CN108330198A - A kind of tissue-derived miRNAs composite amplification systems and its primer special combination for identifying human body fluid - Google Patents
A kind of tissue-derived miRNAs composite amplification systems and its primer special combination for identifying human body fluid Download PDFInfo
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Abstract
The invention discloses a kind of tissue-derived miRNAs composite amplification systems and its primer special combination for identifying human body fluid.Primer combination includes primer combination first and primer combination second：Primer combination first is made of 16 primers, and nucleotide sequence is successively as shown in sequence 21 to sequence 28 and sequence 31 to sequence 38；Primer combination second is made of 8 primers, and nucleotide sequence is successively as shown in sequence 11, sequence 12, sequence 13, sequence 14, sequence 16, sequence 17, sequence 18 or sequence 19.The tissue-derived of human body fluid can effectively be identified using miRNAs composite amplification systems provided by the invention, enormously simplify identification detecting site body fluid or the tissue-derived step of its mottling, can for clear case property, determine suspect and conviction and sentence etc. accurate scientific basis be provided.The present invention is worth with major application.
Description
Technical field
The present invention relates to medical jurisprudence technical fields, and in particular to a kind of tissue-derived miRNAs for identifying human body fluid
Composite amplification system and its primer special combination.
Background technology
Human body fluid (such as venous blood, sperm, menstrual blood, vaginal fluid, saliva, urine and sweat) is that forensic identification is each
More biological material involved in class case identifies that the tissue-derived of human body fluid provides strong evidence for criminal investigation.It passes
The tissue-derived of the identification body fluid of system is mainly based upon enzymatic reaction and immunology detection, and this method is special due to kind or tissue
Anisotropic limitation, most of experiment are to assume experiment and confirmatory test.In addition, for vaginal fluid, menstrual blood and sweat
Identification be all uncertain.With the continuous development of molecular biology field, the detection more and more based on molecule field
Technology be used to identify the tissue-derived of body fluid.A large amount of mRNA labels are employed successfully in venous blood, saliva, sperm, vaginal secretion
The tissue-derived identification of object, urine and sweat.(Xu Y, Xie J, Cao Y, the et al.Development of such as Xu
highly sensitive and specific mRNA multiplex system(XCYR1)for forensic human
Body fluids and tissues identificantion [J] .PLoS One, 2014,9：E100123. needle) is established
To 10 kinds of body fluid such as venous blood, urine, sweat, body fluid identification is carried out by the compound system that 16 sites mRNA form.Song Feng (Song
The Sichuan RNA marker research [D] of phoenix medical jurisprudence Stains of Body identification：Sichuan University, 2016.) pass through reverse transcription-end-point method PCR
The fluorescent complex in 19 sites mRNA established ties up to five kinds of venous blood, sperm, saliva, vaginal fluid and menstrual blood body fluid
Testing result is obtained in sample.
However, mRNA is influenced degradable feature by humidity, ultraviolet light, temperature and environment limits it in medical jurisprudence body fluid
The application range in Identification of The Origin field.MiRNA be it is a kind of be widely present in eukaryocyte be about made of 18-24 nucleic acid in
Source property non-coding microRNA, plays the part of highly important adjustment effect in most biological processes, with tissue specificity,
Molecule is small, copy number is high and not degradable feature, even if its content and molecule knot under extreme temperature, strong acid, basic conditions
Structure has no significant change, and meets the needs of detection degradation sample, becomes the heat for solving medical jurisprudence concern after mRNA
Point；But the feature that its copy number is high, molecular weight is small so that its detection difficulty is larger.
Currently, mainly fluorescent quantitative PCR technique (qPCR) is used to detect based on the miRNAs body fluid identification research carried out
Differential expressions of the miRNAs in each body fluid, and result is analyzed by statistical method.Limited to by detection method,
QPCR need to be directed to each target gene and carry out reverse transcription or amplified reaction.This can not only consume a large amount of sample and reagent, but also
It can aggravate possibility during the reaction, result judgement is caused to obscure.Fluorescent capillary electrophoresis tube can be reacted by one
Multiple miRNA of a variety of body fluid are expanded and detected, for body fluid is identified.(the VANDER such as Vander Meer
MEER D, UCHIMOTO M L, WILLIAMS G.Simultaneous Analysis of Micro-RNA and DNA for
Determining the Body Fluid Origin of DNA Profiles[J].Journal of Forensic
Sciences, 2013,58 (4):967-971.) and Li et al. (LI Y, ZHANG J, WEI W, et al.A strategy for
co-analysis of microRNAs and DNA[J].Forensic Science International：Genetics,
2014,12:The DNA and miRNA of DNA typing result and body fluid qualification result can be obtained simultaneously by 24-29.) having studied extracts point altogether
Analysis method, and in all detecting sample, obtain the characteristic peak of complete DNA typing result and miRNA labels.
Invention content
The technical problem to be solved by the present invention is to how identify the tissue-derived of human body fluid.
In order to solve the above technical problems, present invention firstly provides primer combinations, it may include primer combines first and primer sets
Close second：
Primer combination first may include primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27,
Primer 28, primer 31, primer 32, primer 33, primer 34, primer 35, primer 36, primer 37 and primer 38；
The primer 21 can be following A1) or A2)：
A1) single strand dna shown in the sequence 21 in sequence table；
A2 sequence 21) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 21
The DNA molecular of identical function；
The primer 22 can be following A3) or A4)：
A3) single strand dna shown in the sequence 22 in sequence table；
A4 sequence 22) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 22
The DNA molecular of identical function；
The primer 23 can be following A5) or A6)：
A5) single strand dna shown in the sequence 23 in sequence table；
A6 sequence 23) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 23
The DNA molecular of identical function；
The primer 24 can be following A7) or A8)：
A7) single strand dna shown in the sequence 24 in sequence table；
A8 sequence 24) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 24
The DNA molecular of identical function；
The primer 25 can be following A9) or A10)：
A9) single strand dna shown in the sequence 25 in sequence table；
A10 sequence 25) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 25
There is the DNA molecular of identical function；
The primer 26 can be following A11) or A12)：
A11) single strand dna shown in the sequence 26 in sequence table；
A12 sequence 26) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 26
There is the DNA molecular of identical function；
The primer 27 can be following A13) or A14)：
A13) single strand dna shown in the sequence 27 in sequence table；
A14 sequence 27) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 27
There is the DNA molecular of identical function；
The primer 28 can be following A15) or A16)：
A15) single strand dna shown in the sequence 28 in sequence table；
A16 sequence 28) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 28
There is the DNA molecular of identical function；
The primer 31 can be following A17) or A18)：
A17) single strand dna shown in the sequence 31 in sequence table；
A18 sequence 31) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 31
There is the DNA molecular of identical function；
The primer 32 can be following A19) or A20)：
A19) single strand dna shown in the sequence 32 in sequence table；
A20 sequence 32) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 32
There is the DNA molecular of identical function；
The primer 33 can be following A21) or A22)：
A21) single strand dna shown in the sequence 33 in sequence table；
A22 sequence 33) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 33
There is the DNA molecular of identical function；
The primer 34 can be following A23) or A24)：
A23) single strand dna shown in the sequence 34 in sequence table；
A24 sequence 34) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 34
There is the DNA molecular of identical function；
The primer 35 can be following A25) or A26)：
A25) single strand dna shown in the sequence 35 in sequence table；
A26 sequence 35) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 35
There is the DNA molecular of identical function；
The primer 36 can be following A27) or A28)：
A27) single strand dna shown in the sequence 36 in sequence table；
A28 sequence 36) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 36
There is the DNA molecular of identical function；
The primer 37 can be following A29) or A30)：
A29) single strand dna shown in the sequence 37 in sequence table；
A30 sequence 37) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 37
There is the DNA molecular of identical function；
The primer 38 can be following A31) or A32)：
A31) single strand dna shown in the sequence 38 in sequence table；
A32 sequence 38) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 38
There is the DNA molecular of identical function；
The primer combination second may include primer 11, primer 12, primer 13, primer 14, primer 16, primer 17, primer 18
With primer 19；
The primer 11 can be following B1) or B2)：
B1) single strand dna shown in the sequence 11 in sequence table；
B2 sequence 11) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 11
The DNA molecular of identical function；
The primer 12 can be following B3) or B4)：
B3) single strand dna shown in the sequence 12 in sequence table；
B4 sequence 12) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 12
The DNA molecular of identical function；
The primer 13 can be following B5) or B6)：
B5) single strand dna shown in the sequence 13 in sequence table；
B6 sequence 13) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 13
The DNA molecular of identical function；
The primer 14 can be following B7) or B8)：
B7) single strand dna shown in the sequence 14 in sequence table；
B8 sequence 14) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 14
The DNA molecular of identical function；
The primer 16 can be following B11) or B12)：
B11) single strand dna shown in the sequence 16 in sequence table；
B12 sequence 16) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 16
There is the DNA molecular of identical function；
The primer 17 can be following B13) or B14)：
B13) single strand dna shown in the sequence 17 in sequence table；
B14 sequence 17) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 17
There is the DNA molecular of identical function；
The primer 18 can be following B15) or B16)：
B15) single strand dna shown in the sequence 18 in sequence table；
B16 sequence 18) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 18
There is the DNA molecular of identical function；
The primer 19 can be following B17) or B18)：
B17) single strand dna shown in the sequence 19 in sequence table；
B18 sequence 19) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 19
There is the DNA molecular of identical function.
Any of the above-described primer combination first may also include primer 29, primer 30, primer 39 and primer 40；
The primer 29 can be following A33) or A34)：
A33) single strand dna shown in the sequence 29 in sequence table；
A34 sequence 29) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 29
There is the DNA molecular of identical function；
The primer 30 can be following A35) or A36)：
A35) single strand dna shown in the sequence 30 in sequence table；
A36 sequence 30) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 30
There is the DNA molecular of identical function；
The primer 39 can be following A37) or A38)：
A37) single strand dna shown in the sequence 39 in sequence table；
A38 sequence 39) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 39
There is the DNA molecular of identical function；
The primer 40 can be following A39) or A40)：
A39) single strand dna shown in the sequence 40 in sequence table；
A40 sequence 40) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 40
There is the DNA molecular of identical function.
Any of the above-described primer combination second may also include primer 15 and primer 20；
The primer 15 can be following B9) or B10)：
B9) single strand dna shown in the sequence 15 in sequence table；
B10 sequence 15) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 15
There is the DNA molecular of identical function；
The primer 20 can be following B19) or B20)：
B19) single strand dna shown in the sequence 20 in sequence table；
B20 sequence 20) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 20
There is the DNA molecular of identical function.
The primer combination first specifically can be by the primer 21, the primer 22, the primer 23, the primer 24, institute
State primer 25, the primer 26, the primer 27, the primer 28, the primer 31, the primer 32, the primer 33, institute
Primer 34, the primer 35, the primer 36, the primer 37 and the primer 38 is stated to form.
The primer combination second specifically can be by the primer 11, the primer 12, the primer 13, the primer 14, institute
Primer 16, the primer 17, the primer 18 and the primer 19 is stated to form.
The primer combination first specifically can be by the primer 21, the primer 22, the primer 23, the primer 24, institute
State primer 25, the primer 26, the primer 27, the primer 28, the primer 29, the primer 30, the primer 31, institute
State primer 32, the primer 33, the primer 34, the primer 35, the primer 36, the primer 37, the primer 38, institute
It states primer 39 and the primer 40 forms.
The primer combination second specifically can be by the primer 11, the primer 12, the primer 13, the primer 14, institute
Primer 15, the primer 16, the primer 17, the primer 18, the primer 19 and the primer 20 is stated to form.
The primer combination specifically combines first by the primer and primer combination second forms.
In any of the above-described primer combination first, the primer 21, the primer 23, the primer 25, the primer 27,
The available fluorescence of the primer 29, the primer 31, the primer 33, the primer 35, the primer 37 and the primer 39
Label.
In any of the above-described primer combination first, the primer 21, the primer 23, the primer 25, the primer 27,
5 ' the ends tool of the primer 29, the primer 31, the primer 33, the primer 35, the primer 37 and the primer 39
Body can be marked with FAM.
In order to solve the above technical problems, the present invention also provides a kind of miRNAs composite amplification systems.
MiRNAs composite amplification systems provided by the present invention, it may include miRNAs composite amplification systems first and miRNAs are multiple
Close amplification system second；
The miRNAs composite amplification systems first may include any of the above-described primer combination first；
The miRNAs composite amplification systems second may include primer 11, primer in any of the above-described primer combination second
12, primer 13, primer 14, primer 15, primer 16, primer 17, primer 18, primer 19 or primer 20.
In the miRNAs composite amplification systems first, the primer 21 and the primer 22 are in miRNAs composite amplification systems
Concentration in first can be 0.138 μM.The concentration of the primer 23 and the primer 24 in miRNAs composite amplification system first can
It is 0.036 μM.The concentration of the primer 25 and the primer 26 in miRNAs composite amplification system first can be 0.042 μM.Institute
It can be 0.084 μM to state the concentration of primer 27 and the primer 28 in miRNAs composite amplification system first.The primer 29 and institute
It can be 0.240 μM to state concentration of the primer 30 in miRNAs composite amplification system first.The primer 31 and the primer 32 exist
Concentration in miRNAs composite amplification system first can be 0.288 μM.The primer 33 and the primer 34 are in the compound expansions of miRNAs
Concentration in increasing system first can be 0.036 μM.The primer 35 and the primer 36 are in miRNAs composite amplification system first
Concentration can be 0.120 μM.The concentration of the primer 37 and the primer 38 in miRNAs composite amplification system first can be 0.300
μM.The concentration of the primer 39 and the primer 40 in miRNAs composite amplification system first can be 0.066 μM.
In the miRNAs composite amplification systems second, the primer 11, the primer 12, the primer 13, the primer
14, the concentration tool of the primer 15, the primer 16, the primer 17, the primer 18, the primer 19 or the primer 20
Body can be 0.85 μM.
The miRNAs composite amplification systems first may also include the reagent carried out needed for pcr amplification reaction；It is described " to carry out
Reagent needed for pcr amplification reaction " does not include the primer needed for pcr amplification reaction.
The miRNAs composite amplification systems first specifically can combine first by any of the above-described primer and form.
The miRNAs composite amplification systems first can specifically be combined first by any of the above-described primer and carry out PCR expansions
Increase the reagent composition needed for reaction.
The miRNAs composite amplification systems second specifically can be by the primer 11, primer 12, the primer 13, described
0 group of primer 14, the primer 15, the primer 16, the primer 17, the primer 18, the primer 19 or the primer 2
At.
The present invention also protects the kit containing any of the above-described primer combination；The kit can be used for identifying human body
Liquid it is tissue-derived.
The present invention also protects the preparation of any of the above-described miRNAs composite amplification systems or any of the above-described kit
Method.The preparation method may include the step of individually packing each primer in any of the above-described primer combination.
The present invention also protects X1) or X2)：
X1) any of the above-described primer combination or any of the above-described miRNAs composite amplification systems, are used in preparation
Identify the application in the tissue-derived kit of human body fluid；
X2) any of the above-described primer combination or any of the above-described miRNAs composite amplification systems, in identification human body
Liquid it is tissue-derived in application.
The present invention also protects a kind of tissue-derived method of identification body fluid to be measured, may include following steps successively：
(1) using the total serum IgE of body fluid to be measured as template, be respectively adopted each primer of any of the above-described primer combination second into
Row reverse transcription obtains the cDNA of body fluid to be measured；
(2) after completing step (1), respectively using the cDNA of the body fluid to be measured as template, using any of the above-described primer
It combines first and carries out PCR amplification, obtain pcr amplification product；
(3) after completing step (2), pcr amplification product is subjected to capillary electrophoresis detection, testing result is analyzed and carries out such as
Lower judgement：
If carrying out the peak value of the subsequent products of reverse transcription using primer 12, primer 16 and primer 17 in 3000-
Between 7000rfu, then body fluid to be measured is venous blood；
If carrying out the peak value of the subsequent products of reverse transcription using primer 13, primer 17 and primer 18 in 1000-
Between 7000rfu, then body fluid to be measured is sperm；
If carrying out the peak value of the subsequent products of reverse transcription using primer 17 and primer 19 between 600-3000rfu,
And carried out using primer 11, primer 12 or primer 14 reverse transcription subsequent products peak value not between 600-3000rfu, then
Body fluid to be measured is vaginal fluid；
If the peak value for being carried out the subsequent products of reverse transcription using primer 12, primer 14, primer 17 and primer 19 is existed
Between 600-7000rfu, and carried out using primer 11 reverse transcription subsequent products peak value not between 600-7000rfu,
Then body fluid to be measured is saliva；
If carrying out the peak value of the subsequent products of reverse transcription using primer 11, primer 12, primer 14, primer 17 and primer 19
Between 800-7000rfu, then body fluid to be measured is menstrual blood.
It is any of the above-described described tissue-derived for venous blood, sperm, vaginal fluid, saliva or menstrual blood.
Above, the primer 11, the primer 21 and the primer 22 are designed according to miR214, the nucleotide of miR214
In sequence such as sequence table shown in sequence 1.The primer 12, the primer 23 and the primer 24 are designed according to miR451a,
In the nucleotide sequence of miR451a such as sequence table shown in sequence 2.6 basis of the primer 13, the primer 25 and the primer 2
MiR888-5P is designed, in the nucleotide sequence such as sequence table of miR888-5P shown in sequence 3.The primer 14, the primer 27
It is designed according to miR205-5P with the primer 28, in the nucleotide sequence such as sequence table of miR205-5P shown in sequence 4.It is described to draw
Object 15, the primer 29 and the primer 30 are designed according to miR124-3P, in the nucleotide sequence of miR124-3P such as sequence table
Shown in sequence 5.The primer 16, the primer 31 and the primer 32 are designed according to miR144-5P, the nucleosides of miR144-5P
In acid sequence such as sequence table shown in sequence 6.The primer 17, the primer 33 and the primer 34 are designed according to miR144-3P,
In the nucleotide sequence of miR144-3P such as sequence table shown in sequence 7.The primer 18, the primer 35 and the primer 36
It is designed according to miR891a-5P, in the nucleotide sequence such as sequence table of miR891a-5P shown in sequence 8.The primer 19 described draws
Object 37 and the primer 38 are designed according to miR203-3P, in the nucleotide sequence such as sequence table of miR203-3P shown in sequence 9.Institute
It states primer 20, the primer 39 and the primer 40 to be designed according to miR654-5P, the nucleotide sequence such as sequence of miR654-5P
In table shown in sequence 10.
It is demonstrated experimentally that can effectively identify the tissue of human body fluid using miRNAs composite amplification systems provided by the invention
Source substantially increases identification detecting site body fluid or the tissue-derived science and accuracy of its mottling, can be clear case
Part property determines the accurate scientific basis of the offers such as suspect and conviction and sentence.The present invention is worth with major application.
Description of the drawings
Fig. 1 is the parting collection of illustrative plates of 10 kinds of standard miRNA.
Fig. 2 is sensitivity test collection of illustrative plates.
Fig. 3 is the capillary electrophoresis detection result of section of vein blood sample.
Fig. 4 is the capillary electrophoresis detection result of part semen sample.
Fig. 5 is the capillary electrophoresis detection result of part menstruation blood sample.
Fig. 6 is the capillary electrophoresis detection result of part saliva sample.
Fig. 7 is the capillary electrophoresis detection result of part vaginal fluid sample.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
MiRNeasy Mini Kit are the product of Qiagen companies of Germany.Turbo DNA-freeTMKit is the U.S.
The product of Ambion.ND-2000 spectrophotometrics are calculated as the product of Thermo Fisher companies of Britain.dNTP Mixture
(10mM) and Rnase-free Water are the product of TaKaRa companies.5×First-Strand Buffer、DTT(0.1M)
It is the product of Invitrogen companies with M-MLV reverse transcriptase (200units/ μ L).
Recombinant RNase Inhibitor (10000U/ μ L) are the product of Promega companies.PCR instrument is
The product of eppendorf companies.
Reverse transcriptase primer is synthesized by giving birth to work bioengineering (Beijing) limited liability company, is purified using PAGE modes.
PCR primer is synthesized by precious bioengineering (Dalian) Co., Ltd, is purified using HPLC modes.
Embodiment 1, standard miRNA parting collection of illustrative plates acquisition
One, the synthesis of standard miRNA
10 standard miRNA are synthesized by precious bioengineering (Dalian) Co., Ltd, are purified using HPLC modes.10 marks
The title and nucleotide sequence of quasi- miRNA is as shown in table 1.
Table 1
Two, the acquisition of the cDNA of standard miRNA
Respectively using 10 standard miRNA as template, using corresponding reverse transcriptase primer in table 2 (reverse transcriptase primer title by
The title of its standard miRNA and "-RT " are constituted) reverse transcription is carried out, obtain corresponding cDNA.Reverse transcription reaction is enterprising in PCR instrument
Row.
Table 2
Reverse transcription reaction system is 20 μ L, by 2 μ L dNTP Mixture (10mM), 2.5 5 × First-Strand of μ L
Buffer、1μL DTT(0.1M)、2μL M-MLV reverse transcriptase(200units/μL)、0.2μL
Recombinant RNase Inhibitor (10000U/ μ L), 1 μ L templates (RNA containing 5ng), 1 μ L reverse transcriptions draw
Object and 10.3 μ L Rnase-free Water compositions.A concentration of 0.32 μM in reverse transcription reaction system of reverse transcriptase primer.
Reverse transcription reaction condition：37 DEG C of 60min, 70 DEG C of 5min, 4 DEG C of preservations.
Three, composite amplification
Respectively with cDNA the or Rnase-free Water (negative control) of 10 standard miRNA for template, use is compound
Primer (each PCR primer shown in the row of table 3 the 2nd forms) carries out PCR amplification, obtains pcr amplification product.It is theoretically each
In the size of pcr amplification product such as table 3 shown in the 3rd row.Pcr amplification reaction carries out in PCR instrument.
PCR reaction systems are 10 μ L, by 4 μ 2.5 × Master of L Mix (productions of company of Material Evidence Identification Center, Ministry of Public Security
Product), the cDNA (DNA containing 5ng) of 0.1 μ L standards miRNA, 1 μ L composite primers and 4.9 μ L Rnase-free Water compositions.
In concentration of each PCR primer in PCR reaction systems such as table 3 shown in the 4th row.
PCR reaction conditions：95 DEG C of pre-degeneration 7min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min, 28 cycles；72 DEG C of extensions
5min。
Table 3
Note：FAM is FAM fluorescent markers.
Four, capillary electrophoresis detection
1,1 μ L pcr amplification products, 0.26 μ L Typer500 internal standards and 8.74 μ LHi-Di formamides are mixed, is obtained anti-
Answer system.
2, after completing step 1, reaction system, 95 DEG C of denaturation 5min is taken to be transferred quickly to -20 DEG C of placement 5min, then use
ABI-3130 genetic analyzers carry out capillary electrophoresis detection, and Capillary Electrophoresis is carried out using Genemapper ID v3.2 softwares
Data collection and analysis.Deposition condition：Sample injection time 10s, sample introduction voltage 1kv, working voltage 13.4kv, temperature 60 C, operation
Time 15min.
Experimental result is shown in Fig. 1.The result shows that 10 standard miRNA are successfully detected, peak value 5000-8000rfu it
Between；10 standard miRNA go out peak position and the amplified production size of the 3rd row theoretical calculation in table 3 is completely the same, do not occur non-
Specific peak.
Embodiment 2, sensitivity test
One, composite amplification
The cDNA of the 10 standard miRNA prepared respectively using 1 step 2 of embodiment is template, using composite primer (by table 3
Each PCR primer composition shown in 2nd row) PCR amplification is carried out, obtain pcr amplification product.
PCR reaction systems are 10 μ L, (contain 100ng by the cDNA of 4 μ L 2.5 × Master Mix, 0.1 μ L standards miRNA
DNA, 10ng DNA, 1ng DNA, 0.1ng DNA, 0.01ng DNA or 0.001ng DNA), 1 μ L composite primers and 4.9 μ L
Rnase-free Water compositions.In concentration of each PCR primer in PCR reaction systems such as table 3 shown in the 4th row.
PCR reaction conditions：95 DEG C of pre-degeneration 7min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min, 28 cycles；72 DEG C of extensions
5min。
Four, capillary electrophoresis detection
1,1 μ L pcr amplification products, 0.26 μ L Typer500 internal standards and 8.74 μ LHi-Di formamides are mixed, is obtained anti-
Answer system.
2, after completing step 1, reaction system, 95 DEG C of denaturation 5min is taken to be transferred quickly to -20 DEG C of placement 5min, then use
ABI-3130 genetic analyzers carry out capillary electrophoresis detection, and Capillary Electrophoresis is carried out using Genemapper ID v3.2 softwares
Data collection and analysis.Deposition condition：Sample injection time 10s, sample introduction voltage 1kv, working voltage 13.4kv, temperature 60 C, operation
Time 15min.
If the cDNA of 10 standard miRNA is detected and peak value is between 5000-8000rfu, show PCR reactants
The content of the cDNA of respective standard miRNA in system can be detected.If the cDNA of 10 standard miRNA is not detected
(as lost) shows that the content of the cDNA of the respective standard miRNA in PCR reaction systems cannot be detected.
Experimental result is shown in Fig. 2.The result shows that the sensitivity of the cDNA of 10 standard miRNA of detection is 1ng/ reactants
System.
Embodiment 3, parting map identification human body fluid based on 10 kinds of miRNA it is tissue-derived
One, sample collection
In accordance with Principles in Informed Consent, 50 samples, wherein venous blood sample, sperm sample are collected from unrelated healthy individuals
Sheet, saliva sample, menstruation blood sample and each 10 of vaginal fluid sample.Venous blood sample, which is unrelated healthy individuals, will pass through elbow
The blood collection of taken by venipuncture obtains (50 μ L of each sample) in centrifuge tube.Semen sample is that unrelated healthy individuals will be new
Fresh seminal fluid collecting obtains in aseptic plastic cup.Saliva sample is that the mouth cavity liquid for flowing out unrelated healthy individuals naturally is collected
It is obtained in centrifuge tube.Menstruation blood sample is that 2d or 3d of the unrelated healthy individuals aseptic cotton carrier in the menstrual cycle are acquired.It is cloudy
Road secretion sample is intravaginal wiping of the unrelated healthy individuals medical aseptic cotton swab in the 8d to 28d of menstrual cycle
It obtains.
50 sample standard deviations are stored in -80 DEG C.
Two, the acquisition of cDNA
1, the Total RNA of 50 samples are extracted respectively using miRNeasy Mini Kit, then use Turbo DNA-
freeTMKit is purified (purpose is removal genomic DNA), obtains the Total RNA of 50 samples.
2, after completing step 1, take the Total RNA of (a small amount of) 50 samples into row agarose gel electrophoresis (purpose respectively
For the integrality of the Total RNA of 50 samples of detection).
3, after completing step 2, the Total RNA of 50 samples is taken, are quantified using ND-2000 spectrophotometers.It is fixed
Amount the results are shown in Table 4.
Table 4
4, after completing step 3, respectively using the Total RNA of 50 samples as template, drawn using corresponding reverse transcription in table 2
Object carries out reverse transcription, obtains corresponding cDNA.Reverse transcription reaction carries out in PCR instrument.
Reaction system be 20 μ L, by 2 μ L dNTP Mixture (10mM), 2.5 μ L 5 × First-Strand Buffer,
1μL DTT(0.1M)、2μL M-MLV reverse transcriptase(200units/μL)、0.2μL RecombinantRNase Inhibitor (10000U/ μ L), 1 μ L templates (RNA containing 50ng), 1 μ L reverse transcriptase primers and 10.3 μ L
Rnase-free Water compositions.Reverse transcriptase primer in the reaction system a concentration of 0.85 μM.
The reaction condition of reverse transcription：37 DEG C of 60min, 70 DEG C of 5min, 4 DEG C of preservations.
Three, composite amplification
Respectively with cDNA the or Rnase-free Water (negative control) of 50 samples for template, using composite primer
(each PCR primer shown in the row of table 3 the 2nd forms) carries out PCR amplification, obtains pcr amplification product.Theoretically each PCR expands
Increase production in the size such as table 3 of object shown in the 3rd row.Pcr amplification reaction carries out in PCR instrument.
PCR reaction systems are 10 μ L, by 4 μ L 2.5 × Master Mix, the cDNA (DNA containing 50ng) of 0.5 μ L samples, 3
μ L composite primers and 2.5 μ L Rnase-free Water compositions.Concentration of each PCR primer in PCR reaction systems such as table 3
In the 5th row shown in.
PCR reaction conditions：95 DEG C of pre-degeneration 7min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min, 28 cycles；72 DEG C of extensions
5min。
Four, capillary electrophoresis detection
1,1 μ L pcr amplification products, 0.26 μ L Typer500 internal standards and 8.74 μ LHi-Di formamides are mixed, is obtained anti-
Answer system.
2, after completing step 1, reaction system, 95 DEG C of denaturation 5min is taken to be transferred quickly to -20 DEG C of placement 5min, then use
ABI-3130 genetic analyzers carry out capillary electrophoresis detection, and Capillary Electrophoresis is carried out using Genemapper ID v3.2 softwares
Data collection and analysis.Deposition condition：Sample injection time 10s, sample introduction voltage 1kv, working voltage 13.4kv, temperature 60 C, operation
Time 15min.
The capillary electrophoresis detection result of section of vein blood sample is shown in Fig. 3.The capillary electrophoresis detection of part semen sample
As a result see Fig. 4.The capillary electrophoresis detection result of part menstruation blood sample is shown in Fig. 5.The Capillary Electrophoresis of part saliva sample is examined
It surveys result and sees Fig. 6.The capillary electrophoresis detection result of part vaginal fluid sample is shown in Fig. 7.
The testing result of the peak shape position analysis body fluid sample of 10 standard miRNA in reference implementation example 1.Each pseudo body fluid
The detection result peak shape of 10 Different Individuals is identical, therefore the type of body fluid sample to be measured can be judged with genotyping result：
If the peak value of miR451a, miR144-3P and miR144-5P are in 3000-7000rfu in body fluid sample to be measured
Between, then body fluid sample to be measured is venous blood sample；
If the peak value of miR888-5P, miR144-3P and miR891a-5P are in 1000- in body fluid sample to be measured
Between 7000rfu, then body fluid sample to be measured is semen sample；
If the peak value of miR214, miR451a, miR144-3P, miR205-5P and miR203-3p in body fluid sample to be measured
Between 800-7000rfu, then body fluid sample to be measured is menstruation blood sample；
If the peak value of miR451a, miR144-3P, miR205-5P and miR203-3p exist in body fluid sample to be measured
Between 600-7000rfu, then body fluid sample to be measured is saliva sample；
If the peak value of miR144-3P and miR203-3p waits between 600-3000rfu in body fluid sample to be measured
Survey body fluid sample is vaginal fluid sample.
<110>Material Evidence Identification Center, Ministry of Public Security
<120>A kind of tissue-derived miRNAs composite amplification systems and its primer special combination for identifying human body fluid
<160> 40
<170> PatentIn version 3.5
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Claims (10)
1. primer combines, including primer combination first and primer combine second：
The primer combination first includes primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer
28, primer 31, primer 32, primer 33, primer 34, primer 35, primer 36, primer 37 and primer 38；
The primer 21 is following A1) or A2)：
A1) single strand dna shown in the sequence 21 in sequence table；
A2 sequence 21) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 21 identical
The DNA molecular of function；
The primer 22 is following A3) or A4)：
A3) single strand dna shown in the sequence 22 in sequence table；
A4 sequence 22) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 22 identical
The DNA molecular of function；
The primer 23 is following A5) or A6)：
A5) single strand dna shown in the sequence 23 in sequence table；
A6 sequence 23) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 23 identical
The DNA molecular of function；
The primer 24 is following A7) or A8)：
A7) single strand dna shown in the sequence 24 in sequence table；
A8 sequence 24) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 24 identical
The DNA molecular of function；
The primer 25 is following A9) or A10)：
A9) single strand dna shown in the sequence 25 in sequence table；
A10 sequence 25) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 25
The DNA molecular of congenerous；
The primer 26 is following A11) or A12)：
A11) single strand dna shown in the sequence 26 in sequence table；
A12 sequence 26) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 26
The DNA molecular of congenerous；
The primer 27 is following A13) or A14)：
A13) single strand dna shown in the sequence 27 in sequence table；
A14 sequence 27) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 27
The DNA molecular of congenerous；
The primer 28 is following A15) or A16)：
A15) single strand dna shown in the sequence 28 in sequence table；
A16 sequence 28) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 28
The DNA molecular of congenerous；
The primer 31 is following A17) or A18)：
A17) single strand dna shown in the sequence 31 in sequence table；
A18 sequence 31) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 31
The DNA molecular of congenerous；
The primer 32 is following A19) or A20)：
A19) single strand dna shown in the sequence 32 in sequence table；
A20 sequence 32) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 32
The DNA molecular of congenerous；
The primer 33 is following A21) or A22)：
A21) single strand dna shown in the sequence 33 in sequence table；
A22 sequence 33) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 33
The DNA molecular of congenerous；
The primer 34 is following A23) or A24)：
A23) single strand dna shown in the sequence 34 in sequence table；
A24 sequence 34) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 34
The DNA molecular of congenerous；
The primer 35 is following A25) or A26)：
A25) single strand dna shown in the sequence 35 in sequence table；
A26 sequence 35) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 35
The DNA molecular of congenerous；
The primer 36 is following A27) or A28)：
A27) single strand dna shown in the sequence 36 in sequence table；
A28 sequence 36) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 36
The DNA molecular of congenerous；
The primer 37 is following A29) or A30)：
A29) single strand dna shown in the sequence 37 in sequence table；
A30 sequence 37) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 37
The DNA molecular of congenerous；
The primer 38 is following A31) or A32)：
A31) single strand dna shown in the sequence 38 in sequence table；
A32 sequence 38) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 38
The DNA molecular of congenerous；
The primer combination second includes primer 11, primer 12, primer 13, primer 14, primer 16, primer 17, primer 18 and primer
19；
The primer 11 is following B1) or B2)：
B1) single strand dna shown in the sequence 11 in sequence table；
B2 sequence 11) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 11 identical
The DNA molecular of function；
The primer 12 is following B3) or B4)：
B3) single strand dna shown in the sequence 12 in sequence table；
B4 sequence 12) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 12 identical
The DNA molecular of function；
The primer 13 is following B5) or B6)：
B5) single strand dna shown in the sequence 13 in sequence table；
B6 sequence 13) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 13 identical
The DNA molecular of function；
The primer 14 is following B7) or B8)：
B7) single strand dna shown in the sequence 14 in sequence table；
B8 sequence 14) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 14 identical
The DNA molecular of function；
The primer 16 is following B11) or B12)：
B11) single strand dna shown in the sequence 16 in sequence table；
B12 sequence 16) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 16
The DNA molecular of congenerous；
The primer 17 is following B13) or B14)：
B13) single strand dna shown in the sequence 17 in sequence table；
B14 sequence 17) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 17
The DNA molecular of congenerous；
The primer 18 is following B15) or B16)：
B15) single strand dna shown in the sequence 18 in sequence table；
B16 sequence 18) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 18
The DNA molecular of congenerous；
The primer 19 is following B17) or B18)：
B17) single strand dna shown in the sequence 19 in sequence table；
B18 sequence 19) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 19
The DNA molecular of congenerous.
2. primer combination as described in claim 1, it is characterised in that：The primer combination first further includes primer 29, primer 30, draws
Object 39 and primer 40；
The primer 29 is following A33) or A34)：
A33) single strand dna shown in the sequence 29 in sequence table；
A34 sequence 29) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 29
The DNA molecular of congenerous；
The primer 30 is following A35) or A36)：
A35) single strand dna shown in the sequence 30 in sequence table；
A36 sequence 30) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 30
The DNA molecular of congenerous；
The primer 39 is following A37) or A38)：
A37) single strand dna shown in the sequence 39 in sequence table；
A38 sequence 39) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 39
The DNA molecular of congenerous；
The primer 40 is following A39) or A40)：
A39) single strand dna shown in the sequence 40 in sequence table；
A40 sequence 40) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 40
The DNA molecular of congenerous；
The primer combination second further includes primer 15 and primer 20；
The primer 15 is following B9) or B10)：
B9) single strand dna shown in the sequence 15 in sequence table；
B10 sequence 15) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 15
The DNA molecular of congenerous；
The primer 20 is following B19) or B20)：
B19) single strand dna shown in the sequence 20 in sequence table；
B20 sequence 20) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and there is phase with sequence 20
The DNA molecular of congenerous.
3. primer combination as claimed in claim 1 or 2, it is characterised in that：
In primer combination first, the primer 21, the primer 23, the primer 25, the primer 27, the primer 29,
The primer 31, the primer 33, the primer 35, the primer 37 and the primer 39 use fluorescent marker.
4. a kind of miRNAs composite amplification systems, including miRNAs composite amplification systems first and miRNAs composite amplification system second；
The miRNAs composite amplification systems first includes the combination first of primer described in claims 1 to 3；
The miRNAs composite amplification systems second includes primer 11, primer in the combination second of primer described in claims 1 to 3
12, primer 13, primer 14, primer 16, primer 17, primer 18 or primer 19.
5. miRNAs composite amplification systems as claimed in claim 4, it is characterised in that：The miRNAs composite amplification systems first
In, a concentration of 0.138 μM in miRNAs composite amplification system first of the primer 21 and the primer 22；3 He of the primer 2
A concentration of 0.036 μM in miRNAs composite amplification system first of the primer 24；The primer 25 and the primer 26 exist
A concentration of 0.042 μM in miRNAs composite amplification system first；The primer 27 and the primer 28 are in miRNAs composite amplifications
A concentration of 0.084 μM in system first；The concentration of the primer 31 and the primer 32 in miRNAs composite amplification system first
It is 0.288 μM；A concentration of 0.036 μM in miRNAs composite amplification system first of the primer 33 and the primer 34；It is described
A concentration of 0.120 μM in miRNAs composite amplification system first of primer 35 and the primer 36；The primer 37 and described draw
A concentration of 0.300 μM in miRNAs composite amplification system first of object 38；
In the miRNAs composite amplification systems second, the primer 11, the primer 12, the primer 13, the primer 14, institute
State primer 16, the primer 17, the primer 18 or the primer 19 a concentration of 0.85 μM.
6. the kit containing any primer combination of claims 1 to 3；The kit is used to identify the tissue of human body fluid
Source.
7. the preparation method of kit described in the miRNAs composite amplification systems of claim 4 or 5 or claim 6, including
The step of each primer in any primer combination of claims 1 to 3 is individually packed.
8.X1) or X2)：
X1) any primer combination of claims 1 to 3 or the miRNAs composite amplification systems of claim 4 or 5,
Prepare the application in the tissue-derived kit for identifying human body fluid；
X2) any primer combination of claims 1 to 3 or any miRNAs composite amplifications body of claim 4 or 5
System, identification human body fluid it is tissue-derived in application.
9. a kind of tissue-derived method of identification body fluid to be measured, in turn includes the following steps：
(1) using the total serum IgE of body fluid to be measured as template, each primer of the combination second of primer described in claims 1 to 3 is respectively adopted
Reverse transcription is carried out, the cDNA of body fluid to be measured is obtained；
(2) after completing step (1), respectively using the cDNA of the body fluid to be measured as template, draw using described in claims 1 to 3
Object combines first and carries out PCR amplification, obtains pcr amplification product；
(3) after completing step (2), pcr amplification product is subjected to capillary electrophoresis detection, testing result is analyzed and is sentenced as follows
It is disconnected：
If using primer 12, primer 16 and primer 17 carry out reverse transcription subsequent products peak value 3000-7000rfu it
Between, then body fluid to be measured is venous blood；
If using primer 13, primer 17 and primer 18 carry out reverse transcription subsequent products peak value 1000-7000rfu it
Between, then body fluid to be measured is sperm；
If carrying out the peak value of the subsequent products of reverse transcription using primer 17 and primer 19 between 600-3000rfu, and adopt
Carry out the peak values of the subsequent products of reverse transcription not between 600-3000rfu with primer 11, primer 12 or primer 14, then it is to be measured
Body fluid is vaginal fluid；
If carrying out the peak value of the subsequent products of reverse transcription using primer 12, primer 14, primer 17 and primer 19 in 600-
Between 7000rfu, and carried out using primer 11 reverse transcription subsequent products peak value not between 600-7000rfu, then wait for
Survey body fluid is saliva；
If the peak value for being carried out the subsequent products of reverse transcription using primer 11, primer 12, primer 14, primer 17 and primer 19 is existed
Between 800-7000rfu, then body fluid to be measured is menstrual blood.
10. the method described in kit as claimed in claim 6, application according to any one of claims 8 or claim 9, feature
It is：It is described tissue-derived for venous blood, sperm, vaginal fluid, saliva or menstrual blood.
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