CN103937893A - Forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP (single nucleotide polymorphisms) genetic markers - Google Patents
Forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP (single nucleotide polymorphisms) genetic markers Download PDFInfo
- Publication number
- CN103937893A CN103937893A CN201410162859.2A CN201410162859A CN103937893A CN 103937893 A CN103937893 A CN 103937893A CN 201410162859 A CN201410162859 A CN 201410162859A CN 103937893 A CN103937893 A CN 103937893A
- Authority
- CN
- China
- Prior art keywords
- seq
- sarcophagid
- tetra
- sodium
- parasarcophaga
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
- C12Q1/6869—Methods for sequencing
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP genetic markers. The detection kit comprises amplification primers of sequences shown in SEQ ID NO.1-SEQ ID NO.16 and sequencing primers of sequences shown in SEQ ID NO.17-SEQ ID NO.24. The kit is based on a pyrosequencing platform, so that the whole identification process only needs three hours and the identification results can be quickly obtained. The minimum length of the amplification primers of the kit is only 77bp and the maximum length does not exceed 278bp, so that the kit has advantages in detection of the common insect degrading samples on the forensic scene.
Description
Technical field
The invention belongs to medical jurisprudence technical field, be specifically related to a kind of forensic entomology detection kit of 8 plastosome SNP genetic markers differentiating for forensic entomology Sarcophaga carnaria kind.
Background technology
Death time (Postmortem Interval, PMI) infer and refer to supposition and the judgement apart from dead time at intervals when corpse is found, the aspect such as provide, scope of investigation delimitation, crime time analysis, case are qualitative all significant in case clue.PMI infers it is the key link of criminal case detection, is also medicolegal practice difficult point and study hotspot.Traditionally, legal medical expert according to postmortem phenomena or corpse endogenous material after death Changing Pattern carry out PMI deduction, but putrefaction of dead body progress seriously hampers the application of these methods, also there is dispute in the objectivity of traditional method, repeatability, science in addition.Therefore, set up science, objectively, PMI estimating method repeatably, become medical jurisprudence and study a difficult problem urgently to be resolved hurrily.Forensic entomology utilizes the insect in close relations with the putrefaction of dead body to carry out PMI deduction exactly.In recent years, forensic entomology infers at PMI, and particularly the aspect such as decomposed body PMI deduction embodies good application prospect.
What on corpse, the most often find is the fly class in Diptera (Diptera), therefore fly class is studied maximum with practical application, especially the Flesh flies (Sarcophagidae) in fly class, Calliphoridae (Calliphoridae) and family's Nuscidae (Muscidae).Flesh flies are huge groups in Diptera, and the kind that the whole world has been found at present has more than 2600, exceedes 100 genus.Compare other fly class, Sarcophaga carnaria has the advantage of self uniqueness aspect PMI deduction.First, the larva of Flesh flies many types all has the corpse of having a liking for, this also just sarcophagid be called as the reason of " flesh-fly ".The second, different from other fly class, most of Sarcophaga carnarias are not laid eggs, but directly larviposition is on corpse, and therefore some surfaces have on the corpse of coverture and can find sarcophagid larva.Meanwhile, most scholars think that the modes of reproduction of sarcophagid " viviparity " reduced the growth link of larva, thereby the accuracy that its PMI infers is better than other fly class.The 3rd, some kinds of Sarcophaga can be flown under the special weather conditions such as wet weather, and overcast and rainy arrival corpse fly class is the earliest usually sarcophagid, therefore infer that at the PMI of the plentiful Hunan area sarcophagid of rainfall value is more outstanding.The 4th, in other fly class sluggish early summer and autumn end, sarcophagid but usually can be found on corpse, has filled up the blank of special period.The 5th, Flesh flies Partial Species has the custom of the indoor activity of entering, and therefore on indoor corpse, usually can find the larva of sarcophagid, and this is confirmed by the numerous case reports in home and abroad.The 6th, the sarcophagid individuality of most kinds is larger, more easily causes viewer's attention.
Results of animal confirms that the comparatively common Sarcophaga carnaria kind of China has Sarcophga fuscicauda Boerttcherisca peregrina, fertile palpus Parasarcophaga Parasarcophaga crassipalpi, red tail excrement sarcophagid Sarcophaga africa, yellow palpus Parasarcophaga Parasarcophaga miser, hoary hair's Parasarcophaga Parasarcophaga albiceps, sauce Parasarcophaga Parasarcophaga dux, the black sarcophagid Helicophagella of black tail melanura, wild flax fly Parasarcophaga similis, short angle Parasarcophaga Parasarcophaga brevicornis, red tail draws more than ten kinds such as sarcophagid Ravina pernix
[2,10, 11].The cardinal principle of carrying out PMI deduction according to Sarcophaga carnaria is: utilize the conditions such as Sarcophaga carnaria growth and development characteristics combining environmental temperature, judge more exactly its etap and calculate and grow to this stage institute's elapsed-time standards, and then infer PMI.Under the same conditions, there is notable difference in the developmental rate of different types of Sarcophaga carnaria, for example: 26.7 DEG C of red tail excrement sarcophagids are developed to adult stage needs 10.5 days, seminar's previous experiments is found at same temperature, Sarcophga fuscicauda is developed to adult stage needs 16.7 days, fertile palpus Parasarcophaga needs 15.2 days, and yellow palpus Parasarcophaga needs 23.8 days; And hoary hair's Parasarcophaga, sauce Parasarcophaga, the black sarcophagid of black tail, wild flax fly, short angle Parasarcophaga, red tail draw sarcophagid still to lack the growth data of system because of Identification of Species difficulty, apply in practice so far less.As can be seen here, Identification of Species is to utilize Sarcophaga carnaria to carry out PMI to infer key link fast and accurately.
Sarcophagid adult distinct characteristics, general body body background color is black, has canescence pruinescence, and there is silvery white pruinescence at the chest back side, has black longitudinal grin, and belly back side majority has checkerboard spot, therefore in section's one rank, very easily distinguishes with other section's fly classes such as calliphorid, housefly.Sometimes extremely difficult differentiation between sarcophagid kind and kind, the Morphological Identification of adult and larva has generally acknowledged difficulty, and the larva collecting need to be raised conventionally to turning into adult, to facilitate identifying species.So far only larva, the pupa of Sarcophga fuscicauda and fertile palpus Parasarcophaga have morphology discrimination method, and the larva of all the other kinds does not still have systematic Morphological Identification main points, and even the qualification of female insect is still very difficult.For example: sauce Parasarcophaga male insect differentiates that main points are: mesopodium tibia is without becoming mildewed; The 9th backboard black, brown, minority redness; 7th, 8 synsternites have no chance hair on the neck; Anus caudal lobe is except the leading edge of close end is slightly curve, gradually to end taper, and simultaneously slightly to antecurvature, end point; Front paramere is wide short, and slightly straight, end pawl turns shape; The disconnected shape that cuts of the membranaceous protruding end of adeagus portion, ossified and edge is irregular; Side tegmen body base portion abdomen is prominent to be slightly rectangle, and there is Yi little Jiao front abdomen side, and side sun end of body central authorities are short and small, pleurapophysis bifurcated; And female discriminating main points are considerably less: the 6th backboard interrupts, and two osteocommas are separated by very near; The 8th backboard exists; The 7th web trailing edge is recessed, the little Mao group of two relief angle tools; Third-instar larvae is differentiated even will be by means of Electronic Speculum, and on backboard, clavula can be seen small fold under Electronic Speculum; A pair of rear valve is " D " type.Proved by theoretical and case although sarcophagid is used for the feasibility of PMI deduction, Identification of Species difficulty greatly reduces the using value of sarcophagid in PMI infers.Therefore, find quick, accurate, economic common Sarcophaga carnaria authentication method very necessary.
Sperling in 1994 etc. are used for DNA technique to have a liking for corpse fly class Identification of Species first, and in more than ten years after this, DNA molecular qualification, as the means of supplementing out economy of Morphological Identification method, is accepted by increasing forensic entomology investigator.Studies confirm that in a large number both at home and abroad, Mitochondrial DNA (Mitochondrial DNA, mtDNA) the chtochrome oxidase cozymase in and II (cytochrome oxidase subunits I and II, CO I+CO II) complete sequence (about 2300bp) can realize the Identification of Species of common Sarcophaga carnaria on gene level, even utilizes CO I, CO II, the 16Sribosomal DNA sequence dna (200-300bp) of short-movie section both can complete Partial Species qualification.But current sarcophagid Molecular Identification technology still exists defect.First, although complete sequence analysis differentiates that accuracy rate can reach 99.3%, expense is high, consuming time many, and some special sample are difficult to realize complete sequence amplification; Secondly, conventional 600bp " barcode ", although region qualification effect is higher, has and studies confirm that this region of independent application still exists larger risk in the world; Again, current short fragment sequence is considered to the better selection of Molecular Identification, these only have 200bp left and right fragment to be very easy to amplification, also can successfully amplify target DNA even if placed long-time sample, in special sample qualification, there is obvious advantage (puparium etc.), but its qualification accuracy rate only has 80% left and right, even if the Conjoint Analysis of multi-disc section is still difficult to the accuracy rate that reaches satisfied.Therefore,, on existing Molecular Identification basis, find more quick, accurate, economic Sarcophaga carnaria authentication method very necessary.
Summary of the invention
The present invention is intended to for the present situation of having a liking for corpse fly class and infer sarcophagid species detecting difficulty when the corpse death time, utilize sarcophagid plastosome SNP genetic marker pair with the death time closely-related red tail excrement sarcophagid, Sarcophga fuscicauda, fertile must Parasarcophaga and yellowly must carry out forensic entomology detection by Parasarcophaga, thereby can determine the kind of sample.
In order to achieve the above object, technical scheme provided by the invention is:
The described forensic entomology detection kit based on 8 sarcophagid plastosome SNP genetic markers comprises the sequencing primer of sequence shown in the amplimer of sequence shown in SEQ ID NO.1-SEQ ID NO.16 and SEQ ID NO.17-SEQ ID NO.24.
Wherein, SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.11, shown in SEQ ID NO.13 and SEQ ID NO.15,5 ' of amplimer end is by biotin labeling.
Above-mentioned amplimer and sequencing primer can be used for preparation method medical entomology detection reagent.
Below in conjunction with principle, the invention will be further described:
16 amplimers that amount to that amplimer described in test kit of the present invention has comprised 8 sarcophagid plastosome SNP genetic markers, the nucleotide sequence of each primer is respectively shown in SEQ ID No.1 to SEQ ID No.16 (in table 1):
Table 1 tetra-sodium order-checking amplimer and biotin labeling situation
Test kit of the present invention comprises that 8 sequencing primers check order for tetra-sodium, and the sequence of sequencing primer is as shown in SEQ ID No.17 to SEQ ID No.24 (in table 2):
Table 2 tetra-sodium order-checking sequencing primer
The product that described tetra-sodium sequencing primer can increase taking above-mentioned 8 pairs of amplimers, as template, carries out the sequencing reaction of 8 sarcophagid plastosome SNP genetic markers in tetra-sodium sequenator.
In the specification sheets of test kit of the present invention, also list 8 tetra-sodiums sequence to be detected that checks order, in the time of order-checking, be input in tetra-sodium order-checking software, determine the application of sample order of tetra-sodium sequenator dNTP, each tetra-sodium checks order the nucleotide sequence of sequence to be detected as shown in SEQ ID No.25 to SEQ ID No.32 (in table 3):
The table 3 tetra-sodium sequence to be detected that checks order
In the specification sheets of test kit of the present invention, also listed the haplotype data on 8 SNP locus, comprised red tail excrement sarcophagid, Sarcophga fuscicauda, fertile must Parasarcophaga and yellow must the somatotype result of Parasarcophaga on 8 SNP, four kinds of sarcophagid haplotype data are in table 4:
Haplotype data on 8 SNP locus of four kinds of fly classes of table 4
The medical jurisprudence compound detection test kit that the present invention is based on 8 sarcophagid plastosome SNP genetic markers is the 8 sarcophagid plastosome SNP genetic markers that obtain based on screening, utilizes tetra-sodium order-checking system construction.This test kit by separate 8 pairs of amplimers of packaging, 8 sequencing primers, 8 tetra-sodiums check order sequence to be detected, four kinds of sarcophagids both red tail excrement sarcophagid, Sarcophga fuscicauda, fertile must Parasarcophaga and yellow 8 SNP haplotypes that must Parasarcophaga.
The principle of work of this test kit is first to pass through amplimer, and amplification simultaneously obtains all DNA fragmentations that contain 8 sarcophagid plastosome SNP.Utilize tetra-sodium sequenator purifying workstation by above-mentioned amplified fragments purifying single stranded.Then the single stranded DNA after purifying, sequencing primer and annealing buffer, join in the reagent cabin of containing dNTP, enzyme mixation, substrate mixed solution and carry out tetra-sodium order-checking.Compare according to tetra-sodium sequenator automated provisioning somatotype result and four kinds of sarcophagid haplotype data, thus the kind of judgement sample.
In the present invention, 8 sarcophagid plastosome SNP genetic markers are extremely crucial to the structure of this detection kit.The sarcophagid SNP locus with forensic entomology using value of having reported is at present extremely limited, so first the detection kit that will develop based on sarcophagid SNP just must be summarized and be can be used for the locus that common Sarcophaga carnaria kind is differentiated by a large amount of experiments.According to the sequencing result that data are reported and our unit accumulates for many years in the past, in the present invention, the screening criteria of sarcophagid plastosome SNP genetic marker is: 1) haplotype of SNP composition can be distinguished red tail excrement sarcophagid, Sarcophga fuscicauda, fertile palpus Parasarcophaga and yellow palpus Parasarcophaga; 2) haplotype of SNP composition is stable, conservative in red tail excrement sarcophagid, Sarcophga fuscicauda, fertile palpus Parasarcophaga and yellow palpus Parasarcophaga kind; 3) haplotype of other common Sarcophaga carnarias on these SNP and red tail excrement sarcophagid, Sarcophga fuscicauda, fertile must Parasarcophaga with yellow must Parasarcophaga kind different.
According to the above-mentioned standard of setting up, the present invention filters out 8 sarcophagid plastosome SNP genetic markers altogether for setting up the sarcophagid kind identification system of forensic entomology.Experimental results show that through mass survey, 8 sarcophagid plastosome SNP genetic markers of the present invention are conservative at inner height of the same race, every kind of fly class adopts 10 samples to carry out duplicate detection, simultaneously with Genebank in already present data compare, a variation phenomenon in not finding kind.8 sarcophagid plastosome SNP genetic marker site informations related in test kit are as table 5.
Table 5
Test kit of the present invention has used tetra-sodium sequencing technologies in to the detection of above-mentioned 8 sarcophagid plastosome SNP genetic markers that screen.Tetra-sodium sequencing technologies can the multiple DNA cloning fragments of Simultaneous purification, check order simultaneously, have actual needs convenient, fast, the medical verification of simple operation and other advantages adjustment procedure.The key of tetra-sodium sequencing technologies and difficult point, while being design amplification and sequencing primer, considered following factor: 1) between primer self, primer, between primer and template without obvious hairpin structure, mispairing and dimeric structure; 2) sequencing primer is close to SNP to be detected in principle, avoids the phenomenon of SNP flanking sequence interference detection results; 3) expanding fragment length is controlled at below 280bp.
This test kit utilizes above-mentioned amplimer, has obtained the amplified production that comprises 10 SNP genetic markers, is then purified to single stranded DNA fragment by the tetra-sodium purifying workstation that checks order, and as template, utilizes tetra-sodium sequencing primer, carries out tetra-sodium sequencing reaction.According to the somatotype result of machine demonstration, determine the haplotype of sample, the sarcophagid kind of four kinds of sarcophagid haplotype judgement samples that binding reagents box provides.
More specifically, the component that test kit of the present invention specifically comprises can be:
A) amplimer pair: the amplimer pair of 8 sarcophagid plastosome SNP genetic markers as shown in table 1; The amplimer of 8 sarcophagid plastosome SNP genetic markers is to for obtaining the DNA fragmentation that contains 8 SNP genetic markers.
B) amplified reaction mixed solution: contain PCR buffered soln, MgCl
2, the conventional composition such as dNTPs, archaeal dna polymerase.
C) amplified production purified reagent: contain magnetic bead, 70% ethanolic soln, sex change liquid, binding buffer liquid, distilled water and elutriant; For the product of amplification is carried out to single stranded, so that carry out next step operation.
D) tetra-sodium sequencing primer: 8 tetra-sodium sequencing primers shown in table 2; Be used for carrying out the tetra-sodium corresponding SNP that checks order.
E) the tetra-sodium sequence to be detected that checks order: the sequence to be detected that checks order of 8 tetra-sodiums shown in table 3; Be used for controlling tetra-sodium order-checking application of sample cabin application of sample order.
F) tetra-sodium sequencing reaction reagent: comprise enzyme mixture, substrate mixture, dNTP, annealing buffer.
G) sarcophagid haplotype analysis table: the haplotype of 8 plastosome SNP of red tail excrement sarcophagid, Sarcophga fuscicauda, fertile palpus Parasarcophaga and yellow palpus Parasarcophaga as shown in Table 4; For comparing with tetra-sodium sequencing result, judge sarcophagid kind.
Amplified reaction mixed solution and amplified production purified reagent (removing magnetic bead unexpected) can or be prepared by molecular biology manual by the conventional formula in this area, also can directly use business-like product.Magnetic bead in tetra-sodium sequencing reaction reagent and amplified production purified reagent generally need use business-like product.
As for the template of extracting the DNA in sample to be detected, can use the current various conventional reagent in this area, extract DNA profiling and can carry out with reference to existing ordinary method.
Utilize test kit of the present invention, can analyze Sarcophaga carnaria DNA sample.Analytical procedure comprises the following steps;
1) extract the DNA of sample to be detected, as amplification template;
2) DNA that utilizes above-mentioned amplimer and amplified reaction mixed solution to extract step 1 increases; The loop parameter of described amplified reaction is: 94 DEG C, and 5 minutes; 94 DEG C, 30 seconds, 48 DEG C～58 DEG C (specifically in table 1), 30 seconds, 72 DEG C, 30 seconds, 35 circulations; Then 72 DEG C, 10 minutes, obtain PCR product;
3) the PCR product in step 2 is put in tetra-sodium purifying workstation, single stranded PCR product, specific as follows: in 200 μ L EP pipes, to add 50 μ L PCR products, 47 μ L binding buffer liquid, 3 μ L magnetic beads, vibrate 10 minutes; Use the pump in tetra-sodium sample preparation workstation to hold the magnetic bead in conjunction with DNA fragmentation, in 70% ethanolic soln resident 5 seconds, resident 5 seconds in sex change liquid, resident 5 seconds in rinsing liquid, finally, put into the solution that contains 45 μ L annealing buffers and 3 μ L sequencing primers (SEQ ID No.17 to SEQ ID No.24) in conjunction with the pump of magnetic bead, the switch of turning off pump makes to enter in liquid in conjunction with the magnetic bead of PCR product; Liquid is put into 80 DEG C of thermostat containers, leaves standstill 2min; The tetra-sodium of mentioning in claim 4 sequence to be detected (SEQ ID No.25 to SEQ ID No.32) that checks order is input in tetra-sodium sequenator; Liquid after purifying is put into tetra-sodium sequenator to be detected.
4) be ready to enzyme mixture, substrate mixture and the dNTP that tetra-sodium sequencing reaction is used, join reagent cabin, the amplified production of step 3 is added to and in the annealing buffer that contains sequencing primer, carry out tetra-sodium sequencing reaction.
5) definite sarcophagid kind thereby the haplotype of 10 plastosome SNP of the red tail excrement sarcophagid that the haplotype of 10 SNP that automatically read from tetra-sodium sequenator and this test kit provide, Sarcophga fuscicauda, fertile palpus Parasarcophaga and yellow palpus Parasarcophaga is compared.
Project team early stage Primary Construction Sarcophaga carnaria gene pool, in the best molecule marker process of searching Hunan area Sarcophaga carnaria, find, in the common Sarcophaga carnaria mtDNA sequence of Hunan area, there is informedness single nucleotide polymorphism (single nucleotide polymorphism, SNP), Mega5 software analysis finds that the Haplogroup of setting up by these SNP locus has good sarcophagid Identification of Species and is worth.In the research in insectology field, also find some informational SNP locus, but be not yet applied to Identification of Species.The present invention is expected to solve the problems that exist in the Sarcophaga carnaria Molecular Identification of current Hunan area.The first, the method, based on tetra-sodium sequencing technologies, is obviously better than conventional sequence measurement in speed; The second, the method, by collecting the informedness SNP site in different sequences, has been integrated the advantage of different fragments, and its accuracy is obviously better than conventional molecular assay method; The 3rd, only about 80-100bp of the method expanding fragment length, therefore the preservation to sample, proterties, position etc. very loosely require, and can realize in the case of maximum range and applying.The 4th, the method is lower to sample demand, has standardized program and method, reproducible; The 5th, the method cost is lower, economic and practical.Application tetra-sodium sequencing technologies detects mtDNA Haplogroup, and third generation genetic marker is introduced to sarcophagid Molecular Identification, can realize the common Sarcophaga carnaria Identification of Species in quick, accurate, economic Hunan area.
Compared with prior art, the beneficial effect of sarcophagid plastosome SNP detection kit of the present invention is:
Test kit of the present invention comprises 8 sarcophagid plastosome SNP genetic markers, can effectively be distinguished legal medical expert is inferred to significant red tail excrement sarcophagid, Sarcophga fuscicauda, fertile palpus Parasarcophaga and yellow palpus Parasarcophaga the death time by the haplotype of 8 SNP compositions; And the aspects such as this test kit sensitivity, accuracy, repeatability, specific amplification meet the requirement of forensic dna testing, be effectively supplementing of existing legal medical expert's insect morphology qualification; This test kit carries out based on tetra-sodium order-checking platform, so whole qualification process only needs 3 hours, can obtain fast identification result; The shortest only 77bp of this test kit amplified production length, no longer than 278bp, therefore this test kit has advantage for the detection of the on-the-spot common insect degraded of legal medical expert sample.
Brief description of the drawings
Fig. 1 to 8 is tetra-sodium order-checking and the Sanger method sequencing result figure of the present invention to 8 sites of Sarcophga fuscicauda biological material; In Fig. 1 to 8, the figure A of every width figure is tetra-sodium sequencing result, and figure B is Sanger method sequencing result; The X-coordinate of figure in A is that Y value represents fluorescence intensity according to tetra-sodium enzyme, substrate and the dNTP that sequence application of sample to be detected cabin adds that check order; X-coordinate in figure B is the DNA sequence dna detecting, Y value represents fluorescence intensity.
Embodiment
In embodiment, agents useful for same all uses following reagent and instrument without specified otherwise;
Embodiment 1
The preparation of test kit
For detection of sarcophagid plastosome SNP compound detection test kit can comprise respectively the following reagent of packaging:
A) amplimer pair.Amplimer pair as shown in Table 1, synthetic by TaKaRa Biotechnology company, 8 pairs of synthetic amplimers are configured to 10pM/ μ L with ultrapure water.
B) tetra-sodium sequencing primer.Amplimer pair shown in table 2, synthetic by TaKaRa Biotechnology company, 8 synthetic sequencing primers are configured to 10pM/ μ L with ultrapure water.
C) amplified reaction mixed solution.In the present embodiment, use the PCR reaction mixture 2 of Promaga company ×
green Master Mix (Promerga, Madison, WI, U.S.A.).
C) tetra-sodium order-checking purified reagent.In the present embodiment, use the tetra-sodium order-checking purification kit of QIAGEN company.
D) tetra-sodium sequencing reagent.In the present embodiment, use the PyroMark substrate Mixture tetra-sodium sequencing kit of QIAGEN company.
E) papery specification sheets.Comprise red tail excrement sarcophagid, Sarcophga fuscicauda, fertile palpus Parasarcophaga and yellow palpus Parasarcophaga haplotype data and primer sequence to be measured gives as an addition in specification sheets of the present invention.
Mentioned reagent is made to the forensic entomology sarcophagid detection kit based on sarcophagid plastosome SNP genetic marker by conventional requirements separately after packing respectively, for follow-up experiment.
Use test kit of the present invention to detect 13 kinds of sarcophagids
Use the above-mentioned forensic entomology detection kit based on sarcophagid plastosome SNP genetic marker, we have carried out the detection of 13 kinds of common Sarcophaga carnaria biology samples.Concrete testing process is performed as follows:
A, from 13 kinds of sarcophagid muscle samples, extract genomic dna with test kit, as amplification template;
B, with the DNA profiling in step a, utilize 8 couples of amplimer SEQ ID No.1 to SEQ ID No.16 and amplified reaction mixed solution that sample is distinguished to pcr amplification in following amplification system;
The thermal circulation parameters of amplification
In c, 200 μ L EP pipes, add 50 μ L PCR products, 47 μ L binding buffer liquid, 3 μ L magnetic beads, vibrate 10 minutes; Use the pump in tetra-sodium sample preparation workstation to hold the magnetic bead in conjunction with DNA fragmentation, in 70% ethanolic soln resident 5 seconds, resident 5 seconds in sex change liquid, resident 5 seconds in rinsing liquid, finally contain 45 μ L annealing buffers putting in conjunction with the pump of magnetic bead and add respectively the solution of sequencing primer SEQ ID No.17 to the SEQ ID No.24 that 3 μ L claims 3 mention, the switch of turning off pump makes to enter in liquid in conjunction with the magnetic bead of PCR product; Liquid is put into 80 DEG C of thermostat containers, leaves standstill 2min; Obtain the single stranded DNA template after separation and purification;
D, tetra-sodium sequence SEQ ID to be detected No.25 to SEQ ID No.32 are input in tetra-sodium sequenator;
E, the product that previous step purifying is obtained are put into tetra-sodium sequenator, are ready to enzyme mixture, substrate mixture and dNTP that tetra-sodium sequencing reaction is used, join reagent cabin, carry out tetra-sodium order-checking;
F, tetra-sodium sequenator carry out interpretation to sequencing result automatically, according to specification sheets in four kinds of sarcophagid haplotypes compare, confirm the kind (in table 6) of sarcophagid;
Table 6
(separately there are 2 kinds of calliphorids owing to having detected 16 samples of 13 kinds of sarcophagids, a kind of housefly), taking Sarcophga fuscicauda as example, A figure in Fig. 1 to 8 represents the somatotype result of the 1st to No. 8 Sarcophga fuscicauda samples, all allelotrope that this sample has can be clearly differentiated, thereby the haplotype of this Sarcophga fuscicauda can be judged.And above result is also used sanger sequencing to carry out sequence verification, wherein the sequencing result of 1 to No. 8 sample is shown in respectively the B figure in Fig. 1 to 8, and the base in dash box is SNP site, in full accord with tetra-sodium sequencing result.
According to the sanger sequencing result of 16 samples, we can be divided into these samples 13 kinds of sarcophagids, 2 kinds of calliphorids, a kind of housefly.The tetra-sodium sequencing result of all 16 kinds of fly classes is in table 6, the haplotype that can see red tail excrement sarcophagid, Sarcophga fuscicauda, fertile palpus Parasarcophaga and Huang palpus Parasarcophaga from table is consistent with the haplotype that our specification sheets provides, and repeatability between sample is better, do not belong to together or four kinds of sarcophagid haplotypes of haplotype and this of other kinds of Sarcophaga different.The amplimer that the present invention adopts is described and extends primer and can distinguish four kinds of sarcophagids.
Claims (3)
1. the forensic entomology detection kit based on 8 sarcophagid plastosome SNP genetic markers, it is characterized in that, described test kit comprises the sequencing primer of sequence shown in the amplimer of sequence shown in SEQ ID NO.1-SEQ ID NO.16 and SEQ ID NO.17-SEQ ID NO.24.
2. test kit as claimed in claim 1, is characterized in that, SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.11, shown in SEQ ID NO.13 and SEQ ID NO.15,5 ' of amplimer end is by biotin labeling.
3. amplimer as claimed in claim 1 and the sequencing primer application in preparation method medical entomology detection reagent; Described amplimer sequence is as shown in SEQ ID NO.1-SEQ ID NO.16; Described sequencing primer sequence is as SEQ ID NO.17-SEQ ID NO.24.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410162859.2A CN103937893B (en) | 2014-04-22 | 2014-04-22 | Forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP (single nucleotide polymorphisms) genetic markers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410162859.2A CN103937893B (en) | 2014-04-22 | 2014-04-22 | Forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP (single nucleotide polymorphisms) genetic markers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103937893A true CN103937893A (en) | 2014-07-23 |
| CN103937893B CN103937893B (en) | 2015-05-20 |
Family
ID=51185760
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410162859.2A Expired - Fee Related CN103937893B (en) | 2014-04-22 | 2014-04-22 | Forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP (single nucleotide polymorphisms) genetic markers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103937893B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022639A (en) * | 2017-06-07 | 2017-08-08 | 中南大学 | A kind of other sarcophagid specific DNA gene order in Taiwan |
| KR101861930B1 (en) * | 2016-10-27 | 2018-05-30 | 고려대학교 산학협력단 | SNP marker for classifying species of necrophagous fly and use thereof |
| CN111041109A (en) * | 2020-01-13 | 2020-04-21 | 中南大学 | Molecular marker, primer composition, kit and application for calculating development time of larvae of Boettcherisca peregrina |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605085A (en) * | 2012-03-29 | 2012-07-25 | 广东出入境检验检疫局检验检疫技术中心 | Identification method for L. sativae Blanchard, L. huidobrensis and L. trifolii |
-
2014
- 2014-04-22 CN CN201410162859.2A patent/CN103937893B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605085A (en) * | 2012-03-29 | 2012-07-25 | 广东出入境检验检疫局检验检疫技术中心 | Identification method for L. sativae Blanchard, L. huidobrensis and L. trifolii |
Non-Patent Citations (4)
| Title |
|---|
| SIEW HWA TAN等: "DNA-based characterisation and classification of forensically important flesh flies (Diptera: Sarcophagidae) in Malaysia", 《FORENSIC SCIENCE INTERNATIONAL》 * |
| YADONG GUO等: "Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China based on COI and period gene", 《INT J LEGAL MED》 * |
| 蔡继峰: "苍蝇群落演替及MtDNA分子标记的法医学研究", 《中国优秀博硕士学位论文全文数据库（博士） 医药卫生科技辑》 * |
| 郭亚东: "常见嗜尸性麻蝇分子标记的检测及地区基因库的建立", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101861930B1 (en) * | 2016-10-27 | 2018-05-30 | 고려대학교 산학협력단 | SNP marker for classifying species of necrophagous fly and use thereof |
| WO2018080225A3 (en) * | 2016-10-27 | 2018-08-09 | 고려대학교 산학협력단 | Snp marker for species identification of necrophagous flies and use thereof |
| CN107022639A (en) * | 2017-06-07 | 2017-08-08 | 中南大学 | A kind of other sarcophagid specific DNA gene order in Taiwan |
| CN111041109A (en) * | 2020-01-13 | 2020-04-21 | 中南大学 | Molecular marker, primer composition, kit and application for calculating development time of larvae of Boettcherisca peregrina |
| CN111041109B (en) * | 2020-01-13 | 2021-09-07 | 中南大学 | Molecular marker, primer composition, kit and application for calculating development time of larvae of Boettcherisca peregrina |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103937893B (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103131787B (en) | Forensic medicine compound detection kit based on Y chromosome SNP (single nucleotide polymorphism) genetic marker | |
| CN104046700B (en) | The detection kit of a kind of Rapid identification donkey hide, horse skin and mule skin | |
| CN102174650B (en) | Enterolobium cyclocarpum knot nematode loop-mediated isothermal amplification (LAMP) rapid detection method and application | |
| CN108531610B (en) | Fluorescence multiplex amplification system of 36Y chromosome STR loci and Y-Indel, kit and application thereof | |
| CN103866043B (en) | A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application | |
| CN102433374A (en) | Y-STR locus fluorescent label multiplex amplification system and application thereof | |
| CN111088329B (en) | Fluorescence composite amplification system, kit and application thereof | |
| CN102337345A (en) | Medicolegal composite assay kit based on twenty triallelic SNP (single nucleotide polymorphism) genetic markers | |
| CN103937893B (en) | Forensic entomology detection kit based on eight sarcophagidae mitochondrial SNP (single nucleotide polymorphisms) genetic markers | |
| CN112176074A (en) | Real-time fluorescent PCR primer probe and method for detecting patinopecten yessoensis | |
| CN111808975A (en) | Deer species source identification primer pair of pilose antler product and identification method thereof | |
| CN108315442A (en) | A kind of Huang lip fish microsatellite DNA mark and its screening technique | |
| CN109929936B (en) | Fluorescence labeling multiplex amplification kit for detecting human Y chromosome rapid mutation STR locus and application | |
| CN106701746B (en) | High-throughput malt Purity technology based on Capillary Electrophoresis and SSR marker | |
| CN101712992B (en) | Chlamys nobilis molecular marker with orange shell color and identification method thereof and kit | |
| CN108384884B (en) | Corn SSR molecular markers and application thereof | |
| CN103525933B (en) | A kind of PCR-RFLP method distinguishing grass carp and black carp | |
| CN105713992A (en) | DNA (deoxyribonucleic acid)-bar-code-based identification method of aquatic insects in Chironomidae two genera | |
| CN112695105A (en) | Real-time fluorescence PCR identification method of chlamys farreri | |
| CN1687453A (en) | Idiocratic amplification primer in medicolegal insect flies and authentication method | |
| CN104611440A (en) | Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite | |
| CN101353702A (en) | DNA molecular identification method of Haliotis sieboldii and Haliotis discus Hannai hybrid | |
| RU2642273C1 (en) | Method of differentiating yersinia pestis strains on basic and nonbasic subtypes by pcr method in real time mode | |
| Graham et al. | DNA: an overview | |
| CN113584213B (en) | Hemp SSR molecular markers and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150520 Termination date: 20160422 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |