CN113249501A - Method for identifying cadaver cause of death in water based on bacterial community - Google Patents
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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 a method for identifying cadaver cause of death in water based on a bacterial community. The method is used for identifying whether the carcass in water is drowned or not by sequencing the bacterial communities in the water body sample and the carcass lung tissue sample in water and comparing whether the bacterial communities in the two samples converge or not and whether the bacterial communities in the two samples have the same specific microorganisms or not, and the provided method can be used for forensic medicine detection and has important practical value for identifying the drowned carcass.
Description
Technical Field
The invention belongs to the technical field of forensic medicine identification. And more particularly, to a method for identifying cadaveric causes of death in water based on bacterial communities.
Background
In the practice of forensic identification, the identification of causes of death of cadavers in water is often involved. The method is influenced by the factors of large quantity of water areas, complex water area environment, corpse decay and the like in China, and the cause of death identification of corpses in water is always a difficult problem in forensic science identification. Although diatom inspection is generally accepted as an important auxiliary means for identifying causes of death of corpses in water, inspection materials are easily polluted by the processes of corpse inspection, material taking and inspection, and diatom inspection from the lungs of corpses in water alone is not enough for identifying drowning, and diatom inspection from multiple organs without inspection cannot be eliminated, so that the role of diatom inspection is limited to a certain extent. At present, experts and scholars at home and abroad propose other methods for assisting in identifying the cause of death of the corpses in water, such as chlorophyll (A), histochemistry, blood biochemistry and other detection technologies, but the methods all have certain limitations. Therefore, the detection method which has high sensitivity and high detection rate and can be widely applied to the detection of the cause of death of the corpse in water is provided, and the method has important significance for the current forensic identification practice.
Compared with the traditional diatom morphological examination method, the method for identifying the cause of death of the corpses in water by analyzing microorganisms has the following advantages: the method can be used for deducing whether the cause of death is really drowning; the detection range is wide, and the method can be applied to classification and identification of algae, and can also be used for examination of small and micro plankton and plankton which cannot be subjected to morphological discrimination by using a microscope; and thirdly, the sensitivity is high, and the required sample size is obviously smaller than that of the traditional diatom detection method. Various variant sequences are separated from DNA by using the conserved sequences of water body bacteria, community characteristics can be revealed while qualitative, and a plankton community characteristic change monitoring system in different water areas and the same water area at different time is further established. The diagnosis method is combined with the traditional diatom test method, and has important practical value for drowning identification.
Chinese patent CN 106119384A discloses an aeromonas hydrophila nucleic acid analysis method and application thereof in forensic detection. The method comprises the steps of extracting aeromonas hydrophila nucleic acid from visceral organs of the corpses in water, carrying out PCR amplification on the extracted aeromonas hydrophila nucleic acid, comparing the amplified nucleic acid with the aeromonas hydrophila nucleic acid in a water sample, and analyzing and judging whether the corpses in water are drowned. However, the method may be influenced by a sample, and the aeromonas hydrophila nucleic acid cannot be detected, so that the cause of death in water cannot be identified, and therefore, the method has certain limitations.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing technology and method for identifying the cause of death of the corpse in water and provides a method for identifying the cause of death of the corpse in water based on a bacterial community.
The invention aims to provide a method for identifying cadaver cause of death in water based on bacterial communities.
The above purpose of the invention is realized by the following technical scheme:
the drowned corpse and the non-drowned corpse are characterized in that the drowned person inhales and swallows the water body in the drowned place in the process from the beginning of drowning to death, so the microbial community structure of the drowned corpse sample in the water is consistent with the microbial community structure of the water body in the drowned place, and the specific microbe in the drowned water body can be detected in the corpse sample instead of the microbe in the drowned corpse sample in the water.
The method comprises the following steps of sequencing bacteria in the corpse tissue and the water body sample in water, analyzing and comparing bacterial communities and specific bacteria in the corpse tissue and the water body sample, and deducing whether the corpse in water is drowned or not according to the bacterial communities and the specific bacteria in the corpse tissue and the water body sample:
s1, sampling a water body and corpse tissues at a corpse discovery point;
s2, enriching the bacteria in the water sample obtained in the step S1, and respectively extracting the total DNA of the bacteria and the cadaver tissues in the water sample;
s3, designing a primer according to a bacteria conserved region sequence, connecting a sequencing joint, respectively carrying out PCR amplification by taking the extracted total DNA as a template, purifying, quantifying and homogenizing the product to form a sequencing library, and sequencing the qualified library by quality inspection;
and S4, analyzing the sequencing result, and identifying whether the water corpse is drowned or not by comparing whether the bacterial community in the water sample and the water corpse tissue is convergent or not and whether the water corpse tissue contains common specific bacteria or not.
Preferably, the water sampling amount in step S1 is 1-2L.
Preferably, the cadaver tissue taken in step S1 is a lung tissue mass of a cadaver in water.
More preferably, the removed lung tissue mass is quickly placed into a pre-cooled cryopreservation tube and placed in liquid nitrogen for quick freezing for 1h, and then transferred to a refrigerator at-80 ℃ for storage.
Preferably, in the step S2, the water body sample bacteria are enriched by the suction filtration device until the surface of the filter membrane has a visible covering, and the filter membrane is transferred to a refrigerator at the temperature of minus 80 ℃ for storage.
More preferably, the filter used for suction filtration has a pore size of 0.22. mu.m.
Preferably, the bacterial conserved region sequence of step S3 is 16S rDNA. 16S rDNA is a DNA sequence coding for prokaryotic ribosome small subunit rRNA (16S rRNA), and currently, bacterial diversity research is mainly carried out on a conserved region of a nucleic acid sequence coding for 16S rRNA.
Preferably, the analysis in step S4 is a flora histogram, Bray Curtis analysis and LEfSe analysis of the sequencing results.
The invention has the following beneficial effects:
the invention provides a method for identifying whether a corpse in water is drowned or not based on a bacterial community, which has high sensitivity and small required tissue sample amount and can avoid the condition that PCR (polymerase chain reaction) can not be carried out on single bacterial nucleic acid. The method provided by the invention can be used for forensic detection and has important practical value for drowning identification.
Drawings
FIG. 1 is a histogram of the bacterial flora of lung tissue, gastric contents, duodenal contents and water sample of a drowned rat (A: lung tissue; B: gastric contents; C: duodenal contents; D: water sample).
FIG. 2 shows Bray Curtis analysis of flora of lung tissue, gastric contents, duodenal contents and water samples of drowned rats (A: lung tissue; B: gastric contents; C: duodenal contents; D: water sample).
FIG. 3 is a histogram of lung tissue flora of rat carcasses with and without drowning (A: land death; B: water entry after death; C: drowning; D: water sample).
FIG. 4 shows LEfSe analysis of lung tissue flora of rat carcasses with and without drowning (species without significant difference are yellow, species Biomarker with significant difference is colored following the group, wherein A is land death, B is post-death water entry, C is drowning, D is water sample).
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 selection analysis of optimum test Material
Mature SD rats of 12 weeks are used for simulating drowning, lung tissues, gastric contents, duodenal contents and drowned water body samples of the drowned rats are respectively extracted, total DNA of bacteria is extracted, and PCR amplification is carried out by using primers in 16S rRNA (V3+ V4) region of the bacteria.
The total bacterial DNA is extracted by using a soil microorganism DNA strong extraction kit produced by MOBIO company, the specific steps are carried out according to the instruction, and the extracted DNA sample is stored at the temperature of minus 20 ℃. Using the extracted DNA as a template, a system was prepared as shown in Table 1 using primers for the 16S rRNA (V3+ V4) region of bacteria, and then placed in a PCR apparatus to perform PCR reaction under the reaction conditions shown in Table 2.
The primer sequences of the 16S rRNA (V3+ V4) region were as follows:
Vn F：5'-ACTCCTACGGGAGGCAGCA-3'
Vn R：5'-GGACTACHVGGGTWTCTAAT-3'
TABLE 1 PCR reaction System (50. mu.l)
TABLE 2 PCR reaction conditions
And purifying, quantifying and homogenizing the amplification product to form a sequencing library, performing library quality inspection on the built library, and sequencing the qualified library by using Illumina HiSeq 2500. And drawing a colony histogram according to the sequencing result and carrying out Bray Curtis analysis.
The colony histogram is shown in figure 1, the flora species in lung tissue and water sample are more abundant, the similarity is higher, the duodenum is the second, the stomach content is the lowest, and the influence of drowning liquid on the stomach content is smaller when drowning occurs. The results of the Bray Curtis assay are shown in FIG. 2, where there is some difference in the flora of the lung tissue and the watery flora, but at a close distance. According to the results, the ratio of lung tissues and other tissues and organs of the drowned corpse is the most approximate to that of the water body at the drowned place, and the bacterial community characteristic is the optimal test material extracted from the corpse when the cause of death of the corpse in water is identified.
Example 2 analysis of bacterial community characteristics of cadaver pulmonary tissues of rats with and without drowning
Mature SD rats of 12 weeks are used for simulating drowning, death after death, water and land death, and rat corpse lung tissues and drowning water bodies of different causes of death are sampled.
Sampling a water body sample: and (3) respectively taking 1-2L drowned water samples by using a sterilized emptying type sampler, enriching bacteria in the water body samples by using a suction filtration device, wherein the aperture of the used filter membrane is 0.22 mu m, and performing suction filtration until the surface of the filter membrane is provided with a visible covering. After the enrichment is finished, the filter membrane is taken out by a disposable forceps and is placed into a freezing storage tube to be stored in a refrigerator at the temperature of minus 80 ℃.
And (3) sterilizing the emptying type sampler: after the emptying type sampler is cleaned of dust and oil stain by water and a detergent, the emptying type sampler is washed clean by tap water and then soaked for 8 hours by 10 percent nitric acid. And taking out, washing with tap water for 3-5 times, and fully rinsing with distilled water. Wrapping with tinfoil paper, placing into a high-pressure steam sterilizing pot, sterilizing for 15-30 minutes under the conditions of 103.4kPa and 121.3 ℃, and standing overnight in a 120 ℃ oven after the sterilization is finished.
Sampling lung tissue samples: the lung tissue blocks of the carcasses in water with the body size of 0.5cm by 0.5cm are taken by using disposable sterile forceps and a sterile scalpel, quickly placed into a precooled screw-mouth freezing storage tube with a written number and the temperature of minus 192 ℃, immediately placed in liquid nitrogen for quick freezing for 1 hour and then transferred to a refrigerator with the temperature of minus 80 ℃ for storage.
The steps of extraction of total bacterial DNA, PCR amplification, sequencing and the like are the same as in example 1.
And (3) analyzing a sequencing result: and (4) drawing a flora histogram according to the sequencing result and carrying out LEfSe analysis. Fig. 3 shows a histogram of bacterial colonies in group C, i.e. lung tissue, which are more abundant and similar to bacterial colonies in water samples, and may be caused by the inhalation of large amounts of water during drowning, while the bacterial colonies in group B are more similar to those in group a. The LEfSe analysis results are shown in fig. 4, and group C, which is a bacterial community of lung tissue, has some species that are specific and can be distinguished from other groups.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A method for identifying cadaveric causes of death in water based on bacterial communities, comprising the steps of:
s1, sampling a water body and corpse tissues at a corpse discovery point;
s2, enriching the bacteria in the water sample obtained in the step S1, and respectively extracting the total DNA of the bacteria and the cadaver tissues in the water sample;
s3, designing a primer according to a bacteria conserved region sequence, connecting a sequencing joint, respectively carrying out PCR amplification by taking the extracted total DNA as a template, purifying, quantifying and homogenizing the product to form a sequencing library, and sequencing the qualified library by quality inspection;
and S4, analyzing the sequencing result, and identifying whether the water corpse is drowned or not by comparing whether the bacterial community in the water sample and the water corpse tissue is convergent or not and whether the water corpse tissue contains common specific bacteria or not.
2. The method of claim 1, wherein the water sample volume in step S1 is 1-2L.
3. The method of claim 1, wherein the cadaver tissue harvested in step S1 is a lung tissue mass.
4. The method of claim 3, wherein the removed lung tissue mass is quickly placed in a pre-cooled cryovial and snap frozen in liquid nitrogen for 1 hour, followed by storage in a freezer at-80 ℃.
5. The method as claimed in claim 1, wherein in step S2, the water sample is enriched with bacteria by a suction filtration device until the surface of the filter membrane has a visible covering, and the filter membrane is stored in a refrigerator at-80 ℃.
6. The method as claimed in claim 5, wherein the filter used for suction filtration has a pore size of 0.22. mu.m.
7. The method of claim 1, wherein the bacterial conserved sequence of step S3 is 16S rDNA.
8. The method of claim 1, wherein the analysis of step S4 is a histogram of florae, Bray Curtis analysis, or LEfSe analysis of the sequencing results.
9. Use of the method of claims 1 to 8 in forensic identification.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004046384A1 (en) * | 2002-11-19 | 2004-06-03 | Institute Of Environmental Science And Research Limited | Time of death determination by a micro flora dna profiling method |
| CN106119384A (en) * | 2016-08-04 | 2016-11-16 | 广州市刑事科学技术研究所 | A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects |
| CN108531567A (en) * | 2018-05-10 | 2018-09-14 | 华中农业大学 | A method of the analysis geobiont membrane micro succession of community |
| 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 |
| 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 |
-
2021
- 2021-03-30 CN CN202110354684.5A patent/CN113249501A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004046384A1 (en) * | 2002-11-19 | 2004-06-03 | Institute Of Environmental Science And Research Limited | Time of death determination by a micro flora dna profiling method |
| CN106119384A (en) * | 2016-08-04 | 2016-11-16 | 广州市刑事科学技术研究所 | A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects |
| CN108531567A (en) * | 2018-05-10 | 2018-09-14 | 华中农业大学 | A method of the analysis geobiont membrane micro succession of community |
| 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 |
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| Title |
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| EIJI KAKIZAKI等: "Detection of diverse aquatic microbes in blood and organs of drowning victims: first metagenomic approach using high-throughput 454-pyrosequencing", 《FORENSIC SCIENCE INTERNATIONAL》 * |
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Application publication date: 20210813 |