the present invention.

[0051] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0052] Example 1: mRNA biomarker combination and primer design The mRNA biomarker combination of this invention was obtained by screening the PubMed database, involving two internal reference genes (18S rRNA and ACTB), four human blood-specific mRNA biomarkers (HBA, HBB, ALAS2, and GYPA), and four human menstrual blood-specific mRNA biomarkers (MMP7, MMP10, SFRP4, and STC1). Primers were screened or designed for the above mRNA biomarkers, and single-gene qPCR detection was performed. After temperature gradient and primer concentration control experiments, primers with good amplification efficiency and no non-specific amplification (single melting curve) were retained, while primers that did not meet the requirements were discarded.

[0053] The primer information for the mRNA biomarker combination is shown in Table 1. The primers of this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0054] Table 1

[0055] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer.

[0056] Example 2: qPCR amplification system based on mRNA biomarker combinations After optimizing the reaction conditions, the final concentrations of the forward and reverse primers for each mRNA marker in the qPCR amplification system based on mRNA marker combinations (i.e., specific forward and reverse primer mix) were determined, as shown in Table 2. The annealing temperature was optimized to 58℃ through annealing temperature gradient experiments.

[0057] Table 2

[0058] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer.

[0059] like Figure 1 As shown in the figure, the melting curves of each mRNA marker during qPCR detection clearly show that the target fragments amplified by each mRNA marker are highly specific, with no obvious non-specific amplification peaks. In addition, there are no amplification peaks for NTC (without template control), which verifies the reliability and effectiveness of this detection system.

[0060] Example 3: Application Method of qPCR Amplification System Based on mRNA Marker Combinations 1. RNA extraction, quantification, and reverse transcription of various samples For blood or semen samples, add 200 µL of blood or semen to a 2 mL RNase-free centrifuge tube. For menstrual blood or vaginal secretion samples, cut the tip of a sterile flocked medical swab containing menstrual blood or vaginal secretions and place it into a 2 mL RNase-free centrifuge tube. Then, extract RNA according to the TRIzol™ reagent (Thermo Fisher Scientific, USA) instructions, with an elution volume of 30 µL.

[0061] The extracted RNA was immediately processed according to the Qubit algorithm. TM Quantification was performed using the RNA HS Assay Kit (Thermo Fisher Scientific, USA) on a Qubit 4.0 real-time analyzer (Thermo Fisher Scientific, USA). After quantification, reverse transcription of the RNA was immediately performed according to the instructions of the Fastking gDNA Dispelling RT SuperMix Kit (TIANGEN, China). Random primers were used for reverse transcription, and the obtained cDNA was stored at -20°C.

[0062] 2. Real-time quantitative PCR detection RNA was detected by real-time quantitative PCR according to the instructions of the TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Japan) kit. The reaction system for real-time quantitative PCR is shown in Table 3.

[0063] Table 3

[0064] Real-time quantitative PCR reaction conditions: 95℃ for 30s; 94℃ for 5s, 58℃ for 15s, 72℃ for 30s, for a total of 40 cycles; 95℃ for 15s; 60℃ for 1min; 95℃ for 1s, with a temperature rise / fall rate of 1.6℃ / s. Each amplification batch should include a positive control sample (confirmed human menstrual blood sample) and a negative control sample (ribozyme-free water).

[0065] Example 4: Result Analysis and Identification of qPCR Amplification System Based on mRNA Marker Combinations 1. Results Analysis (1) qPCR platform: The results of qPCR were output through QuantStudio™ Design & Analysis Software v1.5.3 (Thermo Fisher Scientific, USA), namely the Cq value of each sample, which served as key data for subsequent result analysis. When the Cq value of the tested sample was <35.0, it was determined that the tested sample expressed the corresponding mRNA marker; when the Cq value was >35.0, it was determined that the tested sample did not express the corresponding mRNA marker.

[0066] (2) Capillary electrophoresis platform (taking ABI 3500 genetic analyzer as an example): When the sample tested detects a fluorescence peak of a specific fragment of mRNA marker and the peak height is >500 RFU, it is determined that the sample tested expresses the corresponding mRNA specific marker; when the sample tested does not have a fluorescence peak of a specific fragment of mRNA marker, it is determined that the sample tested does not express the corresponding mRNA specific marker; when the sample tested detects a fluorescence peak of a specific fragment of mRNA marker and the peak height is ≤500 RFU, it is impossible to determine whether the sample tested expresses the corresponding mRNA specific marker.

[0067] (3) Targeted high-throughput sequencing platform: When the number of sequencing reads in the specific fragment of the mRNA marker detected in the tested sample is >500, it is determined that the sample expresses the corresponding mRNA-specific marker; when there are no sequencing reads in the specific fragment of the mRNA marker in the tested sample, it is determined that the tested sample does not express the corresponding mRNA-specific marker; when the number of sequencing reads detected in the specific fragment of the mRNA marker in the tested sample is ≤500, it is impossible to determine whether the sample expresses the corresponding mRNA-specific marker.

[0068] 2. Expert Opinion The identification is mainly based on the detection results of multiple human menstrual blood-specific mRNA markers (MMP7, MMP10, SFRP4 and STC1) and human blood-specific mRNA markers to determine whether the tested sample contains human menstrual blood (spots).

[0069] 1) Test results are valid if they meet the following requirements: ① The positive control samples (confirmed human menstrual blood samples) were tested and at least 3 blood-specific mRNA markers were expressed and at least 2 human menstrual blood-specific mRNA markers were detected; ② The negative control (nuclease-free water) sample was tested and no specific mRNA markers were detected.

[0070] 2) The following expert opinions can be issued for the tested samples. The expert opinions are divided into the following four types: ① When the analysis results show the expression of ≤2 human blood-specific mRNA markers, it indicates that the sample does not contain human blood (spots), and the identification opinion is: "It is tended to be believed that the tested sample does not contain human blood (spots)".

[0071] ② If at least 3 human blood-specific mRNA markers are expressed, and no menstrual blood-specific mRNA markers are expressed in any of the tested samples, it indicates that the sample does not contain human menstrual blood (spots), and the assessment opinion is: "It is tended to be considered that the tested sample contains human blood (spots) but does not contain human menstrual blood (spots)".

[0072] ③ When at least three human blood-specific mRNA markers are expressed, but only one human menstrual blood-specific mRNA marker is detected, or when it is impossible to determine whether human menstrual blood-specific mRNA markers are expressed, the expert opinion is: "It is predisposed that the tested sample contains human blood (spots), but no opinion can be given on whether it contains human menstrual blood (spots)."

[0073] ④ If at least 3 human blood-specific mRNA markers are expressed and at least 2 human menstrual blood-specific mRNA markers are detected, the assessment opinion is: "It is presumed that the tested sample contains human menstrual blood (spots)".

[0074] Example 5: qPCR amplification system based on mRNA biomarker combinations for the detection of forensic human menstrual blood, peripheral blood, and non-blood samples. 1. Sample collection This study collected 20 menstrual blood samples, 10 peripheral blood samples, 11 semen samples, and 1 vaginal secretion sample from unrelated volunteers in China. Menstrual blood samples were collected from the vagina of female volunteers using sterile flocked swabs on day 2 of their menstrual cycle. Peripheral blood samples were collected by qualified medical professionals, strictly following the People's Republic of China Health Industry Standards "Guidelines for Venous Blood Sample Collection" (WS / T 661-2020) and "Technical Requirements for Clinical Body Fluid Testing" (WS / T 662-2020). Semen samples were collected by male volunteers using sterile collection cups. Vaginal secretion samples were collected from the upper third of the vaginal wall of female volunteers using sterile flocked swabs rotated between day 8 and day 28 of their menstrual cycle. This study received ethical approval from the Institutional Review Committee of Shanxi Medical University (Approval No.: 2020GLL031) based on the World Medical Association guidelines and the Declaration of Helsinki. All donors participated in this study voluntarily with informed consent. All collection methods complied with relevant guidelines and regulations. After preparation and processing, all samples were stored at -80°C for later use.

[0075] 2. Forensic testing of blood and non-blood samples 2.1 Test Results Table 4 shows the qPCR results of 12 non-blood samples (11 semen samples and 1 vaginal secretion sample) and 10 peripheral blood samples, using the method described in Example 3. A Cq value < 35.0 was marked as positive; a Cq value > 35.0 was marked as negative. The results showed that 18S rRNA and ACTB were stably expressed in all samples. Specifically, all non-blood samples showed expression of fewer than 3 human blood-specific mRNA markers; all peripheral blood samples showed expression of 3 or more human blood-specific mRNA markers.

[0076] Table 4

[0077] Note: In the table, “SE” represents semen sample; “VS” represents vaginal secretion sample; and “PB” represents peripheral blood sample.

[0078] 2.2 Analysis of Result Interpretation Based on Different Gene Combinations and Interpretation Criteria 2.2.1 Calculation Logic of Sample False Positive Rate (1) Single gene detection The test uses any one of the human blood-specific mRNA markers (HBA, HBB, ALAS2, or GYPA). The interpretation rule is: a positive result for this gene indicates the presence of human blood (spots) in the tested sample; a negative result indicates the absence of human blood (spots).

[0079] Single-gene false positive rate calculation:

[0080] Calculation of average misclassification rate for a single gene:

[0081] Definitions: ① False positive sample count: The number of non-human blood (spot) samples that tested positive for this gene, i.e., the number of non-human samples incorrectly identified as containing human blood (spots). ② False negative sample count: The number of human blood (spot) samples that tested negative for this gene, i.e., the number of human blood samples incorrectly identified as not containing human blood (spots). ③ Total sample count = Number of non-human blood (spot) samples + Number of human blood (spot) samples.

[0082] (2) Dual-gene combination detection The test uses any two gene combinations from human blood-specific mRNA markers (HBA+HBB, HBA+ALAS2, HBA+GYPA, HBB+ALAS2, HBB+GYPA, ALAS2+GYPA). The determination rule is: the sample is considered to contain human blood (spots) only if both gene results are positive; otherwise, it is considered not to contain human blood (spots).

[0083] Calculation of false positive rate for dual-gene combinations:

[0084] Calculation of the average misclassification rate for dual-gene combinations:

[0085] Definitions: ① False positive sample count: The number of non-human blood (spot) samples where all results for the dual-gene combination are positive, i.e., the number of non-human samples incorrectly identified as containing human blood (spots). ② False negative sample count: The number of human blood (spot) samples where the target gene combination does not reach the judgment threshold, i.e., the number of human blood samples incorrectly identified as not containing human blood (spots). ③ Total sample count = Number of non-human blood (spot) samples + Number of human blood (spot) samples.

[0086] (3) Three-gene combination detection The test uses any three genes from human blood-specific mRNA markers (there are four combinations: HBA+HBB+ALAS2, HBA+HBB+GYPA, HBA+ALAS2+GYPA, HBB+ALAS2+GYPA). The determination rule is: the sample is considered to contain human blood (spots) only if all three gene results are positive; otherwise, it is considered not to contain human blood (spots).

[0087] Calculation of the false positive rate for three-gene combinations:

[0088] Calculation of the average misclassification rate for three-gene combinations:

[0089] The breakdown is as follows: ① False positive samples: The number of non-human blood (spot) samples where all three gene combinations tested positive, i.e., the number of non-human samples incorrectly identified as containing human blood (spots). ② False negative samples: The number of human blood (spot) samples where the target gene combination did not reach the threshold for detection, i.e., the number of human blood samples incorrectly identified as not containing human blood (spots). ③ Total number of samples = Number of non-human blood (spot) samples + Number of human blood (spot) samples.

[0090] (4) Four-gene combination detection: interpretation of all four genes being positive All genes (HBA, HBB, ALAS2, and GYPA) in the human blood-specific mRNA markers were used for detection. The judgment rule was: the sample was considered to contain human blood (spots) only if all four gene results were positive; otherwise, it was considered not to contain human blood (spots).

[0091] Calculation of the false positive rate for four-gene combinations:

[0092] Definitions: ① False positive sample: The number of non-human blood (spot) samples where all four gene tests are positive, i.e., the number of non-human samples incorrectly identified as containing human blood (spots). ② False negative sample: The number of all human blood (spot) samples where the target gene combination does not reach the judgment threshold, i.e., the number of human blood samples incorrectly identified as not containing human blood (spots). ③ Total number of samples = Number of non-human blood (spot) samples + Number of human blood (spot) samples.

[0093] (5) Four-gene combination test: at least two genes must be positive for interpretation. All four genes (HBA, HBB, ALAS2, GYPA) of human blood-specific mRNA markers were used for detection. The interpretation rule was: if the number of positive genes was ≥ 2 (regardless of which specific genes were positive), the tested sample was considered to contain human blood (spots); if the number of positive genes was < 2, the sample was considered not to contain human blood (spots). This interpretation rule covers the set of interpretation results for specific combinations of "two-gene all-positive," "three-gene all-positive," and "four-gene all-positive."

[0094] Calculation of the false positive rate for at least two genes:

[0095] Definitions: ① False positive sample count: The number of non-human blood (spot) samples with ≥ 2 positive genes, i.e., the number of non-human samples incorrectly identified as containing human blood (spots). ② False negative sample count: The number of all human blood (spot) samples with < 2 positive genes, i.e., the number of human blood samples incorrectly identified as not containing human blood (spots). ③ Total sample count = Number of non-human blood (spot) samples + Number of human blood (spot) samples.

[0096] (6) Four-gene combination test: at least three genes must be positive for interpretation. All four genes (HBA, HBB, ALAS2, GYPA) of human blood-specific mRNA markers were used for detection. The interpretation rule was: if the number of positive genes was ≥ 3 (regardless of which specific genes were positive), the sample was considered to contain human blood (spots); if the number of positive genes was < 3, the sample was considered not to contain human blood (spots). This interpretation rule covers the set of interpretation results for specific combinations of "three genes all positive" and "four genes all positive".

[0097] Calculation of the false positive rate for at least three genes:

[0098] Definitions: ① False positive sample: The number of non-human blood (spot) samples with ≥ 3 positive genes, i.e., the number of non-human samples incorrectly identified as containing human blood (spots). ② False negative sample: The number of human blood (spot) samples with < 3 positive genes, i.e., the number of human blood samples incorrectly identified as not containing human blood (spots). ③ Total sample count = Number of non-human blood (spot) samples + Number of human blood (spot) samples.

[0099] 2.2.2 Comparing sample misclassification rates with the selection of specific gene combinations and interpretation rules Referring to the method described in "2.2.1 Calculation Logic of Sample False Positive Rate" above, the false positive rates of 12 non-blood samples and 10 peripheral blood samples using different gene combinations and interpretation rules were calculated. The results are shown in Table 5. When at least three genes were used for positive interpretation, the false positive rate was 0.00%. Therefore, this invention selects four genes—HBA, HBB, ALAS2, and GYPA—for peripheral blood (spot) detection. Only when the number of positive genes is ≥ 3 can the tested sample be determined to contain human blood (spots); if the number of positive genes is < 3, it is determined not to contain human blood (spots). Based on this, the first identification opinion is given: ① When the analysis results show ≤ 2 human blood-specific mRNA markers expressed, it indicates that the tested sample does not contain human blood (spots), and the identification opinion is: "Tends to believe that the tested sample does not contain human blood (spots)." According to the first identification opinion in Example 4, all non-blood samples were given correct identification opinions.

[0100] Table 5

[0101] 3. Detection of forensic peripheral blood and menstrual blood samples 3.1 Test Results Table 6 shows the qPCR results of 10 peripheral blood samples and 20 menstrual blood samples, including 2 internal reference genes, 4 human blood-specific mRNA markers, and 4 human menstrual blood-specific mRNA markers. A Cq value < 35.0 was marked as positive; a Cq value > 35.0 was marked as negative. The results showed that 18S rRNA and ACTB were stably expressed in all samples. Specifically, all peripheral blood samples showed expression of 3 or more human blood-specific mRNA markers, and less than 2 human menstrual blood-specific mRNA markers; all menstrual blood samples showed expression of 3 or more human blood-specific mRNA markers, and 2 or more human menstrual blood-specific mRNA markers.

[0102] Table 6

[0103] Note: In the table, "PB" represents peripheral blood sample; "MB" represents menstrual blood sample.

[0104] 3.2 Analysis of Result Interpretation Based on Different Gene Combinations and Interpretation Criteria 3.2.1 Comparing sample misclassification rates with the selection of specific gene combinations and interpretation rules Referring to the method described in "2.2.1 Calculation Logic of Sample False Positive Rate" above, the false positive rates of 20 menstrual blood (spot) samples and 10 peripheral blood (spot) samples were calculated using different gene combinations and interpretation rules. The results showed that: single gene detection was 1.67%; double gene combination detection was 3.33%; triple gene combination detection was 5.00%; and quadruple gene combination detection was 6.67% for all four genes being positive, 0.00% for at least two genes being positive, and 0.00% for at least three genes being positive.

[0105] It is evident that when using a four-gene combination test, the false positive rate is 0.00% when using the interpretation rules of at least two positive genes and at least three positive genes. Therefore, four genes—MMP7, MMP10, SFRP4, and STC1—were selected for human menstrual blood (spot) detection, and the blood sample can only be determined to contain human menstrual blood (spots) if at least two human menstrual blood-specific mRNA markers are expressed. Based on this, the identification opinions in Example 4 are given in points 2, 3, and 4: ② If at least three human menstrual blood-specific mRNA markers are expressed, and none of the human menstrual blood-specific mRNA markers are expressed, it indicates that the sample does not contain human menstrual blood (spots), and the identification opinion is: "It is tended to be considered that the tested sample contains human blood (spots) but does not contain human menstrual blood (spots)." ③ If at least three human blood-specific mRNA markers are expressed, but only one human menstrual blood-specific mRNA marker is detected, or if it is impossible to determine whether human menstrual blood-specific mRNA markers are expressed, the expert opinion is: "It is presumed that the tested sample contains human blood (spots), but no indicative opinion can be given regarding whether it contains human menstrual blood (spots)." ④ If at least three human blood-specific mRNA markers are expressed, and at least two human menstrual blood-specific mRNA markers are detected, the expert opinion is: "It is presumed that the tested sample contains human menstrual blood (spots)." According to expert opinion item 4 in Example 4, all peripheral blood samples and human menstrual blood samples were accurately identified.

[0106] Example 6: Effect of replacing human blood-specific mRNA markers In this embodiment, commonly used human blood-specific mRNA markers such as PBGD and SPTB genes (primer information is shown in Table 7) were used in combination with the four human blood-specific mRNAs used in this invention: HBA, HBB, ALAS2 and GYPA genes. These were randomly paired to identify forensic human blood (spots) and non-human blood (spots) samples, and the misclassification rates based on two-gene, three-gene, and four-gene all-positive interpretations were calculated respectively.

[0107] Table 7

[0108] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer.

[0109] 1.1 Replace the HBA and GYPA genes with the PBGD and SPTB genes. Primers for the HBA and GYPA genes were replaced with primers for the PBGD and SPTB genes (both forward and reverse primers were at a final concentration of 0.2 µM). Using the HBB, ALAS2, PBGD, and SPTB genes as human blood-specific mRNA markers, the method described in Example 5 was used to detect forensic human blood (spots) and non-human blood (spots) samples. The false positive rates were as follows: Double gene all-positive: 54.17%; Triple gene all-positive: 78.13%; Quadruple gene all-positive: 100%. 1.2 Replace the HBA gene with the SPTB gene Primers for the SPTB gene (both forward and reverse primers were at a final concentration of 0.2 µM) were used to replace primers for the HBA gene. HBB, ALAS2, GYPA, and SPTB genes were used as human blood-specific mRNA markers. The method described in Example 5 was used to detect forensic human blood (spots) and non-human blood (spots) samples. The false positive rates were as follows: Double gene all-positive: 35.19%; Triple gene all-positive: 36.11%; Quadruple gene all-positive: 33.33%.

[0110] 1.3 Replace the HBB gene with the PBGD gene Primers for the PBGD gene (both forward and reverse primers were at a final concentration of 0.2 µM) were used to replace primers for the HBB gene. HBA, ALAS2, GYPA, and PBGD genes were used as human blood-specific mRNA markers. The method described in Example 5 was used to detect forensic human blood (spots) and non-human blood (spots) samples. The false positive rates were as follows: Double gene all-positive: 70.83%; Triple gene all-positive: 87.50%; Quadruple gene all-positive: 100%.

[0111] 1.4 Conclusion The results showed that when distinguishing between human blood (spot) and non-human blood (spot) samples, the misclassification rate of double / triple / quadrigeneous full-positive interpretation of the human blood-specific mRNA marker combination (HBA, HBB, ALAS2 and GYPA) selected in this invention was significantly lower than that of the other three alternative combinations, verifying its effectiveness and optimality in this application.

[0112] Example 7: Effect of replacing human menstrual blood-specific mRNA markers In this embodiment, commonly used human menstrual blood-specific mRNA markers such as MMP11 and LEFTY2 genes (primer information is shown in Table 8) were used in combination with the four human menstrual blood-specific mRNA markers MMP7, MMP10, SFRP4 and STC1 genes used in this invention. These were randomly paired to identify forensic human peripheral blood (spots) and human menstrual blood (spots) samples, and the misclassification rates based on two-gene, three-gene and four-gene all-positive interpretations were calculated respectively.

[0113] Table 8

[0114] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer.

[0115] 1.1 Replace the MMP7 and STC1 genes with the MMP11 and LEFTY2 genes. Primers for the MMP7 and STC1 genes were replaced with primers for the MMP11 and LEFTY2 genes (both forward and reverse primers were at a final concentration of 0.2 µM). The MMP10, SFRP4, LEFTY2, and MMP11 genes were used as specific mRNA markers for human menstrual blood. The method described in Example 5 was used to detect forensic human peripheral blood (spots) and human menstrual blood (spots) samples. The false positive rates were as follows: Double gene all-positive interpretation: 35.19%; Triple gene all-positive interpretation: 47.22%; Quadruple gene all-positive interpretation: 55.56%.

[0116] 1.2 Replace the STC1 gene with the MMP11 gene Primers for the STC1 gene were replaced with primers for the MMP11 gene (both forward and reverse primers were at a final concentration of 0.2 µM). MMP7, MMP10, SFRP4, and MMP11 genes were used as specific mRNA markers for human menstrual blood. The method described in Example 5 was used to detect forensic peripheral blood (spot) and human menstrual blood (spot) samples. The false positive rates were as follows: Two-gene all-positive interpretation: 28.57%; Three-gene all-positive interpretation: 42.86%; Four-gene all-positive interpretation: 57.14%.

[0117] 1.3 Replace MMP10 and STC1 genes with MMP11 and LEFTY2 genes Primers for the MMP10 and STC1 genes were replaced with primers for the MMP11 and LEFTY2 genes (both forward and reverse primers were at a final concentration of 0.2 µM). The MMP7, SFRP4, LEFTY2, and MMP11 genes were used as specific mRNA markers for human menstrual blood. The method described in Example 5 was used to detect forensic human peripheral blood (spots) and human menstrual blood (spots) samples. The false positive rates were as follows: Double gene all-positive interpretation: 44.44%; Triple gene all-positive interpretation: 58.33%; Quadruple gene all-positive interpretation: 66.67%.

[0118] 1.4 Conclusion The results showed that, in distinguishing between human peripheral blood (spots) and human menstrual blood (spots), the misjudgment rate of double / triple / quadrigeneous positive interpretation of the combination of human menstrual blood-specific mRNA markers (MMP7, MMP10, SFRP4 and STC1) selected in this invention was significantly lower than that of the other three alternative combinations, verifying its effectiveness and optimality in this application.

[0119] Example 8: Effect of increasing specific mRNA markers in human menstrual blood In this embodiment, the MSX1 gene (primer information is shown in Table 9) was added. It was combined with the four human menstrual blood-specific mRNA markers MMP7, MMP10, SFRP4 and STC1 used in this invention to identify forensic human peripheral blood (spots) and human menstrual blood (spots) samples, and the misclassification rates based on two-gene, three-gene and four-gene all-positive interpretations were calculated respectively.

[0120] Table 9

[0121] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer.

[0122] 1.1 Add the determination of the MSX1 gene. Primers for the MSX1 gene were added (both forward and reverse primers had a final concentration of 0.1 µM). MMP7, MSX1, MMP10, SFRP4, and STC1 genes were used as specific mRNA markers for human menstrual blood. The method described in Example 5 was used to detect forensic human peripheral blood (spots) and human menstrual blood (spots) samples. The false positive rates were as follows: Two-gene all-positive interpretation: 26%; Three-gene all-positive interpretation: 36%; Four-gene all-positive interpretation: 44%.

[0123] Adding the fifth menstrual blood-specific mRNA marker, MSX1, significantly increased the false positive rate under the interpretation rules of all two, three, and four genes being positive compared to the four combinations of menstrual blood-specific mRNA markers (MMP7, MMP10, SFRP4, and STC1) of this invention. This result indicates that adding the MSX1 gene did not improve the performance in distinguishing between human peripheral blood (spot) and human menstrual blood (spot) samples. The four combinations of menstrual blood-specific mRNA markers selected in this invention demonstrate superior simplicity and detection efficacy.

[0124] Finally, it should be noted that the above content is only used to illustrate the technical solution of this application, and is not intended to limit the scope of protection of this application. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution created by this invention do not depart from the essence and scope of the technical solution created by this invention.

Claims

1. A combination of mRNA biomarkers, characterized in that, The mRNA biomarker combination includes: an internal reference gene, a human blood-specific mRNA biomarker, and a human menstrual blood-specific mRNA biomarker; the human blood-specific mRNA biomarker consists of HBA, HBB, ALAS2, and GYPA; the human menstrual blood-specific mRNA biomarker consists of MMP7, MMP10, SFRP4, and STC1.

2. The mRNA biomarker combination according to claim 1, characterized in that, The internal reference genes include 18S rRNA and ACTB.

3. A primer composition, characterized in that, The primer composition is used to amplify the mRNA marker combination according to any one of claims 1-2, and the nucleotide sequence of the primer composition is shown in SEQ ID NO. 1-20.

4. The primer composition according to claim 3, characterized in that, In the primer composition, the final concentration of each primer used to amplify the mRNA marker combination is 0.06-0.2 µM.

5. The primer composition according to claim 4, characterized in that, In the primer composition described above: The final concentration of the primers used to amplify 18S rRNA was 0.06 µM; The final concentration of primers used to amplify ACTB was 0.2 µM; The final concentration of primers used to amplify HBA or HBB is 0.075 µM; The final concentration of primers used to amplify ALAS2, GYPA, MMP7, MMP10, SFRP4, or STC1 is 0.2 µM.

6. The use of the mRNA biomarker combination according to any one of claims 1-2 or the primer composition according to any one of claims 3-5 in the preparation of a forensic human menstrual blood or human menstrual blood spot detection kit.

7. A reagent kit for forensic detection of human menstrual blood or human menstrual blood spots, characterized in that, The kit comprises the combination of mRNA markers according to any one of claims 1-2 or the primer composition according to any one of claims 3-5.

8. A method for detecting forensic human menstrual blood or human menstrual blood spots based on a combination of mRNA biomarkers, characterized in that, The method includes using the mRNA marker combination according to any one of claims 1-2, the primer composition according to any one of claims 3-5, or the kit according to claim 7; the method includes the following steps: S1. Extract genomic RNA from the sample and reverse transcribe the RNA into cDNA; S2. Using cDNA as a template, a primer combination is used to specifically amplify the combination of mRNA markers; S3. Sequencing is performed using qPCR, capillary electrophoresis, or targeted high-throughput sequencing platforms. S4. Based on the test results, provide an assessment opinion.

9. The method according to claim 8, characterized in that, The samples include one or more of the following: blood, blood spots, oral cells, semen, sperm spots, bones, hair, saliva, saliva spots, sweat, and amniotic fluid containing fetal cells.

10. The method according to claim 8, characterized in that, The aforementioned expert opinions include: ① tending to believe that the tested sample does not contain human blood or human blood spots; ② tending to believe that the tested sample contains human blood or human blood spots, but does not contain human menstrual blood or human menstrual blood spots; ③ tending to believe that the tested sample contains human blood or human blood spots, but cannot give a preference opinion on whether it contains human menstrual blood or human menstrual blood spots; ④ tending to believe that the tested sample contains human menstrual blood or human menstrual blood spots. The aforementioned identification opinion ① tends to believe that the criterion for determining that the tested sample does not contain human blood or human blood spots is: ≤2 human blood-specific mRNA markers are expressed in the tested sample; The aforementioned identification opinion ② tends to believe that the test sample contains human blood or human blood spots, but does not contain human menstrual blood or human menstrual blood spots. The criteria for this are: in the test sample, at least 3 human blood-specific mRNA markers are expressed, and no human menstrual blood-specific mRNA markers are expressed. The aforementioned identification opinion ③ tends to believe that the tested sample contains human blood or human blood spots. The criteria for determining whether it contains human menstrual blood or human menstrual blood spots are: at least 3 human blood-specific mRNA markers are expressed in the tested sample, and only 1 human menstrual blood-specific mRNA marker is expressed or it cannot be determined whether human menstrual blood-specific mRNA markers are expressed. The aforementioned identification opinion ④ tends to believe that the criteria for determining whether the tested sample contains human menstrual blood or human menstrual blood spots are: at least 3 human blood-specific mRNA markers are expressed in the tested sample, and at least 2 human menstrual blood-specific mRNA markers are expressed.

Images