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Abstract

Introduction

Protocol

Representative Results

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Materials

References

Medicine

DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples

Published: May 3rd, 2024

DOI:

10.3791/66737

1Hainan Vocational College of Political Science and Law, Hainan Gongping Forensic Center, 2Hainan Province Tropical Forensic Engineering Research Center, Department of Forensic Medicine, Hainan Medical University, Hainan Provincial Academician Workstation (Tropical Forensic Medicine), 3Department of Forensic Medicine, Hebei Medical University
* These authors contributed equally

This article describes a protocol for fresh and old necrophilic fly DNA extraction using a modified common DNA extraction kit.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

The inference of the time of death has always been one of the key and difficult issues to be resolved in judicial practice. In the practice of forensic science, for a criminal case, determining postmortem interval plays a crucial role in deducing the time of the crime, locking in the suspect, and narrowing the scope of the investigation. Traditional methods of inferring the time of death are based primarily on early postmortem phenomena, which can generally only be used to infer the time of death within 24 h. However, the long time of death cannot be determined by postmortem phenomena. It is now generally recognized in the forensic science community that forensic ento....

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NOTE: A total of five samples of C. megacephala samples were used in this protocol-three fresh samples (Fresh 1, Fresh 2, and Fresh 3), one sample stored in alcohol for 2 years (Old 1), and one sample stored in dry sealed storage for 2 years protected from light only (Old 2). Samples must be labeled according to the experimental requirements.

1. General sample storage and preparations

  1. Sample selection and preparation
    NOTE: Be sure to work in the fum.......

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We used a microplate reader to measure the values of OD260 and OD280 of the extracted DNA solution and then obtained the OD260/OD280 values to evaluate the purity of the DNA. The OD260/OD280 of all fresh samples and old sample 1 was greater than 2. The OD260/OD280 of old sample 2 had a maximum value of 2.187 although the average was 1.753, and its three measurements fluctuated greatly due to the small (≤0.01) values of both its OD280 and OD280 (Table 1). Based on the value of OD260 obtained, we est.......

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DNA extraction is the most critical link in the molecular identification of necrophilous flies, and its extraction method and the quality of the extracted DNA directly affect subsequent detection. In this experiment, we successfully extracted DNA from necrophilous flies by using a common blood kit, without the need to purchase a special insect DNA extraction kit, which makes insect DNA extraction easier to accomplish.

The surface of flies is rich in chitin, especially in the larval and pupal s.......

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This work is supported by the National Natural Science Foundation of China (82060341,81560304) and by the Academician Innovation Platform Scientific Research Project of Hainan Province (YSPTZX202134).

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NameCompanyCatalog NumberComments
Buffer AEQiagen172026832DNA elution buffer
Buffer ALQiagen172028374lysis buffer
Buffer ATLQiagen172028162tissue lysis buffer
Buffer AW1Qiagen172028760protein removal buffer
Buffer AW2Qiagen57203108desalination buffer
C1-J-2495Taihe BiotechnologyTW21109216forword primer
C1-N-2800Taihe BiotechnologyTW21109217reverse primer
D2000 DNA ladderReal-Times(Beijing) BiotechnologyRTM415Measure the position of electrophoretic bands
DNeasy Mini spin columnQiagen166050343DNA adsorption column
Dry Bath IncubatorMiulabDKT200-2Dused for heating
MIX-30S Mini MixerMiulabMUC881206oscillatory action
Proteinase KQiagen172026218Inactivation of intracellular nucleases and other proteins
Qubit 3.0 FluorometerThermo Fisher Scientific2321611188
Speed Micro-CentrifugeScilogex9013001121centrifuge
Standard#1Thermo Fisher Scientific2342797
Standard#2Thermo Fisher Scientific2342797
Tanon 3500R Gel ImagerTanon16T5553R-455gel imaging
Taq Mix ProMonad00007808-140534PCR Mix
Taq Mix ProMonad00007808-140534PCR Mix
Thermo CyclerZhuhai HemaVRB020Aordinary PCR
Working SolutionThermo Fisher Scientific2342797

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