CN106119384A - A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects - Google Patents
A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects Download PDFInfo
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- CN106119384A CN106119384A CN201610631273.5A CN201610631273A CN106119384A CN 106119384 A CN106119384 A CN 106119384A CN 201610631273 A CN201610631273 A CN 201610631273A CN 106119384 A CN106119384 A CN 106119384A
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- drowned
- nucleic acid
- aeromonas hydrophila
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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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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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
The invention discloses Aeromonas hydrophila and judging that the person of being drowned the most really dies from the application being drowned, and provide a kind of Aeromonas hydrophila method for nucleic acid analysis, with downstream primer, nucleic acid to be analyzed is expanded, amplification of nucleic acid sample is utilized to prepare sample to be tested, utilize genetic analyzer that sample to be tested carries out detection to analyze, the forward primer wherein used in amplification procedure is 5 ' GCCGAGCGCCCAGAAGGTGAGTT 3 ', and downstream primer is 5 ' GAGCGGCTGGATGCGGTTGT 3 '.The method for nucleic acid analysis of the Aeromonas hydrophila provided in the present invention can be used for, in legal medical expert's detection, being drowned in order to judge that the person of being drowned the most really dies from.
Description
Technical field
The present invention relates to forensic science, be specifically related to analysis method and this analysis of a kind of Aeromonas hydrophila nucleic acid
Method is in the application judged in the true cause of the death of the person of being drowned.
Background technology
Prudence inspection case put into practice in, time be commonly encountered the test sensitivity being drowned case.Owing to China's rivers,lakes and seas are numerous, ring
Border is complicated, and the factor impact such as the putrefaction of dead body, and identification of drowning problem is paid close attention to by Forensic Pathology worker always.Although silicon
Algae inspection is as identifying that the important supplementary means being drowned gains universal acceptance, but easily by postmortem, draws materials and checkout procedure is polluted.
In Dan Congshui, the lung detection diatom of corpse is not enough to qualification and is drowned, and multiple organ does not detects diatom and also can not get rid of and be drowned, therefore diatom
The effect of inspection is by a definite limitation.The most many scholars have studied some other auxiliary and identify the method being drowned, such as blood
Liquid biochemistry, histochemistry, the inspection technology such as chlorophyll (A).But all there is certain error and limitation in various methods.Cause
This, comprehensively analyze judgement preferably with the multiple method of inspection when identifying and be drowned.A kind of detection highly sensitive, high can be developed
Being widely used in of rate determines detection method and the technology that drowned person is the most really drowned, contributes to being drowned region, for currently
Legal medical expert's industry has great importance.
Compared with traditional diatom morphological examination method, PCR amplification technique detection microbial DNA labelling is used for being drowned mirror
Fixed mainly have the advantage that 1. detection range is wide, in addition to can be applicable to alga classifying and identifying, it may also be used for little miniature swim
Biological and can not carry out, with microscope, the plankton inspection that form distinguishes；The most highly sensitive, required sample size is significantly less than biography
System diatom examination method；3. can be used for inferring that the cause of the death is the most really for being drowned.Planktonic conserved sequence is utilized to separate from DNA
Go out various types of series of variation, COMMUNITY CHARACTERISTICS can be disclosed the most simultaneously, as set up different waters and same water further
Territory different time plankton change monitoring system, and combine, to identification of drowning then with tradition diatom method of inspection
There is prior practical value.
Summary of the invention
Invent defect and the deficiency that technical problem is that and overcome above-mentioned prior art to be solved, it is provided that a kind of to addicted to water
The analysis method of Aeromonas, and utilize the analysis method to Aeromonas hydrophila in legal medical expert detects, specifically judging
Application in the true cause of the death of the person of being drowned.
Aeromonas hydrophila (Aeromonas hydrophila) it is distributed widely in the various water bodys of nature, it is multiple water
The constitutional pathogenic bacterium of lively thing, this bacterium is vibrionaceae Aeromonas, for Gram-negative brevibacterium, extreme single flagellum, does not has
Having brood cell and pod membrane, normal two of the pathogen just separated from focus is connected.Plain agar plating medium is cultivated
The bacterium colony circle, the smooth of the edge, center projections, yellowish pink, canescence or the lightest pink that are formed are glossy, physically well develop.Addicted to
Hydrophila all can be bred in the range of water temperature 14.0-40.5 DEG C, with 28.0-30.0 DEG C as optimum temperature.PH value is at 6-11
In the range of all can grow；Optimal pH value is 7.27；Aeromonas hydrophila can be deposited in the aquatic of salinity 0 ‰-4 ‰, optimal salinity
It is 0.5 ‰.From the person's of being drowned internal organs, extract Aeromonas hydrophila nucleic acid and expand with round pcr, then carrying out detecting also
Compare with the Aeromonas hydrophila nucleic acid in water sample, by recall rate and the comparison of nucleic acid sequencing result, i.e. can determine whether to drown
The person of dying the most really dies from and is drowned, and concurrently facilitates and finds out real region of being drowned.
The technical problem to be solved is achieved by the following technical programs:
The present invention provide a kind of Aeromonas hydrophila judging that the person of being drowned the most really dies from the application being drowned.
The present invention provides the primer pair of a pair detection Aeromonas hydrophila, and described primer is to including that forward primer and downstream are drawn
Thing, the sequence of forward primer is as shown in SEQ IDNo.1, and the sequence of downstream primer is as shown in SEQ IDNo.2.
The present invention also provides for a kind of Aeromonas hydrophila method for nucleic acid analysis, utilizes above-mentioned primer to carry out nucleic acid to be analyzed
Amplification, utilizes amplified production to prepare sample to be tested, sample to be tested is carried out foranalysis of nucleic acids.Further, forward primer 5 ' end is all
With FAM fluorescent labeling.
The present invention provides a kind of Aeromonas hydrophila method for nucleic acid analysis, and analysis method is: PCR reaction system is 20 μ L,
Include 10 μ L Taq enzyme, each 0.75 μ L of upstream and downstream primer, 7.5 μ L deionized waters, 1 μ L nucleic acid to be analyzed；PCR cycle parameter: 94
DEG C denaturation 10min；94 DEG C of degeneration 40s, respectively at 55 DEG C of 40s that anneal, 72 DEG C extend 40s, totally 35 circulations, finally at 72 DEG C
Continue to extend 10min, cool the temperature to 4 DEG C of preservations to be used, obtain nucleic acid PCR amplified production sample.
The preparation method of sample to be tested is by sample nucleic acid pcr amplification product, Methanamide, ILSCC500 mixing, wherein sample
This nucleic acid PCR product, Methanamide, the volume of ILSCC500 are respectively 1 μ L, 9 μ L, 1 μ L.
The present invention also provides for the application during legal medical expert detects of a kind of above-mentioned Aeromonas hydrophila method for nucleic acid analysis, tool
Body is to be applied to the judgement person of being drowned the most really die from and be drowned.
Further, described application process is: take drowning person's organs and tissues, extracts the nucleic acid in this organs and tissues, makes
With primer as claimed in claim 2 to carrying out PCR amplification, it is carried out detection and analyzes, confirm whether pcr amplification product originates
In Aeromonas hydrophila, judge that the person of being drowned is the most really as being drowned according to the recall rate of Aeromonas hydrophila.
The present invention also provides for a kind of judgement person of being drowned and the most really dies from and be drowned and/or find out the side in region of being really drowned
Whether method, particularly as follows: take drowning person's organs and tissues, contain Aeromonas hydrophila in detection organs and tissues, sentence according to testing result
The disconnected person of being drowned the most really dies from and is drowned, and finds out real region of being drowned.In this determination methods, whether detection internal organs contain
The method having Aeromonas hydrophila can use the Aeromonas hydrophila method for nucleic acid analysis used in the present invention.
There is advantages that
1, the present invention proposes Aeromonas hydrophila and the most really dies from, the judgement person of being drowned, the application being drowned.
2, the present invention provides one to Aeromonas hydrophila method for nucleic acid analysis, it is provided that analyze this nucleotide sequence
Forward primer and downstream primer, and provide suitable amplification method and concrete Amplification simultaneously, and the method is drawn
Use and utilize Aeromonas hydrophila to judge in the application of the real cause of the death of the person of being drowned.
3, present invention also offers the application being applied to by above-mentioned Aeromonas hydrophila method for nucleic acid analysis in legal medical expert's detection,
In the person's of being drowned internal organs, extract Aeromonas hydrophila nucleic acid expanding with round pcr, then carry out detecting and with in water sample
Aeromonas hydrophila nucleic acid compare, by recall rate and the comparison of nucleic acid sequencing result, i.e. whether can determine whether the person of being drowned
Really die from and be drowned, concurrently facilitate and find out real region of being drowned.
Accompanying drawing explanation
Accompanying drawing 1 is Aeromonas hydrophila in the present invention；
Accompanying drawing 2 analyzes Aeromonas hydrophila nucleic acid result (forward primer fluorescent labeling) in internal organs for capillary electrophoresis apparatus detection；
Accompanying drawing 3 is Aeromonas hydrophila nucleic acid amplification product sequencing result in the embodiment of the present invention.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be described in detail.
Embodiment 1 design of primers and primer specificity prove
According to Aeromonas hydrophila (Aeromonas hydrophila) haemolysin gene design go out a pair upstream and downstream and draw
Thing:
Forward primer (as shown in SEQ ID NO.1):
5 '-GCCGAGCGCCCAGAAGGTGAGTT-3 ',
Downstream primer (as shown in SEQ ID NO.2):
5’- GAGCGGCTGGATGCGGTTGT -3’。
The opposite sex checking of embodiment 2 primer pair
Utilizing above-mentioned primer to carry out PCR amplification to the DNA of different samples, forward primer 5 ' is held all with FAM fluorescent labeling, chooses
In multiple water, the DNA of microalgae, common microbiological and different sample carries out specificity verification.
The DNA sample source that in accompanying drawing, label is corresponding is corresponding as follows:
1, Marker 2, chlamydomonas 3, soil algae 4, four row algae 5, Pseudanabaena sp 6, Skeletonema Greville 7, crisp bar algae 8,
Anabaena Azollae Based 9, flat algae 10, woman 11, man 12, boat row algae 13, little ring algae 14, rhombus algae 15,
Shank algae 16, melosira 17, chlorella 18, cyanophyceae 19, silicon whip chrysophyceae 20, Candida albicans 21, addicted to aqueous vapor
Zymomonas mobilis 22, negative control 23, Marker
After amplification, by silver staining, obtain result as shown in Figure 1.From accompanying drawing 1 result, the only No. 21 aobvious sun of sample
Property, the most provable above-mentioned primer can be with specific amplified Aeromonas hydrophila
Embodiment 3 mice group control experiment
Artificially it is drowned the blank group (n of Postmortem submergence group (n=15) and dry death mice at mice group (n=15), mice machinery
=5) the algae DNA extracted in the sample of liver, kidney (body circulation internal organs) expands through this paper primer PCR under the same conditions, and silver staining shows
Band, the judgement of display positive findings is detection, and recall rate the results are shown in Table 1.Through chi-square criterion, enter water group before death each with Postmortem submergence group
Plant positive rate between internal organs and there is significant difference (P < 0.05, table 1).
Table 1 Aeromonas hydrophila circulates internal organs detection number of cases and positive rate at each group of experiment mice body
Be can be seen that by above-mentioned experimental result, utilize the method in the present invention, the DNA in mice organs is expanded, utilize inspection
Go out positive findings to confirm that the method recall rate of the mice cause of the death is up to 93%, it is contemplated that during being drowned, suck the difference of water and individual
Body difference, the method in the present invention of can assert can effectively judge that mice, the most really for being drowned, can assist judgement to be drowned water simultaneously
Territory.
Further, data in embodiment can be seen that, the recall rate in liver is higher than kidney.
Embodiment 4 Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects
Utilize the primer that embodiment designs to a kind of Aeromonas hydrophila method for nucleic acid analysis can be designed, the present embodiment incite somebody to action this
Aeromonas hydrophila method for nucleic acid analysis in invention is applied in legal medical expert's detection, is applied particularly to whether confirmation drowning person is excessive
Die and find corresponding region of being drowned of drowning.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's organs and tissues；
Step 2:
Extract the nucleic acid in this organs and tissues, use the primer in embodiment 1 to carry out PCR amplification；
In step 2, instrument for extracting nucleic acid is utilized to extract the nucleic acid in organs and tissues, the forward primer used in amplification procedure is 5 '-
GCCGAGCGCCCAGAAGGTGAGTT-3 ', downstream primer is 5 '-GAGCGGCTGGATGCGGTTGT-3 '.
Forward primer 5 ' is held all with FAM fluorescent labeling, can realize the fluoroscopic examination in capillary electrophoresis apparatus.
PCR reaction system is 20 μ L, includes 10 μ L Taq enzyme, each 0.75 μ L of upstream and downstream primer, 7.5 μ L deionized waters, 1 μ L
Nucleic acid to be analyzed；PCR cycle parameter: 94 DEG C of denaturations 10min；94 DEG C of degeneration 40s, respectively at 55 DEG C of 40s that anneal, 72 DEG C of extensions
40s, totally 35 circulations, finally continue to extend 10min at 72 DEG C, cool the temperature to 4 DEG C of preservations to be used, obtains nucleic acid PCR and produces
Thing sample.Wherein upstream and downstream primer concentration is 10pmol/ μ L.
The preparation method of sample to be tested is by sample nucleic acid pcr amplification product, Methanamide, ILSCC500 mixing, wherein sample
This nucleic acid PCR product, Methanamide, the volume of ILSCC500 are 1 μ L, 9 μ L, 1 μ L.
Step 3:
The pcr amplification product (sample to be tested) of above-mentioned sample nucleic acid is carried out detection analysis.
In the present embodiment, the instrument that foranalysis of nucleic acids uses is ABI3130XL genetic analyzer, it is possible to use other effectively to divide
Analyzer device.
Capillary electrophoresis apparatus detection is utilized to analyze Aeromonas hydrophila nucleic acid result (forward primer fluorescent labeling) in internal organs
As shown in Figure 2, result shows, the band that PCR primer result is 127bp size of positive sample, i.e. can determine whether in this sample
Containing Aeromonas hydrophila, this sample can determine that as drowning, and is consistent with the known cause of the death.
Pcr amplification product sequencer map is as shown in Figure 3.
Pcr amplification product order-checking is: TGGCTGGATGCGGTTGTGATCCACGTCGTAGCCCATGCCCCAGACGGTGGC
CGTCTTGGAGGAGAGCAGGGACTCCGCGGTCGCGTATTGATCGCGCACCCAGCTGAAACTCACCTTCTGGGCG。
It is last it should be noted that it is only entered by above example in order to the technical scheme of the embodiment of the present invention to be described
Row limits, although the embodiment of the present invention being described in detail with reference to preferred embodiment, and those of ordinary skill in the art
Be to be understood that and still the technical scheme of the embodiment of the present invention can be modified or equivalent, and these amendment or etc.
Also amended technical scheme can not be made the scope of embodiment of the present invention technical scheme is departed from replacement.
SEQUENCE LISTING
<110>Guangzhou City Forensic Science Technology Institute
<120>a kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>primer β-actinF1
<400> 1
GCCGAGCGCCCAGAAGGTGAGTT 23
<210> 2
<211> 20
<212> DNA
<213>primer β-actinR1
<400> 2
GAGCGGCTGGATGCGGTTGT 20
Claims (10)
1. Aeromonas hydrophila is judging that the person of being drowned the most really dies from the application being drowned.
2. the primer pair of a pair detection Aeromonas hydrophila, it is characterised in that described primer is to including that forward primer and downstream are drawn
Thing, the sequence of forward primer is as shown in SEQ IDNo.1, and the sequence of downstream primer is as shown in SEQ IDNo.2.
3. an Aeromonas hydrophila method for nucleic acid analysis, it is characterised in that: utilize primer described in claim 1 to core to be analyzed
Acid expands, and utilizes amplified production to prepare sample to be tested, and sample to be tested is carried out foranalysis of nucleic acids.
4. Aeromonas hydrophila method for nucleic acid analysis as claimed in claim 3, it is characterised in that: forward primer 5 ' is held all with FAM
Fluorescent labeling.
5. Aeromonas hydrophila method for nucleic acid analysis as claimed in claim 3, it is characterised in that: PCR reaction system is 20 μ L, interior
Containing 10 μ L Taq enzyme, each 0.75 μ L of upstream and downstream primer, 7.5 μ L deionized waters, 1 μ L nucleic acid to be analyzed；PCR cycle parameter: 94 DEG C
Denaturation 10min；94 DEG C of degeneration 40s, respectively at 55 DEG C of 40s that anneal, 72 DEG C extend 40s, and totally 35 circulations, finally continue at 72 DEG C
Renew and stretch 10min, cool the temperature to 4 DEG C of preservations to be used, obtain nucleic acid PCR amplified production sample.
6. Aeromonas hydrophila method for nucleic acid analysis as claimed in claim 3, it is characterised in that: the preparation method of sample to be tested is
By sample nucleic acid pcr amplification product, Methanamide, ILSCC500 mixing, wherein sample nucleic acid PCR primer, Methanamide, ILSCC500
Volume be respectively 1 μ L, 9 μ L, 1 μ L.
7. Aeromonas hydrophila method for nucleic acid analysis answering during legal medical expert detects as described in claim 3-6 is arbitrary
With, it is characterised in that: specifically it is applied to the judgement person of being drowned and the most really dies from and be drowned.
Apply the most as claimed in claim 9, it is characterised in that: described application process is: take drowning person's organs and tissues, extracts
Nucleic acid in this organs and tissues, uses primer as claimed in claim 2 to carrying out PCR amplification, it is carried out detection and analyzes, really
Recognize whether pcr amplification product derives from Aeromonas hydrophila, judge that the person of being drowned is the trueest according to the recall rate of Aeromonas hydrophila
Just for being drowned.
9. the judgement person of being drowned the most really dies from and is drowned and/or finds out the method in region of being really drowned, it is characterised in that: take
Whether drowning person's organs and tissues, contain Aeromonas hydrophila in detection organs and tissues, whether judge the person of being drowned according to testing result
Really die from and be drowned, and find out real region of being drowned.
10. method as claimed in claim 9, it is characterised in that: whether described detection internal organs contain the side of Aeromonas hydrophila
Method is with reference to Aeromonas hydrophila method for nucleic acid analysis as described in claim 3-6 is arbitrary.
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| CN107201405A (en) * | 2017-06-16 | 2017-09-26 | 广州市刑事科学技术研究所 | A kind of diatom UPA genetic analysis method and its application in legal medical expert detects |
| CN108950043A (en) * | 2018-07-09 | 2018-12-07 | 无锡中德美联生物技术有限公司 | One kind being drowned related planktonic organism compound amplification detection system and kit |
| CN113249501A (en) * | 2021-03-30 | 2021-08-13 | 广州市刑事科学技术研究所 | Method for identifying cadaver cause of death in water based on bacterial community |
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| CN107201405A (en) * | 2017-06-16 | 2017-09-26 | 广州市刑事科学技术研究所 | A kind of diatom UPA genetic analysis method and its application in legal medical expert detects |
| CN108950043A (en) * | 2018-07-09 | 2018-12-07 | 无锡中德美联生物技术有限公司 | One kind being drowned related planktonic organism compound amplification detection system and kit |
| CN108950043B (en) * | 2018-07-09 | 2022-02-25 | 无锡中德美联生物技术有限公司 | Drowning-related plankton composite amplification detection system and kit |
| CN113249501A (en) * | 2021-03-30 | 2021-08-13 | 广州市刑事科学技术研究所 | Method for identifying cadaver cause of death in water based on bacterial community |
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| Publication number | Publication date |
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| CN106119384B (en) | 2019-12-20 |
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