CN107345246A - A kind of diatom rbcL genetic analysis method and its application in legal medical expert detects - Google Patents
A kind of diatom rbcL genetic analysis method and its application in legal medical expert detects Download PDFInfo
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- CN107345246A CN107345246A CN201710459244.XA CN201710459244A CN107345246A CN 107345246 A CN107345246 A CN 107345246A CN 201710459244 A CN201710459244 A CN 201710459244A CN 107345246 A CN107345246 A CN 107345246A
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- diatom
- drowned
- rbc
- nucleic acid
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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
A kind of diatom of disclosure of the inventionrbcL genes are judging whether the person of being drowned really dies from the application being drowned.A pair of detection diatoms are provided in the present inventionrbcThe primer pair of L genes, also provide a kind of diatomrbcThe analysis method of L genes, diatom nucleic acid is extracted, using in the above-mentioned nucleic acid of primer pair described in claim 2rbcL genes are expanded, and sample to be tested is prepared using amplified production, and foranalysis of nucleic acids is carried out to sample to be tested.Present invention also offers by above-mentioned diatomrbcL genetic analysis method is applied to the application in legal medical expert's detection, and diatom nucleic acid is extracted in the person's of being drowned internal organs and is expanded with round pcr, is then detected and is compared with the diatom nucleic acid in water sample, pass through recall rate and diatomrbcThe comparison of L gene sequencing results, you can judge whether the person of being drowned really dies from and be drowned, while help 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 diatomrbcThe analysis method of L genes and its legal medical expert detect
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
More, environment is complicated, and the factor such as putrefaction of dead body influences, 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 method the most frequently used in medicolegal practice.The method of diatom examination has much at present, 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, eliminate the artificial experimental error observed and counted.
Compared with traditional diatom morphological examination method, PCR amplification techniques detection diatom DNA bar code is used to be drowned mirror
It is fixed mainly to have the advantage that：1. high specificity is detected, available for the diatom examination that form discrimination can not be carried out with microscope；2. spirit
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 diatom
The specificity of DNA bar code, COMMUNITY CHARACTERISTICS can be disclosed qualitative while, such as further establish different waters and same waters
Different time diatom biocoene changing features monitoring system, and be combined with traditional diatom method of inspection, then have to identification of drowning
Prior practical value.
The content of the invention
The technical problems to be solved by the invention be overcome it is above-mentioned in the prior art the defects of and deficiency, there is provided it is a kind of right
DiatomrbcL genes（Large Subunit of Ribulose-1,5-bisphosphate carboxylase
oxygenase, rbcL）The analysis method of general plastid amplification region, and using to diatomrbcThe analysis method of L genes is in method
Application in doctor's detection, the specifically application on the true cause of the death of the person of being drowned is judged.
Chloroplaset ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylase large subunits gene (Large Subunit of Ribulose-1,
5-bisphosphate carboxylase oxygenase, rbcL), it is that chloroplaset carries out photosynthetic key gene piece
Section, the bp of length about 1400, in GenBank,rbcThe information of L sequences is more complete, and it is smaller the probability of introne occur, wide
The general sort research for plant and algae.Diatom is a kind of unicellular organism being distributed widely in water, and cell membrane is by a large amount of
Silicon matter forms, various in the kind of diatom, comes in every shape, and the diatom composition at everywhere water source is all not quite similar.Therefore, inspection is passed through
One of important evidence for judging whether to be drowned before death can not only be turned into by surveying the presence of diatom in each main organs of corpse in water, also may be used
To judging that death place is significant.Judge that the standard be drowned is lungs currently with diatom examination, liver, the group such as kidney
Interior discovery diatom is knitted, and diatom species is consistent with live water sample can be diagnosed as being drowned.
The technique effect of the invention to be reached is realized by following scheme：
A kind of diatom is provided in the present inventionrbcL genes are judging whether the person of being drowned really dies from the application being drowned.
A pair of detection diatoms are provided in the present inventionrbcThe primer pair of L genes, the primer pair include 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 diatom is also provided in the present inventionrbcL genetic analysis methods：Diatom nucleic acid is extracted, utilizes above-mentioned primer pair
To in diatom nucleic acidrbcL genes enter performing PCR amplification, and sample to be tested is prepared using amplified production, and nucleic acid point is carried out to sample to be tested
Analysis.Further, the end of sense primer 5 ' can realize the fluoroscopic examination in HPCE with FAM fluorescence labelings.
A kind of diatom is also provided in the present inventionrbcThe analysis method of L genes, it is specially：PCR reaction systems are 20ml：10
μ L Taq enzymes；Each 0.75 μ L of upstream and downstream primer, concentration are 10 μm of ol/L；7.5 μ L ultra-pure waters；1 μ L DNA profilings.For spy
Different in nature checking test, DNA profiling refer to the genomic DNA of diatom, and for case test experience, DNA profiling refers to sample
Genomic DNA.The genomic DNA of both all contains diatomrbcL genes.
Further, PCR thermal circulation parameters are：94℃ 10 min；94 DEG C of 40 s, 50 DEG C of 1min, 72 DEG C of 1min, altogether
35 circulations, last 72 DEG C, extend 7 min, it is to be used to cool the temperature to 4 DEG C of preservations, produces PCR amplification of nucleic acid samples.
Further, the detection method of the PCR amplification of nucleic acid is capillary electrophoresis detection, and detection method is：By sample
Nucleic acid PCR amplified production, formamide, wherein ILSCC500 mixing, sample nucleic acid pcr amplification product, formamide, ILSCC500
Volume ratio is 1:9:1.
A kind of above-mentioned diatom is also provided in the present inventionrbcApplication of the L genetic analysis method in legal medical expert's detection process, specifically
It is to be 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
Enter performing PCR with the primer pair in the present invention to expand, it is tested and analyzed, confirm whether pcr amplification product derives from diatomrbcL genes, according to diatomrbcWhether the recall rate of L genes judges the person of being drowned really to be drowned.
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, it is specially：Drowning person's organs and tissues are taken, detect in organs and tissues whether contain diatomrbcL genes, sentence according to testing result
Whether the disconnected 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 diatomrbcL genes are judging whether the person of being drowned really dies from the application being drowned.
2nd, provided in the present invention a kind of to diatomrbcThe analysis method of L genes, there is provided analyze the upper of the gene order
Primer and anti-sense primer are swum, and is provided simultaneously and diatomrbcThe amplification method and specific Amplification that L genes are adapted,
And this method is referred to and utilizes diatomrbcL genes are judged in the application of the real cause of the death of the person of being drowned.
3rd, present invention also offers by above-mentioned diatomrbcL genetic analysis method is applied to the application in legal medical expert's detection, is drowning
Diatom nucleic acid is extracted in the person's of dying internal organs and is expanded with round pcr, is then detected and entered with the diatom nucleic acid in water sample
Row compares, and passes through the comparison of recall rate and diatom UPA gene sequencing results, you can and judge whether the person of being drowned really dies from and be drowned,
Contribute to find out region of being really drowned simultaneously.
Brief description of the drawings
Fig. 1 is the Primer-Blast result figures of primer in the present invention；
Fig. 2 detects diatom type strain variation melosira for PCR-CE methods in the present inventionrbcThe capillary electrophoresis detection result of L genes
Figure；
Fig. 3 is the NCBI-BLAST result figures that primer expands diatom type strain amplified production 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
According to diatomrbcL genes design a pair of upstream and downstream primers：
Sense primer（As shown in SEQ ID NO.1）：
5’- CTCAACCATTCATGCG -3’
Anti-sense primer（As shown in SEQ ID NO.2）：
5’- CTGTGTAACCCATAAC -3’
The specificity verification of the primer pair of embodiment 2
The DNA extracted in 29 Plays strains of collection and tissue is expanded respectively using the primer pair in embodiment 1,
And the checking of primer specificity is done, as a result such as table 1 below.Table 1rbcL primers amplification planktonic organism type strain, people's tissue DNA
Capillary electrophoresis detection result
Table1 Capillary electrophoresis test results
Note：+ to be positive ,-it is feminine gender；S1, S2 are different human body tissue extraction sample
Result can be seen that from table, primerrbcL only expands diatom（Rhombus algae, boat-shaped algae, small ring algae, make a variation melosira, shank
Algae, Skeletonema Greville）, to blue-green algae in upper table, green alga, bacterium and tissue amplification are negative findings, the capillary for the melosira that makes a variation
Electrophoresis detection collection of illustrative plates is shown in accompanying drawing 2.Amplified production is sequenced, by sequencing gained sequence in NCBI（https://
blast.ncbi.nlm.nih.gov/Blast.cgi）Carry out BLAST comparisons, comparison result（As shown in Figure 3）It is shown as silicon
Algae.I.e. provable above-mentioned primer can carry out specific amplification to diatom.
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 designs enters performing PCR amplification, carries out capillary electrophoresis analysis to amplified production, it is specified that electrophoresis occurs
Expected amplified production fragment（201bp）, then result judgement is detection, and recall rate the results are shown in Table 2.
The diatom of table 2rbcL genes 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 whether individual difference, the method in the present invention that can assert can effectively judge experiment pig really to be drowned, while can assist to judge
It is drowned place.
The diatom of embodiment 4rbcL genetic analysis method and the application in legal medical expert detects
The primer pair designed using embodiment 1 can design a kind of diatomrbcL genetic analysis methods, this is sent out in the present embodiment
Diatom in brightrbcL genetic analysis method be applied to legal medical expert detection in, be applied particularly to confirm drowning person whether be drowned and
Find region of being drowned corresponding to drowning.The drowning person used in the present embodiment confirms as true drowning personnel.
Legal medical expert's detecting step is as follows：
Step 1：Clip drowning person lungs, liver, renal tissue in fume hood；
Step 2：Extract the genomic DNA in lungs, liver, renal tissue；
Step 3：Enter performing PCR using the primer in embodiment 1 to expand；
In step 3, the sense primer used in amplification procedure is 5 '-CTCAACCATTCATGCG -3 '；Anti-sense primer is
5’- CTGTGTAACCCATAAC -3’.Sense primer 5 ' is held with FAM fluorescence labelings, can be realized in HPCE
Fluoroscopic examination.The HPCE model ABI-3130xl used in embodiment.
PCR reaction systems are 20ml：10 μ L Taq enzymes；Each 0.75 μ L of upstream and downstream primer, concentration are 10 μm of ol/L；7.5
μ L ultra-pure waters；1 μ L DNA profilings.First mixing amplification raw material, DNA profiling are eventually adding amplification system, DNA moulds in the present embodiment
Plate refers to sample genomic DNA.
PCR thermal circulation parameters are：94℃ 10 min；94 DEG C of 40 s, 50 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations,
Last 72 DEG C, extend 7 min, it is to be used to cool the temperature to 4 DEG C of preservations, produces PCR amplification of nucleic acid samples.
Step 4：The PCR nucleic acid for expanding to obtain is subjected to capillary electrophoresis detection, detection method is by sample nucleic acid PCR
Amplified production, formamide, wherein ILSCC500 mixing, sample nucleic acid pcr amplification product, formamide, ILSCC500 volume ratio
For 1:9:1.
The diatom of corpse internal organs in water is detected using PCR-CE methodsrbcL genes, there is positive amplification can confirm as the sample
Contain diatom in this, the sample can determine that as drowning, the results showed that be consistent with the known cause of the death.
Pcr amplification product is sequenced as shown in Figure 4.
Pcr amplification product is sequenced：ACCTACTACAGCACATTGCTTTAGCATACTCAGCACGTTTGTATACTTCTT
CCATAGTAGCAGCTGTGATGTTTAAGTATGAACCTTTAACTTCACCGGTTGCAGCGGATGCACGGTTAATACCTTCT
AAACAATTTAAGAAACGTTCTCTCCAACGCATAAAGAATTGAGA。
It is last it should be noted that above example is only illustrating the technical scheme of the embodiment of the present invention rather than it is entered
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 can still be modified to the technical scheme of the embodiment of the present invention or equivalent substitution, and these modifications or wait
The scope of amended technical scheme disengaging technical scheme of the embodiment of the present invention can not also be made with replacement.
SEQUENCE LISTING
<110>Guangzhou City Forensic Science Technology Institute
<120>A kind of diatom rbcL genetic analysis method and its application in legal medical expert detects
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> DNA
<213>Primer β-actinF1
<400> 1
CTCAACCATTCATGCG 16
<210> 2
<211> 16
<212> DNA
<213>Primer β-actinR1
<400> 2
CTGTGTAACCCATAAC 16
Claims (10)
1. diatomrbcL genes are judging whether the person of being drowned really dies from the application being drowned.
2. a pair of detection diatomsrbcThe primer pair of L 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.
A kind of 3. diatomrbcL genetic analysis methods, it is characterised in that：Diatom nucleic acid is extracted, is drawn using described in claim 2
Thing is in above-mentioned nucleic acidrbcL genes enter performing PCR amplification, and sample to be tested is prepared using amplified production, and nucleic acid is carried out to sample to be tested
Analysis.
4. diatom as claimed in claim 3rbcL genetic analysis methods, it is characterised in that：Sense primer 5 ' is held with FAM fluorescence
Mark, can realize the fluoroscopic examination in HPCE.
5. diatom as claimed in claim 3rbcL genetic analysis methods, it is characterised in that：PCR reaction systems are 20ml：10 μL
Taq enzyme；Each 0.75 μ L of upstream and downstream primer, concentration are 10 μm of ol/L；7.5 μ L ultra-pure waters；1 μ L DNA profilings.
6. diatom as claimed in claim 5rbcL genetic analysis methods, it is characterised in that：PCR thermal circulation parameters are：94℃ 10
min；94 DEG C of 40 s, 50 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations, last 72 DEG C, extend 7 min, cool the temperature to 4 DEG C
Preserve to be used, produce PCR amplification of nucleic acid samples.
7. diatom as claimed in claim 3rbcL genetic analysis methods, it is characterised in that：
The detection method of the PCR amplification of nucleic acid is capillary electrophoresis detection, and detection method is：Sample nucleic acid PCR is expanded and produced
Thing, formamide, ILSCC500 mixing, wherein sample nucleic acid pcr amplification product, formamide, ILSCC500 volume ratio are 1:9:
1。
8. one kind diatom as described in claim 3-7 is anyrbcApplication of the L genetic analysis method in legal medical expert's detection process, its
It is characterised by：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, enter performing PCR using primer pair as claimed in claim 2 and expand, it is tested and analyzed, really
Recognize whether pcr amplification product derives from diatomrbcL genes, according to diatomrbcThe recall rate of L genes judges whether the person of being drowned is true
Just it is being to be drowned.
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：
Drowning person's organs and tissues are taken, detect in organs and tissues whether contain diatomrbcL genes, judge that the person of being drowned is according to testing result
No really die from is drowned, and finds out region of being really drowned.
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Cited By (4)
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| CN108192897A (en) * | 2018-02-11 | 2018-06-22 | 云南省烟草农业科学研究院 | One grows tobacco rbcl genetic fragments and its application |
| CN108265121A (en) * | 2018-01-25 | 2018-07-10 | 四川大学 | Medical jurisprudence based on diatom DNA pyrosequencing spectrum analysis is drowned ground estimating method |
| CN109457017A (en) * | 2018-12-28 | 2019-03-12 | 中国科学院水生生物研究所 | A kind of molecular detecting method of fast quantification frustule density |
| CN110851632A (en) * | 2019-11-12 | 2020-02-28 | 司法鉴定科学研究院 | Drowning place identification system and method based on diatom species classification |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108265121A (en) * | 2018-01-25 | 2018-07-10 | 四川大学 | Medical jurisprudence based on diatom DNA pyrosequencing spectrum analysis is drowned ground estimating method |
| CN108192897A (en) * | 2018-02-11 | 2018-06-22 | 云南省烟草农业科学研究院 | One grows tobacco rbcl genetic fragments and its application |
| CN109457017A (en) * | 2018-12-28 | 2019-03-12 | 中国科学院水生生物研究所 | A kind of molecular detecting method of fast quantification frustule density |
| CN109457017B (en) * | 2018-12-28 | 2023-03-14 | 中国科学院水生生物研究所 | Molecular detection method for rapidly quantifying diatom cell density |
| CN110851632A (en) * | 2019-11-12 | 2020-02-28 | 司法鉴定科学研究院 | Drowning place identification system and method based on diatom species classification |
| CN110851632B (en) * | 2019-11-12 | 2021-07-20 | 司法鉴定科学研究院 | Drowning place identification system and method based on diatom species classification |
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| CN107345246B (en) | 2020-09-29 |
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