DNA Preservation Research Articles

Effects of Lutein on the Body Weights, Hepato-renal DNA and Antioxidant Capacity Following Paraquat-induced Toxicity in Wistar Rats

Introduction: Paraquat is a leading cause of herbicide-related fatalities worldwide. Its high mortality rate is attributed to its inherent generation of ROS-mediated toxicity, and lack of effective treatment till date. Lutein is an antioxidant with free radical scavenging ability, which may contribute to its protective effects and possible DNA preservation. Objective: The study is evaluating the effects of lutein on body weights and hepato-renal antioxidant capacity with DNA quantification following paraquat toxicity in wistar rats. Methods: Thirty-five male Wistar rats (150-180g) rats were randomly divided into five equal groups (A-E). Paraquat toxicity was induced in Groups B-E with 5mg/kg for three days. Group A received normal saline only, while Group B was given paraquat only. Thereafter, Groups C-E received varying doses of lutein (50, 100, 150mg/kg) for 21 days. Their body weights were recorded twice weekly using a digital balance. The animals were sacrificed and their liver with kidneys were excised and processed. The photomicrographs were taken at various magnifications while Image J software was employed for DNA densities. The total antioxidant capacity (TAC) was assessed by Prieto method. Results: There was significant reductions in the body weights, (p=0.00) hepato-renal DNA densities (p=0.00, p=0.01) and antioxidant capacities of the PQ-only group relative to the positive control A in both liver and kidneys (p-0.01, p=0.02). However, the lutein-treated groups had dose-dependent similar features with the control, which had normal histo-morphological feature. The highest dose of lutein showed the best correlation with the control. Conclusion: This study showed significant alterations in body weights, hepato-renal DNA densities and antioxidant capacities especially in the PQ-only group. Meanwhile, the lutein-treated groups, especially at high dosages, showed significant improvements in the indices similar to the control. Hence, lutein may be a useful agent for attenuating paraquat toxicity.

Advancing Precision Oncology: Whole-Exome Sequencing in Endometrial Cancer Liquid-Based Cytology.

Diagnostic strategies for endometrial cancer have been evolving, with cytologic analysis being considered a key method in integrated oncologic diagnostics because of its less invasive nature and adaptability to various assessments. Liquid-based cytology (LBC) has emerged as a promising method for intact DNA preservation; it exhibits improved efficiency in advanced sequencing applications such as next-generation sequencing. However, despite the use of LBC in panel assays, its application in whole-exome sequencing (WES) for comprehensive genomic profiling remains underexplored. To investigate whether molecular classification is possible based on WES using DNA derived from LBC specimens. We combined WES with targeted gene panel analysis to compare genomic findings of LBC and traditional tissue samples obtained from 7 cases of endometrial cancer. We investigated pathogenic mutations, tumor mutational burden, and microsatellite instability, and achieved molecular classification with high accuracy. We found a substantial concordance between LBC and traditional tissue samples in terms of pathogenic mutation detection, with a 95% match in the LBC samples and 94% in the tissue samples. Notably, our results highlight the importance of combining WES with panel-based analysis in identifying the ultramutated status of a case that had been missed during panel analysis. Our findings emphasize the potential of LBC samples in the precise and noninvasive genomic analysis of cases of endometrial cancer and offer a new avenue for developing diagnostic and therapeutic strategies in precision oncology.

Compacted hair in broken teeth reveals dietary prey of historic lions

With recent advances, nuclear genome data for phylogenomic analyses can now be sequenced from minuscule quantities of DNA1 and from specimens that are more than a million years old.2 DNA analysis from hair is a well-established approach3 widely used in forensic science4 and wildlife conservation.5 Hairsamples can be effectively decontaminated6 and can be used to identify the mammalian speciesfrom which the hair was shed.7,8 We aimed to use advances optimized for degraded DNA to systematically identify dietary prey species from hair compacted in the teeth of two Tsavo lions that livedduring the 1890s in Kenya (see description of samples in the STAR Methods and Patterson9 andKerbis Peterhans and Gnoske10 for background on the Tsavo "man-eaters"). Analysis of hair DNA identified giraffe, human, oryx, waterbuck, wildebeest, and zebra as prey and also identified hair thatoriginated from lion. DNA preservation allowed for analyses of complete mitogenome profiles of zebra, giraffe, and lion. Giraffe mitogenomes are phylogeographically partitioned, and we found thatthe lions ate at least two individuals that belong to a subspecies of Masai giraffe (Giraffa tippelskirchi tippelskirchi) typically found in southeast Kenya. The lion mitogenome from a hair sample was identical tothe Tsavo lion endogenous mitogenome and most closely matched other East African lions from Kenyaand Tanzania. Our approach enables a better understanding of the hunting behaviors, diets, and ecology of historical individuals, populations, and species and holds promise for extinct populations and species.

Faecal microbiota and cytokine profiles of rural Cambodian infants linked to diet and diarrhoeal episodes

The gut microbiota of infants in low- to middle-income countries is underrepresented in microbiome research. This study explored the faecal microbiota composition and faecal cytokine profiles in a cohort of infants in a rural province of Cambodia and investigated the impact of sample storage conditions and infant environment on microbiota composition. Faecal samples collected at three time points from 32 infants were analysed for microbiota composition using 16S rRNA amplicon sequencing and concentrations of faecal cytokines. Faecal bacterial isolates were subjected to whole genome sequencing and genomic analysis. We compared the effects of two sample collection methods due to the challenges of faecal sample collection in a rural location. Storage of faecal samples in a DNA preservation solution preserved Bacteroides abundance. Microbiota analysis of preserved samples showed that Bifidobacterium was the most abundant genus with Bifidobacterium longum the most abundant species, with higher abundance in breast-fed infants. Most infants had detectable pathogenic taxa, with Shigella and Klebsiella more abundant in infants with recent diarrhoeal illness. Neither antibiotics nor infant growth were associated with gut microbiota composition. Genomic analysis of isolates showed gene clusters encoding the ability to digest human milk oligosaccharides in B. longum and B. breve isolates. Antibiotic-resistant genes were present in both potentially pathogenic species and in Bifidobacterium. Faecal concentrations of Interlukin-1alpha and vascular endothelial growth factor were higher in breast-fed infants. This study provides insights into an underrepresented population of rural Cambodian infants, showing pathogen exposure and breastfeeding impact gut microbiota composition and faecal immune profiles.

Suitability of undenatured ethanol for DNA and RNA preservation in pig oral fluid and fecal samples used for PCR-based pathogen detection

Suitability of undenatured ethanol for DNA and RNA preservation in pig oral fluid and fecal samples used for PCR-based pathogen detection

Supplementary Material: Suitability of undenatured ethanol for DNA and RNA preservation in pig oral fluid and fecal samples used for PCR-based pathogen detection

Supplementary Material: Suitability of undenatured ethanol for DNA and RNA preservation in pig oral fluid and fecal samples used for PCR-based pathogen detection

An active & passive dual-targeting platform for the precise treatment, process monitoring and effective protection of central nervous system infection

Precise antibacterial activity and effective inflammatory response reduction are critical for successful treatment of Intracranial infection after craniocerebral surgery, which remains an unmet clinical challenge. Herein, an intracranial drug delivery liposome (AFAA-lipo) that possesses dual active&passive targeting capability, direct antibacterial ability, favorable inflammation regulation, and excellent biocompatibility is developed as an antibiotics-free and hardly bacterial-resistance-induced platform for the management of intracranial infection. The surface-anchored specific peptide sequence HRERMS (the oligopeptide designed sourced from β-amyloid precursor protein) makes AFAA-lipo actively penetrate the blood–brain barrier and then perforated and lysed under the stimulation of α-toxin secreted by methicillin-resistant Staphylococcus aureus (MRSA, a severe and widespread intracranial infection type) in the infected site. Our results demonstrate that the AFAA-lipo can eliminate bacteria and biofilms, and reduce inflammatory response through mitochondrial function protection, DNA preservation from damage, TNFR1/NF-κB signaling pathway inhibition, and promotion of microglial polarization towards the anti-inflammatory subtype, ultimately improving post-infection prognosis and survival rate. More interestingly, the pre-intake of AFAA-lipo is capable to reduce MRSA infection risk and alleviate inflammatory response. Furthermore, the near-infrared (NIR) II region imaging fluorescence signal intensity variations of AFAA-lipo is proved the validation to monitor the course of the disease.

Optimal liquid-based DNA preservation for DNA barcoding of field-collected fungal specimens

Preserving fungal tissue DNA in the field is essential for molecular ecological research, enabling the study of fungal biodiversity and community dynamics. This study systematically compares two liquid-based preservation solutions, RNAlater and DESS, for their effectiveness in maintaining macrofungi DNA integrity during field collection and storage. The research encompasses both controlled experiments and real-world field collections. In the controlled experiments, two fungal species were preserved in RNAlater and DESS at different temperatures and durations. DNA extraction success rates were high, but DNA quality and quantity metrics exhibited variations across samples. However, both preservation solutions demonstrated their viability for preserving fungal DNA, with no significant differences between them. In the field-collected macrofungi experiment, 160 paired fungal specimens were preserved in RNAlater and DESS, respectively. Including a drying process to facilitate tissue lysis for DNA extraction significantly impacted the outcomes. RNAlater showed a higher success rate and better DNA quality and quantity compared to DESS. Statistical analysis, including paired and independent t-tests, confirmed significant differences in DNA quality and quantity between the two preservation methods for field-collected samples. This study evaluates RNAlater and DESS for preserving macrofungi DNA in field conditions. Both methods are effective, but RNAlater is superior when a drying step is included in DNA extraction. Researchers can choose based on their specific needs without compromising DNA integrity. These findings advance fungal molecular ecology and DNA preservation strategies in ecological and environmental studies.

Preparation of Biomass-Derived DNA-Chitosan Polyelectrolyte Complex Hydrogels for Economical and Reliable DNA Preservation and Protein Immobilization

Preparation of Biomass-Derived DNA-Chitosan Polyelectrolyte Complex Hydrogels for Economical and Reliable DNA Preservation and Protein Immobilization

Searching for alternative high DNA-yielding bone types for DNA analysis of aged skeletal remains

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Enhancing Salivary DNA Preservation via DNase I Inactivation: Role of Temperature and EDTA

Background Deoxyribonuclease I (DNase I) catalyzes the cleavage of double-stranded DNA into its end products. This enzyme requires cations for its activity. In addition, DNase activity is influenced by indirect DNase inhibitors (i.e. EDTA) and temperature. EDTA is an indirect DNase I inhibitor. Therefore, DNase inactivation is crucial for DNA preservation, especially for salivary samples, and the downstream application of genetic techniques using non-blood samples. Saliva is a complex mixture contaminated with oral microbes and contains DNase which is harmful for DNA preservation. This study aims to preserve salivary DNA through temperature and EDTA treatment. Methods This is an experimental study. Salivary DNA was extracted using the spin-column method. DNA degradation assays were carried out using either 5 µL of salivary supernatant or 2.5 mg/mL of DNase I which were incubated at -20°C, 2-8°C, room temperature, 40°C, and 50°C; and added with 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM EDTA for 60 minutes. After incubation, the results were visualized using agarose gel electrophoresis. Results The average concentration of salivary DNA was 61.70 µg/mL (30.50-109.05) and the purity was 1.893 µg/mL (1.800-2.005). Incubation at -20°C, 2-8°C, and room temperature degraded salivary DNA but incubation at 40 and 50°C did not completely degrade DNA. Addition 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM EDTA to 5 µL of salivary supernatant or 2.5 mg/mL DNase I preserved salivary DNA. Conclusions Temperature of 40 and 50°C, and 0.125 mM EDTA can inactivate DNase and preserve salivary DNA.

A Comparison Study of Cryopreservation of Seminal Fluid between Flinders Technology Associates (FTA-card) and Swab for Multiplex Short Tandem Repeats (STR) Analysis

Background Whatman™ FTA (Flinders Technology Associates) is a form of card soaked in chemicals that denatures proteins while protecting DNA and ensuring the safe handling of dried body fluid spots (blood, semen, and saliva) and buccal cells. To this day, these cards are still infrequently used in forensic science. Therefore, reference samples including biological material may be collected on FTA cards for genetic analysis that have been widely used for DNA preservation and analysis, particularly in forensic sciences and genetic studies. The goal of the study was to evaluate the effects of cryopreservation on DNA quantitative and short tandem repeats (STR) profiling by comparing the yield and quality of DNA extracted before and after cryopreservation using FTA card and swabs in the forensic analysis. The mean total DNA concentration and the purity of seminal fluid from FTA-cards and swabs before and after sperm cryopreservation were measured by using nano-drop spectrophotometer (93.688 ± 12.5) (90.94 ± 13.9) ng/ µl; ( 221.07 ± 20.97)( 135.47 ± 19.6) ng/ µl respectively. Also a highly significant difference (p<0.001) was detected when the results of concentration DNA on FTA card were compared with dilution using wash media before and after sperm cryopreservation. Whereas significant difference (p<0.05) was observed between DNA concentration on FTA card with and without dilution after sperm cryopreservation. STR loci were present in the majority of data, particularly in FTA-card and swab samples where results explained that 100% of STR profiles were present in all 15 loci, both before and after sperm cryopreservation. In contrast to the conventional method of storing and analyzing STR profiling, it can be concluded that seminal fluid samples taken by FTA card and swabs can be taken and stored for a time by cryopreservation, leads to maintaining DNA integration in the field of criminal research.

Spatially and temporally precise microbiome profiling in the small intestine using the SIMBA capsule with X-ray tracking

IntroductionFew minimally invasive options for sampling the small intestinal (SI) luminal fluid exist to study the SI microbiota in health and disease. To address the lack of tools and methods to study GI regions that are difficult to access, Nimble Science developed a fully autonomous and passive sampling method, the Small Intestine MicroBiome Aspiration (SIMBA™) capsule, for convenient, high-quality, and reliable sampling to study the diet-microbiota interactions in the SI. MethodsThe sealing efficacy and microbial DNA preservation capacity of the SIMBA capsules was first validated through in vitro simulation assays. Then, a clinical study was conducted with 20 healthy participants to validate the in vivo use of SIMBA capsules to reliably capture samples for SI microbiome analysis before and after an intervention (NCT04489329). Briefly, participants ingested the capsules at baseline and 7 days later, with a probiotic capsule containing a blend of L. rhamnosus R0011 and B. longum R0175. Following baseline SIMBA capsule ingestion, multiple low-dosage x-ray scans were performed to track the sampling location. Fecal samples corresponding with the baseline and intervention capsule were analyzed for comparison. ResultsThe SIMBA capsules’ performance in vitro demonstrated the potential for contamination-free sampling with preservation of the microbial communities. Within the clinical study, the capsules performed safely and reliably for collection of SI content. X-ray tracking confirmed that 97.2% of the capsules completed sample collection in the SI regions before reaching the colon. Importantly, our data showed that the capsules sampled in the right area of the intestines and that baseline SIMBA microbiome profile is significantly different from fecal microbiome profile. SIMBA successfully detected a concurrent probiotic intervention in the small intestine, which was not detectable using stool samples. DiscussionsThe high accuracy of sampling location and sealing efficacy of the SIMBA capsules makes them potentially useful research tools in clinical trials for studying diet-microbiota interactions in health and disease, and perhaps eventually for the clinical diagnosis of GI tract conditions affecting the SI such as SIBO.

Novel buffer for long-term preservation of DNA in biological material at room temperature.

The collection and preservation of biological material before DNA analysis is critical for inter alia biomedical research, medical diagnostics, forensics and biodiversity conservation. In this study, we evaluate an in-house formulated buffer called the Forensic DNA Laboratory-buffer (FDL-buffer) for preservation of biological material for long term at room temperature. Human saliva stored in the buffer for 8years, human blood stored for 3years and delicate animal tissues from the jellyfish Pelagia noctiluca comb jelly Beroe sp., stored for 4 and 6years respectively consistently produced high-quality DNA. FDL-buffer exhibited compatibility with standard organic, salting out and spin-column extraction methods, making it versatile and applicable to a wide range of applications, including automation.

Metagenomic and paleopathological analyses of a historic documented collection explore ancient dental calculus as a diagnostic tool

Dental calculus is a microbial biofilm that contains biomolecules from oral commensals and pathogens, including those potentially related to cause of death (CoD). To assess the utility of calculus as a diagnostically informative substrate, in conjunction with paleopathological analysis, calculus samples from 39 individuals in the Smithsonian Institution’s Robert J. Terry Collection with CoDs of either syphilis or tuberculosis were assessed via shotgun metagenomic sequencing for the presence of Treponema pallidum subsp. pallidum and Mycobacterium tuberculosis complex (MTBC) DNA. Paleopathological analysis revealed that frequencies of skeletal lesions associated with these diseases were partially inconsistent with diagnostic criteria. Although recovery of T. p. pallidum DNA from individuals with a syphilis CoD was elusive, MTBC DNA was identified in at least one individual with a tuberculosis CoD. The authenticity of MTBC DNA was confirmed using targeted quantitative PCR assays, MTBC genome enrichment, and in silico bioinformatic analyses; however, the lineage of the MTBC strain present could not be determined. Overall, our study highlights the utility of dental calculus for molecular detection of tuberculosis in the archaeological record and underscores the effect of museum preparation techniques and extensive handling on pathogen DNA preservation in skeletal collections.

Stability, enrichment, and quantification of total and HPV16-specific IgG present in first-void urine

First-void urine (FVU) samples, containing human papillomavirus (HPV)-specific IgG from female genital tract secretions, provide a non-invasive option for disease monitoring and vaccine impact assessment. This study explores the utility of FVU for IgG quantification, exploring stability and compatibility with DNA preservation methods, alongside various IgG enrichment methods. Healthy female volunteers provided FVU and serum samples. FVU was collected with or without urine conservation medium (UCM) and stored under different conditions before freezing at −80 °C. Four IgG enrichment methods were tested on FVU samples. All samples were analyzed using three total human IgG quantification assays and an in-house HPV16-specific IgG assay. Samples stored with UCM buffer had higher total and HPV16-specific IgG concentrations (p ≤ 0.01) and IgG remained stable for at least 14 days at room temperature. Among IgG enrichment methods, Amicon filtration (AM) and AM combined with Melon Gel purification (AM-MG) provided similar HPV16-IgG concentrations, correlating strongly with serum levels. Protein G magnetic beads methods were incompatible with time-resolved fluorescence-based assays. This study highlights FVU as a reliable and convenient sample for IgG quantification, demonstrating stability for at least 14 days at room temperature and compatibility with UCM DNA preservation. It emphasizes the need to select appropriate IgG enrichment methods and confirms the suitability of both AM and AM-MG methods, with a slightly better performance for AM-MG.

DNA preservation in compact and trabecular bone

Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)–epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process.

Environmental Factors Affecting the Concentration of DNA in Blood and Saliva Stains: A Review

DNA evidence has now become an essential part of forensic investigations since it offers vital information for person identification and crime resolution. However, the biological material is affected by some environmental factors which may impact the DNA in biological samples. This may affect the correctness and reliability of forensic DNA analysis. This review is related to the influence of various environmental conditions on the stability and degradation of DNA in biological stains including blood and saliva stains. The common factors that affect DNA are temperature, humidity, exposure to sunlight, and type of substrate. The information is crucial to improve forensic DNA analysis and forensic protocol optimization. The DNA stability and integrity in biological materials, such as blood and saliva stains, are indispensable for forensic DNA analysis. Environmental influences, however, significantly affect DNA concentration and may jeopardize forensic analysis. The present review explores various environmental factors for their effect on DNA stability in blood and saliva stains. While DNA degradation is slowed but not completely prevented by low temperatures, it is accelerated by high temperatures. Risks of contamination arise from the promotion of microbial growth and DNA breakdown by humidity. DNA photodamage brought on by sunlight exposure results in strand breakage and cross-linking. DNA stability is also influenced by the type of substrate used; porous surfaces, such as cloth, are better at keeping fluids than non-porous ones, such as glass. Maintaining the integrity of DNA evidence requires an understanding of these variables. The present studies will help to create sophisticated DNA preservation methods for use in forensic DNA examination. The study emphasizes the requirement of improvement in forensic DNA analysis skills, related to the preservation of DNA pieces of evidence and the possible effect of environmental factors.

Advancing responsible genomic analyses of ancient mollusc shells.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111-6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.

DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

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.