CN107201405A - A kind of diatom UPA genetic analysis method and its application in legal medical expert detects - Google Patents
A kind of diatom UPA genetic analysis method and its application in legal medical expert detects Download PDFInfo
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- CN107201405A CN107201405A CN201710459243.5A CN201710459243A CN107201405A CN 107201405 A CN107201405 A CN 107201405A CN 201710459243 A CN201710459243 A CN 201710459243A CN 107201405 A CN107201405 A CN 107201405A
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- diatom
- drowned
- upa
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
Judging whether the person of being drowned really dies from the application being drowned the invention discloses a kind of diatom UPA genes.The primer pair of a pair of detection diatom UPA genes is provided in the present invention, a kind of analysis method of diatom UPA genes is also provided, extract diatom nucleic acid, expanded using UPA genes in the above-mentioned nucleic acid of primer pair described in claim 2, sample to be tested is prepared using amplified production, foranalysis of nucleic acids is carried out to sample to be tested.Further, used probe is 5 ' CGGAGGCGTACAAAG 3 ', and the end of probe 5 ', with FAM fluorescent reporter groups mark, 3 ' ends are marked with NFQ MGB fluorescent quenching groups.Present invention also offers the application being applied to above-mentioned diatom UPA genetic analysis method during legal medical expert detects, diatom nucleic acid is extracted in the person's of being drowned internal organs and is expanded with round pcr, then detected and be compared with the diatom nucleic acid in water sample, pass through the comparison of recall rate and diatom UPA gene sequencing results, it can determine whether whether the person of being drowned really dies to be drowned, while helping to find out region of being really drowned.
Description
Technical field
The present invention relates to forensic science, and in particular to a kind of analysis method of diatom UPA genes and its legal medical expert detection
In application.
Background technology
Medical jurisprudence inspection case practice in, when be commonly encountered the test sensitivity for being drowned Related Cases.Because China's rivers,lakes and seas are many
Many, environment is complicated, and the factor influence such as the putrefaction of dead body, and the cause of the death identification of corpse is always the difficult point in medicolegal practice in water
One of.For how to judge that corpse is to enter water before death or after death throw corpse to enter water in water, educational circles still suffers from dispute.Diatom examination is always
It is most common method in medicolegal practice.The method of current diatom examination has a lot, but domestic conventional diatom examination method master
To be detected based on diatom morphology.However as the development of Protocols in Molecular Biology, existing scholar attempts to detect by PCR
The cause of the death identification that the specific DNA bar code of diatom is drowned.DNA bar code refers to the core using short standard DNA sequence
Thuja acid diversity carries out the identification of species, and the algae that morphology is difficult to differentiate between can be avoided by carrying out diatom examination using DNA bar code
Class, eliminates manual observation and the experimental error counted.
Compared with traditional diatom morphological examination method, fluorescent quantitative PCR detection diatom DNA bar code is used to drown
Dead identification mainly has the advantage that：1. high specificity is detected, available for the diatom examination of form discrimination can not be carried out with microscope；
2. sensitivity is high, and required sample size is significantly less than traditional diatom method of inspection；3. it is drowned place and time available for deduction.Utilize silicon
The specificity of algae DNA bar code, can disclose COMMUNITY CHARACTERISTICS qualitative while, such as further set up different waters and same water
Domain different time diatom biocoene changing features monitoring system, and be combined with traditional diatom method of inspection, to identification of drowning then
There is prior practical value.
The content of the invention
The technical problems to be solved by the invention are to overcome above-mentioned defect of the prior art and not enough there is provided one kind is right
Diatom UPA genes（Universal Plastid Amplicon）, general plastid amplification region) analysis method, and using pair
Application of the analysis method of diatom UPA genes in legal medical expert detects, is specifically the application on the true cause of the death of the person of being drowned is judged.
UPA genes, the fragment is the V structure domain of chloroplaset 23S rRNA genes, length about 370bp.The sequencing of UPA genes
Efficiency and amplification efficiency are all very high, and with higher resolution ratio, are adapted to the research of diatom molecular classification.Diatom is a kind of extensive point
The unicellular organism in water is distributed in, cell membrane is made up of a large amount of silicon matters, various in the kind of diatom, is come in every shape, everywhere water
The diatom composition in source is all not quite similar.Therefore, the presence by detecting diatom in each main organs of corpse in water can not only turn into
Judge whether one of important evidence for being drowned before death, also can be to judging that death place is significant.Examined currently with diatom
It is lungs to test the standard for judging to be drowned, and liver, kidney etc. organizes interior discovery diatom, and diatom species is consistent with live water sample
It is diagnosed as being drowned.
The technique effect of the invention to be reached is realized by following scheme：
A kind of diatom UPA genes are provided in the present invention and are judging whether the person of being drowned really dies from the application being drowned.
The primer pair of a pair of detection diatom UPA genes is provided in the present invention, the primer pair includes sense primer and downstream
Primer, the sequence of sense primer is as shown in SEQ IDNo.1, and the sequence of anti-sense primer is as shown in SEQ IDNo.2.
A kind of analysis method of diatom UPA genes is also provided in the present invention, diatom nucleic acid is extracted, utilizes claim 2
UPA genes are expanded in the above-mentioned nucleic acid of primer pair, and sample to be tested is prepared using amplified production, and core is carried out to sample to be tested
Acid analysis.Further, used probe is 5 '-CGGAGGCGTACAAAG-3 ', and the end of probe 5 ' is with FAM fluorescence report bases
Group's mark, 3 ' ends are marked with NFQ-MGB fluorescent quenching groups.
A kind of fluorescent quantitative PCR determination method of diatom UPA genes is also provided in the present invention, is specially：Expand
It is 20ml to increase detection reaction system：Premix Ex Taq reagent 10ml, concentration is 10 μm of ol/L forward primer 0.4ml, concentration
For 10 μm of ol/L reverse primer 0.4ml, ROX Reference Dye II0.2ml, probe 0.8ml, DNA profiling 2ml, sterilizing
Distilled water 6.2ml.For specificity verification experiment, DNA profiling refers to the genomic DNA of diatom, for case test experience,
DNA profiling refers to sample genome DNA.The genomic DNA of both all contains diatom UPA genes.
Further, PCR reaction conditions are：Stage1：95 DEG C of pre-degeneration, 30s, heating rate 20 DEG C/s, 1cycle；
Stage 2：PCR reacts 95 DEG C, 5s, 20 DEG C/s of heating rate, 60 DEG C, 34s, heating rate 20 DEG C/s, 40 cycles；Moving back
Fiery step carries out fluorescence signal collection.
The preparation method of sample to be tested is：The dead's entity internal organs are taken, the base in internal organs is extracted using nucleic acid extraction kit
Because group DNA carries out fluorescent quantitative PCR detection, it is to treat to extract the genomic DNA in internal organs using nucleic acid extraction kit
Test sample sheet.
A kind of above-mentioned diatom UPA gene by fluorescence quantitative PCR determination methods are also provided in the present invention to detect in legal medical expert
Application in journey, is specifically drowned applied to whether the judgement person of being drowned really dies from.
Further, described application process is：Drowning person's organs and tissues are taken, the nucleic acid in the organs and tissues is extracted, makes
Fluorescent quantitative PCR detection and analysis is carried out with primer pair as claimed in claim 2, confirms whether sample to be tested contains diatom
Whether whether UPA genes, judge the person of being drowned really to be drowned according to the detection of diatom UPA genes.
The present invention also provides whether a kind of judgement person of being drowned really dies from the side for being drowned and/or finding out real region of being drowned
Method, be specially：Take in drowning person's organs and tissues, detection organs and tissues and whether contain diatom UPA genes, judged according to testing result
Whether the person of being drowned, which really dies from, is drowned, and finds out region of being really drowned.
The present invention has advantages below：
1st, the present invention proposes whether diatom UPA genes really die from the application being drowned in the judgement person of being drowned.
2nd, there is provided a kind of analysis method to diatom UPA genes, there is provided analyze the upper of the gene order in the present invention
Primer and anti-sense primer are swum, and provides the fluorescent quantitative PCR detection parameter being adapted with diatom UPA genes simultaneously, and
This method is referred in the application that the real cause of the death of the person of being drowned is judged using diatom UPA genes.
3rd, present invention also offers the application being applied to above-mentioned diatom UPA genetic analysis method during legal medical expert detects, drowning
Diatom nucleic acid is extracted in the person's of dying internal organs and is tested and analyzed with fluorescent quantitative PCR, is then carried out with the diatom nucleic acid in water sample
Compare, pass through the comparison of recall rate and diatom UPA gene sequencing results, you can judge whether the person of being drowned really dies from and be drowned, together
When help to find out region of being really drowned.
Brief description of the drawings
Fig. 1 is the Primer-Blast result figures of primer in the present invention；
Fig. 2 is the amplification curve result figure of fluorogenic quantitative detection diatom type strain UPA genes in the present invention；
Fig. 3 expands the NCBI-BLAST result figures of diatom type strain amplified production for primer in the present invention
Fig. 4 is detection internal organs amplified production sequencer map in the present invention.
Embodiment
The present invention will be described in detail with reference to the accompanying drawings and examples.
The design of primers of embodiment 1 and primer specificity are proved
A pair of upstream and downstream primers are designed according to the UPA genes of diatom：
Sense primer（As shown in SEQ ID NO.1）：
5’-CAGTGAGATACCACCCTTGTAATGTT-3’
Anti-sense primer（As shown in SEQ ID NO.2）：
5’-TCTAGATACGATTTCTAACCGCTCAGA-3’
The specificity verification of the primer pair of embodiment 2
Real-time fluorescence quantitative PCR detection is carried out to the DNA of different samples using above-mentioned primer and probe, the end of probe 5 ' is glimmering with FAM
Light reporter group is marked, and 3 ' ends are marked with NFQ-MGB fluorescent quenching groups, choose five kinds of Common diatoms, two kinds of human body fungal components
Primer specificity checking is done with a variety of non-diatomaceous algae DNA.
1:Small ring algae；2：Rhombus algae；3：Boat-shaped algae；4：Shank algae；5：Make a variation melosira；6：Escherichia coli；7：Bifid bar
Bacterium；8：Aeromonas salmonicida；9：Aeromonas veronii；10：Aeromonas sobria；11：Chlorella pyrenoidosa；12：Common bead
Algae；13：Scenedesmus obliquus；14：Crescent algae fibre；15：Nostoc；16：Anabena；17：Dan Qizao；18：Soft and fine synnema algae；19：
The malicious microcystic aeruginosa of production；20：Malicious microcystic aeruginosa is not produced；21：Ultra-pure water（Negative control）
Amplification curve diagram as shown in Figure 2 is obtained after amplification.Expanded by the visible only five diatom samples of accompanying drawing 2, it is right
Other bacteriums and non-diatomaceous algae do not have amplification curve, and amplified production is sequenced, by sequencing gained sequence in NCBI
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) carry out BLAST comparisons, comparison result（Accompanying drawing 3）It is shown as
Diatom.I.e. provable above-mentioned primer can carry out specific amplification to diatom.
This method, which only needs to quantitative fluorescent PCR, can carry out diatom examination, by observing amplification curve（Standard PCR does not have
Amplification curve）Diatom can be determined whether（Amplification curve plays peak and Ct values and is less than 35 as diatoms）.
The animal groups control experiment of embodiment 3
Artificially it is drowned experiment pig group（n=15）, experiment pig machinery at Postmortem submergence group（n=15）With the blank pair of dry death experiment pig
According to group（n=5）Lungs, liver, kidney（Body circulation internal organs）Sample in the genomic DNA that extracts passed through under identical amplification system
The primed probe that the present embodiment 1 is designed carries out real-time fluorescence quantitative PCR amplification, and amplification curve and Ct values occurs in regulation（Cycle
threshold）It is positive amplification less than 35, result judgement is detection, and recall rate the results are shown in Table 1.
The diatom UPA genes of table 1 detect number of cases and positive rate in each group experimental animal body circulation internal organs
It can be seen that by above-mentioned experimental result, using the method in the present invention, the DNA in experiment porcine tissues expanded, utilized
Positive amplification result confirms the method recall rate of the experiment pig cause of the death up to 93%, it is contemplated that the difference of water is sucked during being drowned
And individual difference, it can assert that the method in the present invention can effectively judge whether experiment pig is really drowned, while can assist to judge
It is drowned place.
The diatom UPA genetic analysis method of embodiment 4 and the application in legal medical expert detects
The primer pair designed using embodiment can be designed the present invention in a kind of diatom UPA genetic analysis methods, the present embodiment
In diatom UPA genetic analysis method be applied to legal medical expert detection in, be applied particularly to confirm drowning person whether be to be drowned and seek
Look for excessive corresponding region of being drowned.The drowning person used in the present embodiment confirms as true drowning personnel.
Legal medical expert's detecting step is as follows：
Step one：Clip drowning person lungs, liver, renal tissue in fume hood；
Step 2：Extract the genomic DNA in lungs, liver, renal tissue；
Step 3：Implementation fluorescent quantitative PCR is carried out using the primer and probe in embodiment 1；
In step 3, the sense primer used in amplification procedure is 5 '-CAGTGAGATACCACCCTTGTAATGTT-3 '；Under
Trip primer is 5 '-TCTAGATACGATTTCTAACCGCTCAGA-3 '；Taqman probes are 5 '-CGGAGGCGTACAAAG-3 '；
The end of probe 5 ' is marked with FAM fluorescent reporter groups, and 3 ' ends are marked with NFQ-MGB fluorescent quenching groups, it is possible to achieve glimmering
Real_time quantitative detection in Fluorescent Quantitative PCR instrument.
Real time fluorescent quantitative augmentation detection reaction system is 20ml：PCR reaction systems are 20ml：Premix Ex Taq
（Probe qPCR）Reagent（2×）10ml, concentration is 10 μm of ol/L forward primer 0.4ml, and concentration is the reverse of 10 μm of ol/L
Primer 0.4ml, ROX Reference Dye II（50×）0.2ml, probe 0.8ml, DNA profiling 2ml, sterilize distilled water
6.2ml.Above-mentioned 2 ×/50 × implication be used in reagent concentration be it is final its 2/50 times of concentration in amplification system.
First mixing amplification raw material, DNA profiling is eventually adding amplification system.For specificity verification experiment, what DNA profiling referred to
It is the genomic DNA of diatom, for case test experience, DNA profiling refers to sample genomic DNA.The genome of both
DNA all contains diatom UPA genes.
Reaction condition is: Stage 1：95 DEG C of pre-degeneration, 30s, heating rate 20 DEG C/s, 1 cycle；Stage 2：PCR
95 DEG C of reaction, 5s, 20 DEG C/s of heating rate, 60 DEG C, 34s, heating rate 20 DEG C/s, 40 cycles；Carried out in annealing steps
Fluorescence signal collection.
In the present embodiment, the instrument that Real time PCR is used is the Flex of QuantStudio 7, can also use it
He effectively implements quantitative fluorescence analysis instrument.
The diatom UPA genes of corpse internal organs in water are detected using Real time PCR instrument, with positive amplification
Confirm as containing diatom in the sample, the sample can determine that as drowning, as a result show to be consistent with the known cause of the death.
Pcr amplification product is sequenced as shown in Figure 4.
Pcr amplification product is sequenced：AGTGAG ATACCACCCT TGTAATGTTA GATTTCTAAC TTTGAATCAT
TATCTGGTCA AAGAACAGTT TCAGGCGGGC AGTTTGACTG GGGCGGTCGC CTCCCAAATG GTGACGGAGG
CGTACAAAGG TTTCCTCTGA GCGGTTAGAA ATCGTATCTA GA。
It is last it should be noted that above example is only entered to the technical scheme that illustrates the embodiment of the present invention rather than to it
Row limitation, although the embodiment of the present invention is described in detail with reference to preferred embodiment, one of ordinary skill in the art
It should be understood that the technical scheme of the embodiment of the present invention can still be modified or equivalent substitution, and these are changed or waited
It can not also make the scope of amended technical scheme disengaging technical scheme of the embodiment of the present invention with replacing.
SEQUENCE LISTING
<110>Guangzhou City Forensic Science Technology Institute
<120>A kind of diatom UPA genetic analysis method and its application in legal medical expert detects
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213>Primer β-actinF1
<400> 1
CAGTGAGATACCACCCTTGTAATGTT 26
<210> 2
<211> 27
<212> DNA
<213>Primer β-actinR1
<400> 2
TCTAGATACGATTTCTAACCGCTCAGA 27
Claims (10)
1. diatom UPA genes are judging whether the person of being drowned really dies from the application being drowned.
2. the primer pair of a pair of detection diatom UPA genes, it is characterised in that：The primer pair includes sense primer and downstream is drawn
Thing, the sequence of sense primer is as shown in SEQ IDNo.1, and the sequence of anti-sense primer is as shown in SEQ IDNo.2.
3. a kind of diatom UPA genetic analysis methods, it is characterised in that：Diatom nucleic acid is extracted, primer described in claim 2 is utilized
UPA genes in above-mentioned nucleic acid are expanded, sample to be tested is detected using fluorescent quantitative PCR, core is carried out to sample to be tested
Acid analysis.
4. diatom UPA genetic analysis methods as claimed in claim 3, it is characterised in that：Used probe be 5 '-
CGGAGGCGTACAAAG-3 ', and the end of probe 5 ', with FAM fluorescent reporter groups mark, 3 ' ends are with NFQ-MGB fluorescent quenching groups
Mark.
5. diatom UPA genetic analysis methods as claimed in claim 3, it is characterised in that：Augmentation detection reaction system is 20ml：
Premix Ex Taq reagent 10ml, concentration is 10 μm of ol/L forward primer 0.4ml, and concentration is 10 μm of ol/L reverse primer
0.4ml, ROX Reference Dye II0.2ml, probe 0.8ml, DNA profiling 2ml, sterilizing distilled water 6.2ml.
6. diatom UPA genetic analysis methods as claimed in claim 5, it is characterised in that：PCR reaction conditions are：Stage 1：In advance
95 DEG C of denaturation, 30s, heating rate 20 DEG C/s, 1 cycle；Stage 2：PCR reacts 95 DEG C, 5s, heating rate 20 DEG C/s, 60
DEG C, 34s, heating rate 20 DEG C/s, 40 cycles；Fluorescence signal collection is carried out in annealing steps.
7. diatom UPA genetic analysis methods as claimed in claim 3, it is characterised in that：The preparation method of sample to be tested is：Take dead
Person's entity internal organs, the genomic DNA in internal organs is extracted using nucleic acid extraction kit, and internal organs are extracted using nucleic acid extraction kit
In genomic DNA, the gene DNA of extraction is sample to be tested as sample to be tested.
8. a kind of application of the diatom UPA genetic analysis method in legal medical expert's detection process as described in claim 3-7 is any, it is special
Levy and be：It is applied particularly to the judgement person of being drowned and whether really dies from be drowned.
9. application as claimed in claim 8, it is characterised in that：Described application process is：Drowning person's organs and tissues are taken, are extracted
Nucleic acid in the organs and tissues, carries out fluorescent quantitative PCR detection and analysis, really using primer pair as claimed in claim 2
Recognize whether sample to be tested contains diatom UPA genes, whether judge whether the person of being drowned is really excessive according to the detection of diatom UPA genes
Die.
Judge whether the person of being drowned really dies from the method for being drowned and/or finding out real region of being drowned 10. a kind of, it is characterised in that：
Take in drowning person's organs and tissues, detection organs and tissues and whether contain diatom UPA genes, whether the person of being drowned is judged according to testing result
Real die from is drowned, and finds out region of being really drowned.
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| CN113796878B (en) * | 2021-09-10 | 2024-04-19 | 高阳 | Application of energy spectrum technology based on virtual anatomy in drowning case |
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Application publication date: 20170926 |