CN114525349A

CN114525349A - Method for identifying semen by using digital PCR and special kit thereof

Info

Publication number: CN114525349A
Application number: CN202210240063.9A
Authority: CN
Inventors: 孙启凡; 石慧霞; 季安全; 魏孙祥; 胡胜; 赵一霞
Original assignee: Institute of Forensic Science Ministry of Public Security PRC
Current assignee: Institute of Forensic Science Ministry of Public Security PRC
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2022-05-24

Abstract

The invention discloses a method for identifying semen by using digital PCR and a special kit thereof. The invention successfully establishes a system for detecting miR-891a-5p and reference gene RNU6b by double digital PCR, thereby obtaining an accurate and sensitive semen detection method based on the digital PCR technology, realizing 100% accurate identification of semen, and accurately judging mixed spots, trace semen and simulated case semen spot samples. Therefore, the invention has good application prospect and is expected to be widely applied to forensic actual cases. The invention has important application value.

Description

Method for identifying semen by using digital PCR and special kit thereof

Technical Field

The invention belongs to the field of forensic medicine, and particularly relates to a method for identifying semen by using digital PCR and a special kit thereof.

Background

The accurate determination of the body fluid speckle tissue attribute left behind at a case site is of great significance to the determination of case properties, reconstruction of crime scenes and the like, while the fine speckles are biological evidences which are common in forensic cases, particularly cases such as rape, lewd and the like, and are of great importance to the accuracy and the qualification of the cases. The traditional fine spot detection method comprises a chemical method, an enzyme catalysis method, a spectrum method, an immunology method and the like, and has the limitations of high requirement on the integrity of a detected material, different sensitivity and specificity, unstable detection result, influence on subsequent DNA detection and the like. MicroRNA (miRNA) is non-coding small-molecule RNA with the length of 18-25 bases, has the advantages of high stability, strong conservation, good tissue specificity, compatibility with DNA synchronous analysis and the like, and is an important tool for forensic body fluid identification.

Real-time quantitative PCR (RT-qPCR) is considered as a gold standard for detecting the expression and quantification of miRNA, and is a mainstream detection method for identifying body fluid based on miRNA at present, but the method needs to scientifically and accurately select reference genes for relative quantitative analysis or prepare a standard curve through a standard product with known concentration for absolute quantification. On the other hand, the qPCR needs to design an independent detection system aiming at different target genes, so that the composite detection difficulty is high, and the wide application of the qPCR in forensic practice is virtually limited. A large number of researches show that miR-891a-5p has good semen specificity, but the detection methods used at present are all RT-qPCR relative quantification methods, so that certain false positive and false negative probabilities exist, and the repeatability and consistency of different researches are poor.

The digital PCR is a method which is developed gradually in recent years and can carry out absolute quantitative detection on trace nucleic acid samples, the principle is that a sample to be detected is divided into tens of thousands of microdroplets, each microdroplet does not contain or contains at least one nucleic acid target molecule to be detected, after independent PCR reaction, each microdroplet is detected one by one, and finally, the copy concentration of the target molecule to be detected is given according to the Poisson distribution principle and the proportion of positive microdroplets. The digital PCR is a quantitative method with high sensitivity and high accuracy, has good repeatability particularly for the detection result of low-concentration nucleic acid molecules, and can be used for the absolute quantitative detection of miRNA. Compared with qPCR, the digital PCR has stronger tolerance to PCR inhibitors, less dependence on PCR efficiency and capability of multiplex detection. However, to date, digital PCR technology has not been introduced into the field of forensic body fluid identification.

In addition, most studies currently use only freshly prepared laboratory samples for detection and analysis, but considering the particularity of forensic actual work and the complexity of forensic-related examination materials, body fluid spots left behind in crime scenes often have the characteristics of trace amount and degradation, and may be mixed with other body fluids to different degrees. Therefore, the detection of mixed spots and trace degraded samples is always the key and difficult point of forensic body fluid identification, and an accurate, sensitive and efficient precise spot detection technology is urgently needed.

Disclosure of Invention

The invention aims to accurately, sensitively and efficiently identify semen or seminal plaques.

The invention firstly protects a kit for identifying semen, which can comprise a primer pair 891, a reverse transcription primer 891, a hydrolysis probe 891, a primer pair RNU6b, a reverse transcription primer RNU6b and a hydrolysis probe RNU6 b;

primer pair 891 can consist of forward primer-891F and reverse primer-891R;

the 5 'end of the hydrolysis probe 891 is provided with a fluorescence label A, and the 3' end is provided with a non-fluorescence quenching group;

the primer pair RNU6b can be composed of a forward primer RNU6b-F and a reverse primer RNU6 b-R;

the 5 'end of the hydrolysis probe RNU6b is provided with a fluorescent label B, and the 3' end is provided with a non-fluorescent quenching group;

the nucleotide sequence of the reverse transcription primer 891 can be shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the forward primer-891F can be shown as SEQ ID NO: 3 is shown in the specification;

the nucleotide sequence of the reverse primer-891R can be shown as SEQ ID NO: 4 is shown in the specification;

the nucleotide sequence of the hydrolysis probe 891 can be as shown in SEQ ID NO: 5 is shown in the specification;

the nucleotide sequence of the reverse transcription primer RNU6b can be shown as SEQ ID NO: 7 is shown in the specification;

the nucleotide sequence of the forward primer RNU6b-F can be shown as SEQ ID NO: 8 is shown in the specification;

the nucleotide sequence of the reverse primer RNU6b-R can be shown as SEQ ID NO: 9 is shown in the figure;

the nucleotide sequence of the hydrolysis probe RNU6b is shown as SEQ ID NO: shown at 10.

In the kit, the fluorescent marker A can be a HEX fluorescent marker. The fluorescent label B can be a FAM fluorescent label. The non-fluorescent quencher may be a quencher of the MGB probe.

The kit may specifically comprise the primer pair 891, the reverse transcription primer 891, the hydrolysis probe 891, the primer pair RNU6b, the reverse transcription primer RNU6b and the hydrolysis probe RNU6 b.

The invention also protects the application of any one of the kits, which can be d1) or d 2):

d1) identifying or aiding in identifying semen;

d2) detecting or assisting to detect whether the sample to be detected contains semen or not;

the use is for the diagnosis and treatment of non-diseases.

The invention also provides a method for identifying or assisting in identifying whether the sample to be detected contains semen, which comprises the following steps:

(a1) obtaining copy concentrations of miR-891a-5p and miR-891a-5p/RNU6b ratios in a plurality of semen samples and a plurality of non-semen samples; the ratio of miR-891a-5p/RNU6b is the ratio of miR-891a-5p copy concentration and RNU6b copy concentration;

the non-semen sample is at least one of menstrual blood, peripheral blood, saliva and vaginal secretion;

(a2) carrying out ROC curve analysis on the copy concentration of miR-891a-5p and the miR-891a-5p/RNU6b ratio obtained in the step (a1) to obtain an optimal cutoff value of miR-891a-5p for identifying semen and an optimal cutoff value of miR-891a-5p/RNU6b ratio for identifying semen; then, the following judgment is made:

if the copy concentration of miR-891a-5p of the sample to be detected is lower than that of the negative control sample, the sample to be detected does not contain semen;

if the copy concentration of miR-891a-5p of the sample to be detected is higher than that of the negative control sample and is greater than the optimal cutoff value of miR-891a-5p, the sample to be detected contains semen;

if the copy concentration of the miR-891a-5p of the sample to be detected is higher than that of the negative control sample and is the optimal cutoff value of miR-891a-5p as follows: when the log2(miR-891a-5p/RNU6b) of the sample to be detected is more than log2 (the optimal cut-off value of the ratio of miR-891a-5p/RNU6b), the sample to be detected contains semen; when the log2(miR-891a-5p/RNU6b) of the sample to be detected is less than or equal to log2 (the optimal cut-off value of the ratio of miR-891a-5p/RNU6b), the sample to be detected does not contain semen;

the method is useful for diagnosis and treatment of non-diseases.

In the above method, the method for obtaining the copy concentration of miR-891a-5p and the copy concentration of RNU6b can comprise the following steps:

(1) taking total RNA of a sample, and carrying out reverse transcription by adopting a reverse transcription primer 891 and a reverse transcription primer RNU6b to obtain cDNA of the sample;

(2) performing digital PCR by using cDNA of the sample obtained in the step (1) as a template and using a hydrolysis probe 891, a forward primer-891F, a reverse primer-891R, a hydrolysis probe RNU6b, a forward primer RNU6b-F and a reverse primer RNU6b-R to obtain microdroplet data; obtaining miR-891a-5p copy concentration and RNU6b copy concentration according to microdroplet data; the copy concentration of miR-891a-5p is lower than 0.1 copies/mu L and is regarded as undetected, and the copy concentration is recorded as 0 copies/mu L;

the nucleotide sequence of the hydrolysis probe 891 can be as shown in SEQ ID NO: 5 is shown in the specification; the 5 'end of the hydrolysis probe 891 is provided with a fluorescence label A, and the 3' end is provided with a non-fluorescence quenching group;

the nucleotide sequence of the hydrolysis probe RNU6b can be shown as SEQ ID NO: 10 is shown in the figure; the hydrolysis probe RNU6b has a fluorescent label B at the 5 'end and a non-fluorescent quencher at the 3' end.

In the above method, the fluorescent marker A may be a HEX fluorescent marker. The fluorescent label B can be a FAM fluorescent label. The non-fluorescent quencher may be a quencher of the MGB probe.

In any of the methods described above, the system in which digital PCR is performed may have a concentration of 250nmol/L for hydrolysis probe 891 and hydrolysis probe RNU6b, and 900nmol/L for forward primer-891F, reverse primer-891R, forward primer RNU6b-F, and reverse primer RNU6 b-R.

In any of the above methods, the reaction procedure for performing digital PCR may specifically be 95 ℃ for 10 min; 30s at 94 ℃, 60s at 59.8 ℃, 30s at 72 ℃ and 45 thermal cycles; 10min at 98 ℃; keeping at 4 ℃.

In any of the above methods, the negative control sample may be prepared by:

(a1) preparing a reaction system; the reaction system contains dNTP mix, RT Buffer, RNase Inhibitor, reverse transcription primer 891, reverse transcription primer RNU6b, total RNA of a sample and enucleated enzyme water;

(a2) and carrying out reverse transcription on the reaction system to obtain a negative control sample.

In the step (a1), the reaction system is specifically 15. mu.L, and consists of 0.15. mu.L dNTP mix (100mM total), 1.5. mu.L 10 × RT Buffer, 0.19. mu.L RNase Inhibitor (20U/. mu.L), 0.75. mu.L aqueous solution of reverse transcription primer 891 (1. mu. mol/L), 0.75. mu.L aqueous solution of reverse transcription primer RNU6b (1. mu. mol/L), 5. mu.L sample total RNA and 5.66. mu.L water for enucleation enzyme.

In the (a2), the reaction conditions may be: 30min at 16 ℃; 30min at 42 ℃; 5min at 85 ℃; keeping at 4 ℃.

The invention also protects the application of substances for detecting the copy concentration of miR-891a-5p and the copy concentration of RNU6b in a sample to be detected, and the application can be d1) or d 2):

d1) identifying or aiding in identifying semen;

the use is for the diagnosis and treatment of non-diseases.

In the above application, the substance for detecting the copy concentration of miR-891a-5p and the copy concentration of RNU6b in the sample to be tested may be any one of the primer pair 891, any one of the reverse transcription primer 891, any one of the hydrolysis probe 891, any one of the primer pair RNU6b, any one of the reverse transcription primer RNU6b, and any one of the hydrolysis probe RNU6 b.

The invention realizes the double detection of miR-891a-5p and RNU6b by designing TaqMan MGB probes with different fluorescence modifications at the 5' end, detects 107 samples of 5 body fluids (including semen, peripheral blood, menstrual blood, saliva and vaginal secretion) in a training set, evaluates the semen identification capability of the training set through non-parameter inspection and ROC curve analysis, and prepares mixed spots, trace semen and simulated case semen spots in different proportions to test the accuracy of the system. Results show that the expression level of miR-891a-5P in semen is obviously higher than that of other body fluids (P is less than 0.0001) and 100% accurate discrimination can be realized; semen plaques (including semen plaques mixed with 1: 100 of other 4 body fluids, 0.008 mu L of micro semen, 14d placed outdoors, 24h of ultraviolet irradiation, washed semen plaques and the like) under all test conditions detect and are accurately identified as semen specific components miR-891a-5 p. Compared with qPCR, the digital PCR technique has the following advantages: 1) the nucleic acid molecules are absolutely quantified, so that the problems of normalization and calibration are solved; 2) the accuracy, the repeatability and the sensitivity are higher when the trace target nucleic acid is detected; 3) is relatively insensitive to potential PCR inhibitors and is less susceptible to amplification efficiency. In addition, in order to ensure the effectiveness of the whole system, the body fluid specific miRNA and the positive control are subjected to double digital PCR detection in the same reaction system for the first time, so that the false negative and false positive probabilities caused by the problems of the sample in the processes of RNA extraction, reverse transcription and ddPCR are avoided. Meanwhile, the composite detection can greatly save samples, simplify the experimental process, reduce the detection cost and be beneficial to the application and popularization of actual case work.

In conclusion, the invention successfully establishes a system for detecting the miR-891a-5p and the reference gene RNU6b by using the double digital PCR, thereby obtaining an accurate and sensitive semen detection method based on the digital PCR technology, realizing 100% accurate identification of semen, and accurately judging the mixed stain, the trace semen and the simulated case semen stain samples. Therefore, the invention has good application prospect and is expected to be widely applied to actual cases of legal medical experts.

Drawings

FIG. 1 is a one-dimensional diagram of annealing temperature gradient amplification of the digital PCR detection system shown in FIG. 1.

FIG. 2 is a ROC curve analysis.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, Digital PCR reactions were carried out on a QX200 AutoDG Droplet Digital PCR System (Bio-Rad, USA) including an automated Droplet generator, PX1^TMHeat sealing apparatus, C1000Touch^TMThermal cycler and QX200^TMA droplet analyzer.

In the following examples, peripheral blood samples were collected by venipuncture into EDTA anticoagulant blood collection tubes. Saliva samples were obtained by collecting volunteer saliva in centrifuge tubes and fasting for 1h before sample collection. Menstrual blood samples were collected from volunteers at menses 2d or 3d with sterile cotton swabs. The semen sample is obtained by collecting fresh semen of a volunteer in a semen collecting cup; volunteers were abstinent for 2 days prior to sample collection. Vaginal secretions are obtained using tampons (at least one week after sexual intercourse). All samples were collected and prepared and stored at-80 ℃ until use. The sample collection was reviewed by the ethical committee of the department of public security material evidence accreditation center and all volunteers signed informed consent.

Example 1 preparation of a kit for the identification of semen

The inventor of the invention prepares a kit for identifying semen, which consists of a primer pair 891, a reverse transcription primer 891, a hydrolysis probe 891, a primer pair RNU6b, a reverse transcription primer RNU6b and a hydrolysis probe RNU6 b. Primer pair 891 consists of forward primer-891F and reverse primer-891R. The hydrolysis probe 891 has a HEX fluorescent label at the 5 'end and a quencher of the MGB probe at the 3' end. The primer pair RNU6b consists of a forward primer RNU6b-F and a reverse primer RNU6 b-R. The 5 'end of the hydrolysis probe RNU6b has FAM fluorescent label, and the 3' end has a quenching group of MGB probe.

The reverse transcription primer 891, the hydrolysis probe 891, the forward primer-891F, the reverse primer-891R, the reverse transcription primer RNU6b, the hydrolysis probe RNU6b, the forward primer RNU6b-F and the reverse primer RNU6b-R were synthesized by Biotech Ltd.

The nucleotide sequences of miR-891a-5p and reference gene RNU6b, primers for reverse transcription, forward primers and reverse primers for amplification and hydrolysis probes are shown in Table 1.

TABLE 1

Note: the underlined part is the base of the hydrolysis probe bound to the region of the RNA sequence itself.

Example 2 establishment of a method for identifying semen Using digital PCR

First, experiment method

1. Extraction of total RNA from test sample

(1) The total RNA of the sample to be tested is extracted by using miRNeasy Mini Kit (Qiagen, Germany), and the RNA elution volume is unified to 30 mu L, so as to obtain the total RNA of the sample to be tested.

(2) The concentration and purity (OD260/280) of total RNA in the test samples were determined using a Nanodrop 2000c (Thermo Fisher Scientific, USA).

2. Obtaining cDNA of sample to be tested

(1) Taking total RNA of a sample to be tested according to

The procedure of the MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) specification was adopted to perform Reverse Transcription using the Reverse Transcription primer 891 and the Reverse Transcription primer RNU6b in the Kit prepared in example 1, and cDNA of the sample to be tested was obtained.

The reaction system was 15. mu.L, consisting of 0.15. mu.L dNTPmix (100mM total), 1.00. mu.L Multiscript^TMRT enzyme (50U/. mu.L), 1.5. mu.L 10 XTT Buffer, 0.19. mu.L RNase Inhibitor (20U/. mu.L), 0.75. mu.L reverse transcription primer 891 aqueous solution (1. mu. mol/L), 0.75. mu.L reverse transcription primer RNU6b aqueous solution (1. mu. mol/L), 5. mu.L total RNA of a sample to be tested, and 5.66. mu.L enucleated enzyme water.

The reaction conditions are as follows: 30min at 16 ℃; 30min at 42 ℃; 5min at 85 ℃; keeping at 4 ℃.

(2) According to the method of the step (1), the Multiscribes in the reaction system are added^TMAnd replacing RT enzyme with enucleated enzyme water, and keeping other steps unchanged to obtain a negative control template.

3. Micro-drop digital PCR amplification reaction

(1) Preparing a reaction system

The reaction system is 24 mu L and consists of 12 mu L ddPCR Supermix for Probes (No dUTP), 1.6 mu L cDNA of a sample to be detected, a hydrolysis probe 891, a forward primer-891F, a reverse primer-891R, a hydrolysis probe RNU6b, a forward primer RNU6b-F, a reverse primer RNU6b-R and enucleate enzyme water; in the reaction system, the concentration of the hydrolysis probe 891 and the hydrolysis probe RNU6b was 250nmol/L, and the concentration of the forward primer-891F, the reverse primer-891R, the forward primer RNU6b-F, and the reverse primer RNU6b-R was 900 nmol/L.

(2) After step (1) is completed, the reaction is transferred to a 96-well plate, droplets are generated in an automated droplet generator, and another 96-well plate is placed to receive the generated droplets.

(3) And (3) after the step (2) is finished, covering a membrane on the 96-well plate for receiving the generated microdroplet, sealing the membrane, and carrying out PCR amplification reaction to obtain a PCR amplification product.

Reaction procedure: 10min at 95 ℃; 30s at 94 ℃, 60s at 56-62 ℃, 30s at 72 ℃ and 45 thermal cycles; 10min at 98 ℃; keeping at 4 ℃. The temperature rise and fall speed is 2.0 ℃/s.

(4) After the step (3) is completed, taking out the 96-well plate and placing the 96-well plate in a QX200^TMDroplet data were read on a droplet analyzer and data collection and analysis were performed using QuantaSoft v 1.7.4. Reactions comprising more than 10000 microdroplets were considered valid reactions and used for data analysis. miR-891a-5p copy concentration lower than 0.1 copies/. mu.L is regarded as undetectable, and the copy concentration is recorded as 0 copies/. mu.L.

And replacing the cDNA of the sample to be detected with a negative control template according to the steps, and taking the cDNA as a negative control without changing other steps.

4. Optimization of annealing temperature

Using C1000Touch^TMThe thermal cycler temperature gradient function optimizes the PCR amplification annealing temperature (62-56 ℃). Setting an annealing temperature gradient includes: 62 deg.C, 61.6 deg.C, 60.9 deg.C, 59.8 deg.C, 58.4 deg.C, 57.3 deg.C, 56.5 deg.C, 56 deg.C.

5. Establishment of method for identifying semen by digital PCR

Double Digital PCR detection of miR-891a-5p and RNU6b is carried out on 107 samples to be detected, and the double Digital PCR detection comprises 67 semen spot samples (58 semen and 9 semen spots) and 40 non-semen spot samples (10 peripheral blood, 10 menstrual blood, 10 saliva and 10 vaginal secretion) to obtain negative control and copy concentrations of miR-891a-5p and RNU6b of each sample to be detected (automatically generated by a QX200 AutoDG Droplet Digital PCR System). Wherein 42 parts of semen is 10 muL, 16 parts of semen is 300 muL, 9 parts of seminal spots are 50 muL, 6 parts of peripheral blood is 10 muL, 4 parts of peripheral blood is 500 muL, 6 parts of saliva is 10 muL, 4 parts of saliva is 2mL, and menstrual blood and vaginal secretion are 2cm cut²A sanitary napkin.

ROC curve analysis is carried out on the miR-891a-5p and the miR-891a-5p/RNU6b ratio to determine the identification Ability (AUC) and the optimal cut-off value of the detection system.

6. Data analysis

Seminal plaques were analyzed for differences in expression from other common body fluids using GraphPad Prism 9 software for nonparametric (Mann-Whitney U test) and ROC curve analysis, with P < 0.05 indicating that the differences were statistically significant.

Second, experimental results

1. Optimization of dual ddPCR detection system

The optimization experiment of annealing temperature is shown in figure 1(a is RNU6 b; b is miR-891a-5 p; annealing temperatures of A08-H08 are 62 ℃, 61.6 ℃, 60.9 ℃, 59.8 ℃, 58.4 ℃, 57.3 ℃, 56.5 ℃ and 56 ℃ respectively). The annealing temperature was optimized to 59.8 deg.c (D08) depending on the separation status of the two detection channel positive and negative droplets.

2. Establishment of method for identifying semen by digital PCR

(1) In the research, 107 samples of 5 body fluids are used for establishing a method for identifying semen by digital PCR, and the detection results of the RNA concentration, the purity, the miR-891a-5p copy concentration, the RNU6b copy concentration and the miR-891a-5p copy concentration/RNU 6b copy concentration of each body fluid type sample are shown in Table 2. The copy concentration of miR-891a-5p of the negative control is below 0.1 copies/mu L.

The results showed that all samples of 5 body fluids had positive concentrations of RNU6b copies; all semen samples can detect miR-891a-5p and the expression level is high (1.70-2890.00 copies/mu L); the majority of non-semen samples (34/40) did not detect miR-891a-5p, and a minority of non-semen samples (6/40) were able to detect miR-891a-5p but at very low miR-891a-5p copies concentrations (< 0.69 copies/. mu.L) and only present at higher total RNA concentrations in the samples. By nonparametric test (Mann-Whitney U test), the miR-891a-5P and miR-891a-5P/RNU6b ratio (namely miR-891a-5P expression after internal reference normalization) are all obviously higher than other body fluids in semen, and the P value is less than 0.0001, so that the significant difference is realized.

TABLE 2 expression levels of miR-891a and RNU6b in 5 body fluid samples

Note: a indicates that the highest value of the copy number of RNU6b in this body fluid type sample exceeds the digital PCR detection limit (i.e., all droplet FAM channels are positive).

(2) The ROC curve analysis is shown in FIG. 2(a is miR-891a-5 p; b is miR-891a-5p/RNU6b ratio): the capacity of miR-891a-5p and miR-891a-5p/RNU6b to distinguish semen from non-semen is 100% (AUC ═ 1), namely, the semen sample can be accurately identified by absolute quantification or relative expression of miR-891a-5 p. The optimal cutoff value of miR-891a-5p is 1.195 copies/mu L; the optimal cutoff value for the miR-891a-5p/RNU6b ratio was 0.000548, i.e., log2(miR-891a-5p/RNU6b) ═ 10.834.

Accordingly, the identification principle of the method for identifying semen by digital PCR established above is as follows: 1) if the copy concentration of the miR-891a-5p in the sample is lower than that of the negative control, the sample does not contain semen; 2) if the copy concentration of miR-891a-5p in the sample is higher than that of the negative control and is more than 1.195 copies/mu L, the sample contains semen; 3) if the copy concentration of miR-891a-5p of the sample is higher than that of the negative control and is less than 1.195 copies/mu L, calculating the ratio of miR-891a-5p/RNU6b by combining an internal reference gene to obtain the relative expression quantity, and if log2(miR-891a-5p/RNU6b) > -10.834, the sample contains semen; if the log2(miR-891a-5p/RNU6b) is less than or equal to-10.834, the sample does not contain semen.

Example 3, application of the method established in example 2

First, application of the method established in example 2 in detection of mixed spots

1. Semen samples (semen sample 1, semen sample 2 or semen sample 3) and 200 μ L of non-semen samples (peripheral blood sample, menstrual blood sample, saliva sample or vaginal secretion sample) were mixed according to 1: 10. 1: 50 or 1: 100 to obtain a mixed sample. Wherein 200 μ L vaginal secretion sample is obtained from 2.5cm²A sanitary napkin.

2. The mixed sample is used as a sample to be detected, and the copy concentration of miR-891a-5p and log2(miR-891a-5p/RNU6b) of the mixed sample are detected according to the method established in the example 2.

The results are shown in Table 3. The results show that all mixed samples can detect miR-891a-5p and are identified as containing semen (miR-891a-5p is more than 1.195 copies/mu L). log2(miR-891a-5p/RNU6b) is gradually reduced along with the reduction of semen components in the mixing ratio, which indicates that the stability of RNU6b in a sample is better, and the applicability of RNU6b as an internal reference gene is reflected to a certain extent.

TABLE 3

Note: the value 1 is miR-891a-5p (copies/. mu.L), and the value 2 is log2(miR-891a-5p/RNU6 b).

Second, application of the method established in example 2 in detection of micro-scale samples

1. Taking 1 mu L semen sample (semen sample 1, semen sample 2 or semen sample 3), and sequentially carrying out gradient dilution by 2 times with sterile water for 8 gradients to obtain a micro sample. Taking 1 μ L micro sample for detection, that is, the amount of semen is 1 μ L, 0.5 μ L, 0.25 μ L, 0.125 μ L, 0.063 μ L, 0.031 μ L, 0.016 μ L, 0.008 μ L.

2. And (3) taking the trace sample obtained in the step (1) as a sample to be detected, and detecting the copy concentration of miR-891a-5p and log2(miR-891a-5p/RNU6b) of the trace sample according to the method established in the embodiment 2.

The results are shown in Table 4. The results show that the digital PCR quantitative results of RNU6b and miR-891a-5p show obvious reduction rules along with the reduction of the amount of the spermatic fluid, and the total RNA concentration measured by the Nanodrop 2000c has no obvious rule. As reported in other studies, the method of Nanodrop and the like has poor quantitative accuracy on low-concentration RNA samples, and the study further proves that false negative can be caused if subsequent detection is carried out according to inaccurate quantitative results. Therefore, the method of the invention adopts a constant RNA volume to perform RT-dPCR and utilizes a positive control to perform normalization for the subsequent miRNA semen spot identification research, rather than relying on an inaccurate quantitative method, so that the analysis is simplified, and the miRNA identification method is beneficial to the miRNA laboratory application and implementation of actual cases. When the amount of the semen is reduced to 0.063 mu L, the semen can still be identified by the copy concentration of miR-891a-5 p; when the amount of the semen is reduced to 0.008 mu L, although the copy concentration of miR-891a-5p does not reach the standard of judging semen to be positive, the sample can be identified as the semen after the sample amount is normalized by log2(miR-891a-5p/RNU6 b).

TABLE 4

Third, application of the method established in the embodiment 2 in detecting the sample of the simulated case fine spots

1. 50 μ L of semen sample (semen sample 1, semen sample 2 or semen sample 3) was placed on a cotton swab and air dried.

2. After the step 1 is finished, placing the sample for 0d, 4d, 8d or 14d in the outdoor natural open air condition to obtain an outdoor placing sample; placing under an ultraviolet lamp for irradiation for 0h, 4h, 8h or 24h to obtain an ultraviolet lamp irradiation sample; soaking the washed sample in a laundry detergent of 'blue moon' for 10min, and then washing the washed sample for 20s by using tap water to obtain a washed sample; standing for 10min to obtain an unwashed sample.

The sample placed outdoors, the sample irradiated by the ultraviolet lamp and the washed sample are all the sample simulating case fine spots.

3. And (3) taking the simulated case seminal vesicle sample and the unwashed sample obtained in the step (2) as samples to be detected, and detecting the copy concentration and log2(miR-891a-5p/RNU6b) of miR-891a-5p of the simulated case seminal vesicle sample and the unwashed sample according to the method established in the embodiment (2).

The results are shown in Table 4. The specific component miR-891a-5p of the semen placed outdoors is remarkably reduced at 4d and gradually reduced along with time gradient, and the copy concentration of the internal reference gene RNU6b is more stable than that of miR-891a-5 p; compared with an outdoor placed sample, the change of the copy concentration of the seminal plaque miR-891a-5p irradiated by the ultraviolet lamp on the sample along with the time gradient is small, and the copy concentration is obviously reduced only in the first gradient (from 0h to 4 h); after washing, the miR-891a-5p copy concentration of the sample is obviously reduced. According to the identification principle of the method for identifying semen by digital PCR in example 2, all the samples with simulated degradation (outdoor placed sample, ultraviolet lamp irradiated sample, washed sample) were judged to contain semen.

TABLE 5

The above results show that semen can be accurately identified by using the semen identification kit prepared in example 1 and the method established in example 2, and that the semen identification kit has extremely high accuracy.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

<110> material evidence identification center of public security department

<120> method for identifying semen by using digital PCR and special kit thereof

<160>10

<170> PatentIn version 3.5

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Claims

1. A kit for identifying semen comprises a primer pair 891, a reverse transcription primer 891, a hydrolysis probe 891, a primer pair RNU6b, a reverse transcription primer RNU6b and a hydrolysis probe RNU6 b;

the primer pair 891 consists of a forward primer-891F and a reverse primer-891R;

the primer pair RNU6b consists of a forward primer RNU6b-F and a reverse primer RNU6 b-R;

the nucleotide sequence of the reverse transcription primer 891 is shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the forward primer-891F is shown as SEQ ID NO: 3 is shown in the specification;

the nucleotide sequence of the reverse primer-891R is shown as SEQ ID NO: 4 is shown in the specification;

the nucleotide sequence of the hydrolysis probe 891 is shown as SEQ ID NO: 5 is shown in the specification;

the nucleotide sequence of the reverse transcription primer RNU6b is shown as SEQ ID NO: 7 is shown in the specification;

the nucleotide sequence of the forward primer RNU6b-F is shown as SEQ ID NO: 8 is shown in the specification;

the nucleotide sequence of the reverse primer RNU6b-R is shown as SEQ ID NO: 9 is shown in the figure;

2. The kit of claim 1, wherein: the fluorescence label A is a HEX fluorescence label; the fluorescent label B is a FAM fluorescent label; the non-fluorescence quenching group is a quenching group of the MGB probe.

3. The use of the kit of claim 1 or 2 as d1) or d 2):

d1) identifying or assisting in identifying semen;

the use is for the diagnosis and treatment of non-diseases.

4. A method for identifying or assisting in identifying whether a sample to be detected contains semen or not comprises the following steps:

the method is useful for diagnosis and treatment of non-diseases.

5. The method of claim 4, wherein: the method for obtaining the copy concentration of the miR-891a-5p and the copy concentration of the RNU6b comprises the following steps:

the nucleotide sequence of the hydrolysis probe 891 is shown as SEQ ID NO: 5 is shown in the specification; the 5 'end of the hydrolysis probe 891 is provided with a fluorescence label A, and the 3' end is provided with a non-fluorescence quenching group;

the nucleotide sequence of the hydrolysis probe RNU6b is shown as SEQ ID NO: 10 is shown in the figure; the 5 'end of the hydrolysis probe RNU6b has a fluorescent label B, and the 3' end has a non-fluorescent quencher group.

6. The method of claim 5, wherein: the fluorescence label A is a HEX fluorescence label; the fluorescent label B is a FAM fluorescent label; the non-fluorescence quenching group is a quenching group of the MGB probe.

7. The method of claim 5, wherein: in the digital PCR system, the concentration of hydrolysis probe 891 and hydrolysis probe RNU6b was 250nmol/L, and the concentration of forward primer-891F, reverse primer-891R, forward primer RNU6b-F and reverse primer RNU6b-R was 900 nmol/L.

8. The method of claim 5, wherein: the reaction program for carrying out digital PCR is 95 ℃ for 10 min; 30s at 94 ℃, 60s at 59.8 ℃, 30s at 72 ℃ and 45 thermal cycles; 10min at 98 ℃; keeping at 4 ℃.

9. The method of claim 4, wherein: the preparation method of the negative control sample comprises the following steps:

10. The application of the substances for detecting the copy concentration of miR-891a-5p and the copy concentration of RNU6b in the sample to be detected is d1) or d 2):

d1) identifying or aiding in identifying semen;

the use is for the diagnosis and treatment of non-diseases.