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Idiocratic amplification primer in medicolegal insect flies and authentication method

A technique for amplifying primers and specificity, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc. It can solve problems such as fragment length differences, achieve the effect of shortening the cycle and improving work efficiency

Inactive Publication Date: 2007-03-07
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are sliding and unequal exchange phenomena in the process of DNA replication of SSR, which makes their repeat numbers vary greatly among different varieties or individuals, which easily leads to differences in the length of the primer binding site and the fragment length between the two binding sites.

Method used

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  • Idiocratic amplification primer in medicolegal insect flies and authentication method
  • Idiocratic amplification primer in medicolegal insect flies and authentication method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, the acquisition of species-specific primers

[0060] Using ISSR universal primers for Lucilia sericata, Aldrichina grahami, Chrysomya megacephala, Parasarcophaga crassipalpis and Muscadomestica DNA samples were amplified by PCR on a PCR instrument (Flexigene Thermal Cycler), wherein the collection of DNA samples was carried out with reference to the method of Zheng Z-M et al. (Zheng Z-M, Huang G, Journal ofShanghai Normal University (Natural Science Edition).No.1, Vol .30, 2002);

[0061] Primer 1: CACACACACACAGT

[0062] The reaction system (15 μl) for PCR amplification is as follows:

[0063] 10×PCR buffer 1.5μl

[0064] Primer 0.6μl (10pmol / μl)

[0065] 10mM dNTPs 0.25μl

[0066] wxya 2 O 10.0 μl

[0067] Taq DNA polymerase 0.15μl (5U / μl)

[0068] Template DNA 2.0μl (10ng / μl)

[0069] Wherein the composition of 10×PCR reaction buffer is as follows:

[0070] 200mM Tris-HCl;

[0071] 100mM (NH 4 ) 2 SO 4 ;

[0072] 100mM KCl;

[0073] 1% T...

Embodiment 2

[0094] Embodiment 2, verification of primer species specificity

[0095] Three pairs of necrophagous fly specific amplification primers obtained in Example 1 were used to carry out the DNA samples (collection method the same as in Example 1) in the PCR instrument respectively to the DNA samples of three fly species of Chrysocephala botany, Sarcophagus spp. and Musca domestica. Carry out PCR amplification on (Flexigene Thermal Cycler) (the concrete condition of amplification is the same as embodiment 1).

[0096] The obtained PCR amplification products were detected by electrophoresis on 1.0% Agarose gel, observed and photographed under ultraviolet light, and the results are shown in FIG. 2 .

[0097] From the results in Figure 2 (weak bands below 100bp in the figure are primer dimers, which do not hinder the identification results), it can be seen that primer C only amplifies DNA fragments of corresponding sizes in Chrysocephala chinensis, and in other fly species Then there ...

Embodiment 3

[0099] Embodiment 3, the preparation of detection kit

[0100] According to the results of Example 2, the following three pairs of species-specific amplification primers obtained in Example 1:

[0101] SCAR marker C (Chrysomya megacephala):

[0102] Forward primer 5′-AATCGGTTACTTGCAATCTCA-3′

[0103] Reverse primer 5′-CGACAGTTGATGTTGTTTTCGTTTA-3′

[0104]SCAR marker D (Parasarcophaga crassipalpis)

[0105] Forward primer 5′-CCACTGAACAAAGCGACG-3′

[0106] Reverse primer 5′-TTTTCTTCGCACGGCTTT-3′

[0107] SCAR marker E (Musca domestica)

[0108] Forward primer 5′-AATGCTATCTGCCCCTCTTG-3′

[0109] Reverse primer 5′-CATTCCCACTTGTACTTTCGTT-3′

[0110] Preparation of rapid test kits for Chrysanthemum cephalexus, M. cerevisiae and Musca domestica.

[0111] The preparation kit uses the following common reagents in conventional PCR:

[0112] 10×PCR buffer

[0113] dNTPs (10mM)

[0114] wxya 2 o

[0115] Taq DNA polymerase (5U / μl)

[0116] Among them, the composition of 10×b...

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Abstract

This invention discloses an idiocratic amplification primer in medicolegal insect flies. We utilize this method to authenticate the fly species by this primer, and obtain solution box for authentication. The amplification primer special can finish the authentication within 3 hours; as a result it can be use to fast authentication.

Description

technical field [0001] The invention belongs to the technical field of biological detection and forensic identification, and in particular relates to forensic insect fly-specific amplification primers and identification methods. Background technique [0002] Forensic entomology is an emerging interdisciplinary subject that has been studied more in recent years. It uses the characteristics of necrophilia of certain flies to provide necessary clues and basis for forensic identification by studying its occurrence, developmental duration and geographical distribution characteristics. . Insects play an important role in the material cycle of dead corpses. According to their growth and succession on the corpse, the time of death of the corpse can be inferred more accurately. In the process of solving a case, the time of death is an important basis for dividing the range of suspects. This method will assist forensic doctors in criminal investigation to quickly and accurately ident...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 吴虹何琳苗雪霞黄勇平
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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