Extraction of PCR amplifiable DNA from oil, fat and meat samples of reticulated python (Malayopython reticulatus) for genetics and forensics applications

Virgin olive oil is made from diverse cultivars either mixed or single. Those ensure different tastes and typicity, and these may be also enhanced by the region of production of cultivars. The different olive oil labels correspond to their chemical composition and acidity. Labels also may correspond to a protected origin indication, and thus, such oils contain a given composition in cultivars. To verify the main cultivars used at the source of an olive oil sample, our method is based on DNA technology. DNA is present in all olive oil samples and even in refined oil, but the quantity may depend on the oil processing technology and oil conservation conditions. Thus, several supports were used to retain DNA checking different techniques (silica extraction, hydroxyapatite, magnetic beads, and spun column) to prepare DNA from variable amounts of oil. At this stage, it was usable for amplification through PCR technology and especially with the magnetic beads, and further purification processes were checked. Finally, the final method used magnetic beads. DNA is released from beads in a buffer. Once purified, we showed that it did not contain compounds inhibiting PCR amplification using SSR primers. Aliquot dilution fractions of this solution were successfully routinely used through PCR with different SSR primer sets. This enables confident detection of eventual alien alleles in oil samples. First applied to virgin oil samples of known composition, either single cultivars or mixtures of them, the method was verified working on commercial virgin oil samples using bottles bought in supermarkets. Last, we defined a protocol starting from 2 x 40 mL virgin olive oil, and DNA was prepared routinely in about 5 h. It was convenient to genotype together several loci per sample to check whether alleles were in accordance with those of expected cultivars. Thus, forensic applications of our method are expected. However, the method needs further improvement to work on all oil samples.

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.

An improved method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of glycidyl fatty acid esters in oils was developed. The method incorporates stable isotope dilution analysis (SIDA) for quantifying the five target analytes: glycidyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3). For the analysis, 10 mg sample of edible oil or fat is dissolved in acetone, spiked with deuterium labelled analogs of glycidyl esters and purified by a two-step chromatography on C18 and normal silica solid phase extraction (SPE) cartridges using methanol and 5% ethyl acetate in hexane, respectively. If the concentration of analytes is expected to be below 0.5 mg/kg, 0.5 g sample of oil is pre-concentrated first using a silica column. The dried final extract is re-dissolved in 250 μL of a mixture of methanol/isopropanol (1:1, v/v), 15 μL is injected on the analytical C18 LC column and analytes are eluted with 100% methanol. Detection of target glycidyl fatty acid esters is accomplished by LC-MS/MS using positive ion atmospheric pressure chemical ionization operating in Multiple Reaction Monitoring mode monitoring 2 ion transitions for each analyte. The method was tested on replicates of a virgin olive oil which was free of glycidyl esters. The method detection limit was calculated to be in the range of 70-150 μg/kg for each analyte using 10 mg sample and 1-3 μg/kg using 0.5 g sample of oil. Average recoveries of 5 glycidyl esters spiked at 10, 1 and 0.1 mg/kg were in the range 84% to 108%. The major advantage of our method is use of SIDA for all analytes using commercially available internal standards and detection limits that are lower by a factor of 5-10 from published methods when 0.5 g sample of oil is used. Additionally, MS/MS mass chromatograms offer greater specificity than liquid chromatography-mass spectrometry operated in selected ion monitoring mode. The method will be applied to the survey of glycidyl fatty acid esters in food products on the Canadian market.

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

Morphological and chemical profiling for forensic hair examination: A review of quantitative methods

Fatty acid composition and distribution of human milk fat (HMF), from mothers over different lactating periods in Guangzhou, China, were analyzed. The universal characteristics were consistent with previously reported results although the fatty acid content was within a different range and dependent on the local population (low saturated fatty acid and high oleic acid for Guangdong mothers' milk fat). Based on the composition of the total and sn-2 fatty acids of mature milk fat, an efficient evaluation model was innovatively established by adopting the "deducting score" principle. The model showed good agreement between the scores and the degree of similarity by assessing 15 samples from different sources including four samples of HMF, eight samples of human milk fat substitutes (HMFSs) and infant formulas, and three samples of fats and oils. This study would allow for the devolvement of individual human milk fat substitutes with different and specific fatty acid compositions for local infants.

Discrepancies in the analysis of 3‐chloropropane‐1,2‐diol (3‐MCPD) esters can be explained by the hypothesis that in some refined oils significant amounts of fatty acid esters of glycidol (glycidyl esters) are present in addition to 3‐MCPD esters. Glycidyl esters were separated from triacylglycerols by gel permeation chromatography (GPC) and detected by gas chromatography‐mass spectrometry (GC‐MS). Six samples of palm oil and palm oil‐based fats were analyzed by GPC and GC‐MS. In chromatograms of all samples, significant peaks, retention time and mass spectra in conformity with self‐synthesized glycidyl palmitate and glycidyl oleate were detectable. Quantification of individual glycidyl esters was not possible because of a lack of pure standards. Concentration of ester‐bound glycidol in different samples of fats and oils was estimated using an indirect difference method. Glycidyl esters could be detected only in refined, but not in crude or native, fats and oils. The highest concentrations were detected in palm oil and palm oil‐based fats. In a palm oil sample, glycidyl ester concentration varied according to different deodorization parameters, temperature, and time, while 3‐MCPD ester concentration was relatively constant, indicating that mitigation of glycidyl esters possibly may be achieved by optimizing refining parameters.

The impact of spin coupling signal loss on fat content characterization in multi-echo acquisitions with different echo spacing

Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction-DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis.

A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

Detection procedures for castor oil in genuine and treated groundnut oils were screened and false turbidity was noticed when ammonium molybdate/sulfuric acid reagents were added to rancid groundnut oil. Successively bleached and neutralized rancid groundnut oil samples do not respond to the tests. Turbidity did not appear for groundnut oils containing 10 and 20% castor oil and even for pure castor oil sample. Thin layer chromatography (TLC) was found to be quite effective for most of the treated, adulterated, genuine, old and fresh oil and fat samples, but showed a streaking, when applied to rancid groundnut oil. However, streaking could be greatly reduced and TLC could be successfully performed with bleached and neutralized, rancid groundnut oil samples.

Background: Species specification of the meat is a highly important field of quality control management in meat consumption and industry. Meat forgery with undeclared sources has become a crucial trouble in many countries as well as in Iraq. It means that raw consumed meat or meat derivatives may include different meat genus; so, the components of meat are not regular like the known marker. The diet has sever effect on public health, religious factor, food safety principles, fair-trades and consumers desire. Aim of the study: The current study was aimed to analysis the species of adulterated meat using molecular technique that targets Mitochondrial Cytochrome cyt b gene by Multiplex PCR for detection of meat species and authentication that provide many forensic and judicial applications for several populations throughout the world. Materials and Methods: The following seventy (70) typical meat and meat derivatives were gathered from neighborhood super markets and eateries in the province of Kerbala: 25 cow, 10 buffalo, 25 sheep, and 10 goats. For meat samples, DNA extraction was done. Next, using species-specific primers of the Mitochondrial Cytochrome cyt b gene for cattle, sheep, goats, and buffalo, the collected DNA was amplified using the Multiplex PCR method. Additionally, the presence of Staphylococcus aureus, one of the microorganisms most commonly linked to food illness, was typically examined in all of the meat samples. Results: Multiplex PCR technique resulting in species-specific products of the amplicon sizes for the DNA prepared samples with the lengths 472bp., 124bp., 585bp., and 330bp. for the flesh of cow, buffalo, sheep, and goats, in that order. In addition to this analysis technique indicated that8 (44.4) % of examined cattle meat samples were mixed with buffalo meat and10 (55.6) % of examined sheep meat samples were mixed with goat meat. As well as, most of examined (normal and mixed) meat samples were contaminated with Staphylococcus aureus bacteria. Discussion: It has been demonstrated that the highly conserved Mitochondrial Cytochrome cyt b gene primers, when used in the multiplex PCR methodology, provide a sensitive, straightforward, adaptable, and dependable method for the investigation of meat samples and meat products. Conclusions: The current study's conclusions state that the “mitochondrial cytochrome cyt b gene” is a useful marker for differentiating between normal and adulterated meat. It also states that Staphylococcus aureus was found in 16/52 (30.76%) of the normal meat samples and 12/18 (66.7%) of the adulterated meat samples, with a significant importance of P=0.046.

Scanning probe microscopy (SPM) describes a relatively new field of imaging technology that utilizes high resolution, real-time, near-field, three-dimensional microscopes. One of its most successful progenitors is the atomic force microscope (AFM), also known as the scanning force microscope (SFM), a technology capable of attaining high resolution under physiologically relevant conditions by feeling a sample’s surface with a sharp probe. Careful control of the AFM probe force under liquids and/or with vibrating probe techniques (e.g., “intermittent contact” or “tapping” modes) can be employed to reduce sample damage. Such techniques are especially useful for imaging soft biological specimens. The same control over the probe can also be used to manipulate biological structures, such as dissection and extraction of DNA from chromosomes. Because the AFM probe responds to numerous near-field forces, various imaging modes can yield different types of surface information. Simultaneous monitoring of amplitude and phase changes in a vibrating probe can concurrently map both surface topography and chemistry. The ability of the AFM probe to distinguish between different molecular species may make it a useful tool in forensics. In particular, the precise ability of the AFM to identify regions of interest (through a combined optical/atomic force microscope) and excise specific sequences of DNA, could expedite DNA identification processes.KeywordsAtomic Force MicroscopeScanning Tunneling MicroscopeScan Probe MicroscopyAtomic Force Microscope ProbeContact Atomic Force MicroscopeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology.