DNA Extraction Protocol Research Articles

RT-qPCR Protocol Optimization Assessment with Propidium Monoazide for Viable and Non-Viable Pathogen Detection

Real time PCR or RT-qPCR (including reverse transcriptase PCR) is a gold standard in diagnostic testing for pathogens such as bacteria and viruses that contain RNA or DNA for amplification. As a validated molecular assay technique, RT- qPCR has been instrumental in microbial detection because of its high throughput, quantification of DNA or RNA samples as well as quick turnaround especially when compared with culturing methods that can take multiple days to generate result. While the advancement of PCR has had significant impact for diagnosis of diseases such as COVID- 19through its high sensitivity and specificity, the test also brings with it its own challenges of false positive and false negative results. This is normally attributed to PCR's susceptibility to contamination or inhibitors. However, there are other conditions like DNA extraction techniques and protocols in PCR methodology that can impact the accuracy of the PCR results as well. This paper, therefore, aims to synthesize factors that contribute to false positive and negatives in PCR results, improvements to protocols that includes the use of DNA intercalating dyes such as Propidium Monoazide (PMA) in PCR assay to improve DNA extraction and discuss the importance of an optimized PCR assay for diagnostic testing efficacy from a social and economic standpoint.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS (EEHV) IN FREE-RANGING AFRICAN ELEPHANTS (LOXODONTA AFRICANA) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants.

DNA extraction protocol impacts ocular surface microbiome profile.

The aim of this study is to provide a reference frame to allow the comparison and interpretation of currently published studies on 16S ribosomal ribonucleic acid amplicon sequencing of ocular microbiome samples using different DNA extraction protocols. Alongside, the quantitative and qualitative yield and the reproducibility of different protocols has been assessed. Both eyes of 7 eligible volunteers were sampled. Five commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome and 2 host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration. The V3-V4 region of the 16S rRNA gene was targeted using Illumina MiSeq sequencing. The DADA2 pipeline in R was used to perform the bio-informatic processing and taxonomical assignment was done using the SILVA v132 database. The Vegdist function was used to calculate Bray-Curtis distances and the Galaxy web application was used to identify potential metagenomic biomarkers via linear discriminant analysis Effect Size (LEfSe). The R package Decontam was applied to control for potential contaminants. Samples analysed with PowerSoil, RNeasy and NucleoSpin had the highest DNA yield. The host DNA depletion kits showed a very low microbial DNA yield; and these samples were pooled per kit before sequencing. Despite pooling, 1 of both failed to construct a library.Looking at the beta-diversity, clear microbial compositional differences - dependent on the extraction protocol used - were observed and remained present after decontamination. Eighteen genera were consistently retrieved from the ocular surface of every volunteer by all non-pooled extraction kits and a comprehensive list of differentially abundant bacteria per extraction method was generated using LefSe analysis. High-quality papers have been published in the field of the ocular surface microbiome but consensus on the importance of the extraction protocol used are lacking. Potential contaminants and discriminative genera per extraction protocol used, were introduced and a reference frame was built to facilitate both the interpretation of currently published papers and to ease future choice - making based on the research question at hand.

Field-based recombinase polymerase amplification and lab-based qPCR assays for detection of Helicoverpa armigera.

Helicoverpa armigera (Hübner) is a major crop pest native to Europe, Asia, Australia, and Africa which has recently invaded South America and has caused billions of dollars in agricultural losses. Because of challenges in differentiating between H. armigera and Helicoverpa zea (Boddie), a closely related species native to North and South America, genetic tests have previously been developed to detect H. armigera DNA in pooled samples of moth legs. In this study, a field-based recombinase polymerase amplification (RPA) assay using a lateral flow strip and a qPCR melt curve assay were developed for specific detection of H. armigera DNA in pooled moth samples. In addition, a crude DNA extraction protocol for whole moths was developed to allow rapid preparation of DNA samples. The RPA field test was able to detect ≥ 10 pg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of 999 H. zea equivalents. The qPCR assay was able to detect ≥ 100 fg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of up to 99,999 H. zea equivalents. Both RPA and qPCR assays detected H. armigera in the crude DNA extracted in the field from a pool of one H. armigera moth and 999 H. zea moths. These newly developed molecular assays to detect H. armigera will contribute to large-scale surveillance programs of H. armigera.

Modified Phenol/Chloroform-Free DNA Isolation from Yeast and Other Fungi by Non-Enzymatic Salting Out Method.

The most commonly used fungal DNA extraction techniques require enzymatic digestion or hazardous chemicals. Here, we describe a quick non-enzymatic salt-out method that we developed for isolating high-quality DNA from various fungal species. DNA was extracted from four species of typical fungi, including Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, and Aspergillus flavus. Results that we obtained from agarose gel electrophoresis and spectrophotometry verified the integrity, high quality, and purity of the extracted DNAs. Also, PCR amplification and restriction enzyme digestion confirmed the efficiency of the extracted DNAs in molecular biology applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Yeast and fungal cultivation for DNA extraction Basic Protocol 2: Modified salting out DNA extraction protocol Support Protocol 1: PCR amplification of extracted genomes Support Protocol 2: Digestion with restriction enzyme.

Point-of-Care Diagnosis of Malaria Using a Simple, Purification-Free DNA Extraction Method Coupled with Loop-Mediated Isothermal Amplification-Lateral Flow.

We propose a protocol suitable for point-of-care diagnosis of malaria utilizing a simple and purification-free DNA extraction method with the combination of loop-mediated isothermal amplification assay and lateral flow (LAMP-LF). The multiplex LAMP-LF platform developed here can simultaneously detect Plasmodium knowlesi, P. vivax, P. falciparum, and Plasmodium genus (for P. malariae and P. ovale). Through the capillary effect, the results can be observed by the red band signal on the test and control lines within 5 min. The developed multiplex LAMP-LF was tested with 86 clinical blood samples on-site at Hospital Kapit, Sarawak, Malaysia. By using microscopy as the reference method, the multiplex LAMP-LF showed 100% sensitivity (95% confidence interval (CI): 91.4 to 100.00%) and 97.8% specificity (95% CI: 88.2% to 99.9%). The high sensitivity and specificity of multiplex LAMP-LF make it ideal for use as a point-of-care diagnostic tool. The simple and purification-free DNA extraction protocol can be employed as an alternative DNA extraction method for malaria diagnosis in resource-limited settings. By combining the simple DNA extraction protocol and multiplex LAMP-LF approach, we aim to develop a simple-to-handle and easy-to-read molecular diagnostic tool for malaria in both laboratory and on-site settings.

Sequence-Based Characterization of Microalgal Microbiomes: Impact of DNA Extraction Protocol on Yield and Community Composition

The bacterial communities associated with microalgae are vital for the growth and health of the host, and engineering algal microbiomes can enhance the fitness of the algae. Characterization of these microbiomes mostly relies on sequencing of DNA, which can be extracted with an array of protocols that potentially impact DNA quantity and quality and thus potentially affect subsequent analyses of microbiome composition. Here, we extracted DNA from Isochrysis galbana, Tetraselmis suecica, and Conticribra weissflogii microbiomes using four different protocols. DNA yield and quality was greatly impacted by the choice of extraction protocol, whereas microbiome composition determined by 16S rRNA gene amplicon sequencing was only impacted to a minor degree, with microalgal host species being the main determinant of microbiome composition. The I. galbana microbiome was dominated by the genus Alteromonas, whereas the microbiome associated with T. suecica was dominated by Marinobacteraceae and Rhodobacteraceae family members. While these two families were also prevalent in the microbiome associated with C. weissflogii, Flavobacteriaceae and Cryomorphaceae were also highly dominant. Phenol-chloroform extraction resulted in higher DNA quality and quantity compared to commercial kits; however, because they have other advantages such as high throughput and low toxicity, commercial kits can be employed to great benefit for the characterization of microalgal microbiomes. IMPORTANCE Microalgae are very important as primary producers in the ocean, but also as forthcoming sustainable producers of biotechnologically interesting compounds. Accordingly, the bacterial microbiomes associated with microalgae are attracting increasing attention due to their effects on the growth and health of microalgae. Since most members of these microbiomes cannot be cultured, knowledge about community composition is best obtained using sequencing-based methods. This study evaluates the impact of DNA extraction methods on DNA quantity and quality along with sequence-based characterization of the bacterial microbiome composition of three microalgae: Isochrysis galbana, Tetraselmis suecica, and Conticribra weissflogii.

Sample Collection, DNA Extraction, and Library Construction Protocols of the Human Microbiome Studies in the International Human Phenome Project.

The online version contains supplementary material available at 10.1007/s43657-023-00097-y.

Optimized method for dna extraction and PCR amplification in aroeira tree

Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of β-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

Quality analysis of genomic DNA and authentication of fisheries products based on distinct methods of DNA extraction.

Molecular genetic techniques are an effective monitoring tool, but high-quality DNA samples are usually required. In this study, we compared three different protocols of DNA extraction: NaCl (saline); phenol-chloroform and commercial kit (Promega)-from three biological tissues of five individuals of Lutjanus purpureus under two methods of storage. The evaluated items included DNA concentration and purity, processing time and cost, as well as the obtaining of functional sequences. The highest average values of DNA concentration were obtained using the saline procedure and the commercial kit. Pure DNA was only obtained using the saline protocol, evaluated by the ratio of 260/280. The saline and phenol-chloroform protocols were the least expensive methods. The commercial kit costs are counterbalanced by the short time required. The procedure based on phenol-chloroform presented the worst results regarding DNA yield and the time required to perform all steps. The saline and commercial kit protocols showed similar results concerning the amount and quality of extracted DNA. Therefore, the final choice should be based on the available financial resources and the available time for carrying out each procedure of DNA extraction.

Small‐organelle‐enriched metagenomics: An improved method for environmental DNA‐based identification of marine plankton

AbstractUnderstanding marine biodiversity is of paramount importance. Recently, we developed a polymerase chain reaction (PCR)‐free small‐organelle‐enriched metagenomics (SoEM) method with enhanced performance in species identification compared to multimarker DNA metabarcoding. However, this method has a problem of applicability for field surveys, for example, the need to filter large volumes of seawater. Here, we propose an improved version of the original SoEM method (SoEM pioneer version [SoEM‐pv]) for marine biodiversity assessment. There are three major contributions to this proposed SoEM method (SoEM v1.0): (1) presentation of PCR‐free protocols for seawater environmental DNA analysis; (2) provision of widely applicable optimal DNA extraction protocols that can be applied to small volumes (approximately 2 liters) of coastal waters; and (3) description of easy‐to‐use bioinformatics pipelines for analysis of the SoEM data. To validate the accuracy and sensitivity of SoEM v1.0, we evaluated the performance of the taxonomic capture capacity from seawater samples and found that, given the sample volume of 2 liters, (1) SoEM v1.0 had an approximately 5‐ to 10‐fold higher DNA extraction yield than the SoEM‐pv; (2) SoEM v1.0 had the highest taxonomic diversity compared to the other methods, 1.6‐fold higher than PCR‐based multi‐locus DNA metabarcoding and 1.2‐fold higher than shotgun metagenomic sequencing; and (3) the number of species identified did not increase proportionally with increasing sample size from 2 to 20 liters. We suggest that SoEM v1.0 is a promising and feasible method for marine eukaryotic diversity studies and expect it to be welcomed by biological oceanographers and coastal resource managers.

Optimizing insect metabarcoding using replicated mock communities.

1: Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundances, and biomass. 2: Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization?; and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? 3: We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalizes better across species for homogenates. 4: We conclude that a non-destructive, mild lysis approach shows the highest promise for presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.

Experimentation in teaching Genetics: DNA extraction from natural products

Brazil is a country that has a vast wealth of fruit species and using natural products as a tool for teaching has been reported as an interesting possibility for student learning. Objective: The objective of this work is to present a DNA extraction protocol using natural products in Science teaching. Methodology: The methodological proposal consists of the extraction of genetic material using accessible and low-cost materials, namely: a) passion fruit; b) Tomato; c) Papaya; d) Banana; e) Onion; f) Garlic, g) Grape and h) Kiwi. Initially, pectin can be obtained, a complex carbohydrate that appears as a whitish structure rich in bubbles that forms at the top of the alcoholic phase, and the DNA molecule, compacted together with histonic and non-histonic proteins, visualized macroscopically. Results: The extraction of DNA from natural products aims to facilitate and instigate the teaching and learning of Genetics, both in high school and elementary school students, serving as an integrating axis for discussion and problematization of themes related to the structure and function of acids nucleic. In addition, it can allow for interdisciplinarity with other disciplines and opens up the possibility for learning based on students' daily lives.

Bronchoalveolar lavage fluid polymerase chain reaction for invasive pulmonary aspergillosis among high-risk patients: a diagnostic meta-analysis

BackgroundPolymerase chain reaction (PCR) assays are perceived to facilitate the diagnosis of fungal infections. However, due to lack of standardization, the value of bronchoalveolar lavage (BAL) fluid PCR in diagnosis of invasive pulmonary aspergillosis (IPA) remains unclear.MethodsWe conducted a systematic meta-analysis to evaluate the accuracy of BAL fluid PCR in IPA diagnosis among high-risk patients. All studies involving patients at risk for IPA were included. The sensitivity, specificity, positive and negative likelihood ratios of BAL fluid PCR were summarized for diagnosis of proven/probable IPA, or proven IPA only. Potential heterogeneity was assessed by subgroup analyses and meta-regression.ResultsForty-one studies involving 5668 patients were analyzed. The summary sensitivity, specificity, positive and negative likelihood ratios of BAL fluid PCR for proven/probable IPA were 0.75 (95% CI = 0.67–0.81), 0.94 (95% CI = 0.90–0.96), 11.8 (95% CI = 7.7–18.1) and 0.27 (95% CI = 0.20–0.36), respectively. Whereas for proven IPA only, sensitivity and specificity were 0.91 (95% CI = 0.68–0.98) and 0.80 (95% CI = 0.74–0.85) in fourteen studies involving 2061 patients. Significant heterogeneity was present due to the underlying disease, antifungal treatment and differences in DNA extraction techniques and choice of PCR assay. Compared to patients with hematological malignancies (HM) and hematopoietic stem cell/solid organ transplantation (HSCT/SOT), sensitivity was higher in the population with disease such as chronic obstructive pulmonary disease, solid tumor, autoimmune disease with prolonged use of corticosteroids, etc. (0.88 vs. 0.68, P < 0.001), which was related to the concurrent use of antifungal prophylaxis among patients with HM and HSCT/SOT.ConclusionBAL fluid PCR is a useful diagnostic tool for IPA in immunocompromised patients and is also effective for diagnosing IPA in patients without HM and HSCT/SOT. Furthermore, standard protocols for DNA extraction and PCR assays should be focused on to improve the diagnostic accuracy.Trial registration PROSPERO, registration number CRD42021239028.

Automated and parallel microfluidic DNA extraction with integrated pneumatic microvalves/pumps and reusable open-channel columns.

A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.

Optimized DNA isolation from marine sponges for natural sampler DNA metabarcoding

AbstractMarine sponges have recently been recognized as natural samplers of environmental DNA (eDNA) due to their effective water filtration and their ubiquitous, sessile, and regenerative nature. However, laboratory workflows for metabarcoding of sponge tissue have not been optimized to ensure that these natural samplers achieve their full potential for community survey. We used a phased approach to investigate the influence of DNA isolation procedures on the biodiversity information recovered from sponges. In Phase 1, we compared three treatments of residual ethanol preservative in sponge tissue alongside five DNA extraction protocols. The results of Phase 1 informed which ethanol treatment and DNA extraction protocol should be used in Phase 2, where we assessed the effect of starting tissue mass on extraction success and whether homogenization of sponge tissue is required. Phase 1 results indicated that ethanol preservative may contain unique and/or additional biodiversity information to that present in sponge tissue, but blotting tissue dry generally recovered more taxa and generated more sequence reads from the wild sponge species. Tissue extraction protocols performed best in terms of DNA concentration, taxon richness, and proportional read counts, but the non‐commercial tissue protocol was selected for Phase 2 due to cost‐efficiency and greater recovery of target taxa. In Phase 2 overall, we found that homogenization may not be required for sponge tissue and more starting material does not necessarily improve taxon detection. These results combined provide an optimized DNA isolation procedure for sponges to enhance marine biodiversity assessment using natural sampler DNA metabarcoding.

An improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity.

Shorea robusta (Dipterocarpaceae), commonly known as Sal, is an economically and culturally important timber species, known to contain a wide spectrum of polyphenols, polysaccharides, and other secondary metabolites in the tissues, which can interfere with the extraction of high-quality genomic DNA. In order to screen simple sequence repeat (SSR) markers and carry out other DNA-based analyses for this species in our laboratory, a high-throughput DNA extraction methodology was needed. Hence, we have optimized a simple, rapid, safe, and reliable high-throughput protocol for DNA extraction suitable for both fresh and dry leaves. The standardized protocol delivered good DNA yield of ∼1500 µg from 1 g of leaf tissue, with purity indicated by a 260 nm/280 nm absorbance ratio ranging from 1.70 to 1.91, which validated the suitability of extracted DNA and revealed reduced levels of contaminants. Additionally, the protocol that we developed was found to be suitable for polymerase chain reaction (PCR) amplification using microsatellite markers. Genome-wide characterization with SSR markers has been established in S. robusta, which further validates the protocol and its usefulness in DNA-based studies across the genus and/or family.

Revealing the Tick Microbiome: Insights into Midgut and Salivary Gland Microbiota of Female Ixodes ricinus Ticks.

The ectoparasite Ixodes ricinus is an important vector for many tick-borne diseases (TBD) in the northern hemisphere, such as Lyme borreliosis, rickettsiosis, human granulocytic anaplasmosis, or tick-borne encephalitis virus. As climate change will lead to rising temperatures in the next years, we expect an increase in tick activity, tick population, and thus in the spread of TBD. Consequently, it has never been more critical to understand relationships within the microbial communities in ticks that might contribute to the tick's fitness and the occurrence of TBD. Therefore, we analyzed the microbiota in different tick tissues such as midgut, salivary glands, and residual tick material, as well as the microbiota in complete Ixodes ricinus ticks using 16S rRNA gene amplicon sequencing. By using a newly developed DNA extraction protocol for tick tissue samples and a self-designed mock community, we were able to detect endosymbionts and pathogens that have been described in the literature previously. Further, this study displayed the usefulness of including a mock community during bioinformatic analysis to identify essential bacteria within the tick.

Comparison of DNA extraction methods on CITES-listed timber species and application in species authentication of commercial products using DNA barcoding

Quality and quantity of DNA extracted from wood is important for molecular identification of wood species, which can serve for conservation of wood species and law enforcement to combat illegal wood trading. Rosewood (Dalbergia and Pterocarpus) and agarwood (Aquilaria) are the most commonly found hardwood in timber seizure incidents. To monitor international trade of timber and commercial wood products and to protect these endangered wood species from further population decline, in this study, we have compared three extraction protocols for DNA extraction from 12 samples of rosewood and agarwood timber logs, and later applied the best DNA extraction protocol on 10 commercial wood products claimed to be rosewood and agarwood. We also demonstrated the applicability of DNA mini-barcoding with multi-loci combination with reference library for identifying the species of timber and commercial wood products. We found that a silica column-based method with guanidine thiocyanate-containing binding buffer served the best in DNA extraction from different parts of wood in all three genera with good quality and quantity. Single barcode region ITS2 or multi-loci combinations including ITS2 barcode region generally provide better discriminatory power for species identification for both rosewood and agarwood. All 10 products were identified to species-level using multi-loci combination. In terms of accuracy in labelling, 80% of them were labelled correctly. Our work has shown the feasibility of extracting good quality of DNA from authentic wood samples and processed wood products and identifying them to species level based on DNA barcoding technology.

The influence of storage conditions and total DNA extraction protocol on the results of molecular analysis of the European spruce bark beetle (Ips typographus L.)

One of the key steps of the molecular identification of bark beetles is obtaining a sufficient quantity of high-quality DNA extract. In this study, we investigated the influence of different storage procedures for Ips typographus (L.) specimens and various DNA extraction protocols on the quantity and quality of DNA intended for use in molecular diagnostics. Adult beetles were frozen at -20 °C, either dry or in ethanol. We tested four different protocols for DNA extraction. We compared the quantity of extracted DNA and assessed its quality with PCR and Sanger sequencing. Different storage protocols had no significant effect on the quantity of DNA extracted. However, freezing specimens in ethanol provided higher-quality DNA for molecular applications. Only two of the extraction protocols produced sequenceable amplicons, and the difference in the amount of extracted DNA between them was not significant. We propose the optimal combination of storing specimens in ethanol at -20°C and using the Nucleospin Insect DNA extraction kit from Macherey Nagel, enabling a timeefficient identification process.