Efficient DNA Extraction Research Articles

A rapid quarantine approach for the pathogenic and invasive Phytophthora species associated with imported fruits in China.

Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor-saving techniques are necessary and anticipated. A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR-RFLP-CE) technology-based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR-RFLP-CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. We provide a quick, practical, and high-throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry.

Detection of extracellular antibiotic resistance genes in river water: Application of ultrafiltration-magnetic beads method

Antibiotic resistance genes (ARGs) are widespread contaminants that pose significant threats to public health. Rivers play a crucial role in the dissemination of ARGs within the aquatic environment. However, there are limitations in the current research on the differentiation of intracellular ARGs (iARGs) and extracellular ARGs (eARGs) in river water. In this study, we developed a method combining ultrafiltration and adsorption of silica-hydroxy magnetic beads for efficient extraction of extracellular DNA (eDNA) from river water. The conditions of adsorption, washing, desorption, and pretreatment were optimized to enhance eDNA recovery. By using only 90 mL of water sample, our method could collect sufficient eDNA for subsequent detection of eARGs through qPCR analysis. The eDNA recovery rate ranged from 51.4% to 69.8%. The occurrence of five prevalent ARGs (tetC, sulI, blaTEM, ermB, qnrS) as well as integrase gene intl1 were investigated in both iDNA and eDNA extracted from river water samples collected from two tributaries of the Pearl River. Our results revealed that the absolute abundance levels of eARGs ranged from 10−1 to 105 copies/mL, which were significantly higher than those observed for iARGs ranging from 10−1 to 104 copies/mL. Moreover, there was a significant difference in contamination profiles for ARGs between two tributaries. The ultrafiltration-magnetic beads method overcomes challenges associated with low efficiency extraction when working with water samples containing low nucleic acid concentrations. This approach provides an improved technique for extracting eARGs from river water while also generating valuable data supporting assessments related to eARG contamination in such environments.

Demonstration of potential DNA contamination introduced by laboratory consumables using Fluorescein

The development of increasingly efficient DNA extraction and profiling kits has increased the amount of allelic information obtained from trace DNA samples, but also inadvertently, increased the detection of DNA contamination. This study aimed to evaluate the potential of DNA transfer using fluorescein, fluorescent under an alternate light source, in the use of a range of forensically relevant DNA profiling consumables. An evaluation of two pre-lysis methods adopting three different sample tubes, some with deliberate seal damage, showed the PrepFiler™ Automated Forensic DNA Extraction Kit caused leakage and crusting when the rim of the PrepFiler™ LySep column was compromised, but no leakage was observed under the same conditions using the Investigator STAR Lyse&Prep kit. The AutoLys tube showed minimal leakage using the PrepFiler™ chemistry. A DNA extract tube with an external thread, similar to the AutoLys tube, showed no leakage after fridge or freezer storage. However, it highlighted that a centrifugal spin does not guarantee all the DNA will pool at the base of the tube. A comparison of adhesive plate sealing films to 8-well strip caps for sealing 96-well PCR plates showed the adhesive plate sealing films presented a lower risk of DNA transfer, largely due to the adhesion of dispersed liquid on the sticky surface of the film. Overall, this study highlighted a number of variables that may be considered in the development of more refined contamination minimisation protocols in respect to increased sensitivities of DNA profiling.

Efficiency comparison of DNA extraction kits for analysing the cockle gut bacteriome

Cockles play a vital ecological role and provide valuable ecosystem services globally. However, the performance, production, and safe consumption of cockles are significantly influenced by their gut-associated bacteriome. Accurate understanding of gut-bacteriome interactions, and surveillance of pathogenic bacteria loads in cockles, rely on efficient DNA extraction methods that yield high-quality and representative bacterial DNA. Despite this importance, reliable extraction methods for cockles are currently overlooked. Therefore, we evaluated the performance of five DNA extraction kits (E.Z.N.A.® Soil DNA; FastDNA® Spin; DNeasy PowerSoil Pro; QIAamp PowerFecal DNA; ZymoBIOMICS™DNA Miniprep) in terms of DNA quality, yield, bacterial community structure (analysed by using denaturating gradient gel electrophoresis; DGGE), and bacteriome composition (analysed by 16S rRNA gene sequencing) in Cerastoderma edule gut. The DNeasy kit provided the highest purity and quantity of bacterial DNA, while the PowerFecal and Zymo kits exhibited reduced extraction efficiency. DGGE profiles revealed significant variability between the tested kits (R = 0.512; mean P = 0.011), but the FastDNA kit under-represented the bacterial community in cockles’ gut. Based on alpha diversity, the DNeasy kit outperformed the others and successfully detected all abundant genera found with the alternative kits. Our findings indicate that the DNeasy kit is an efficient DNA extraction method, enabling a molecular representation of the gut-associated bacteriome in C. edule. These results contribute to the development of effective techniques for studying the cockle gut bacteriome and its ecological implications.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

A Novel Method of Extraction and Purification of Bacterial DNA for PCR Detection of MecA, 16SrRNA, VanB

Bacteria are the main pest of nosocomial infections in healthcare facilities around the world. The aim of this study is to modify and minimize the amount of time, while still having no influence on the extraction's purity of DNA the method of extracting the DNA molecule and compare it with other methods that take longer to extract and purify DNA. This study examined two processes for isolating DNA from Gram-positive bacterial species: a boiling method and an enzymatic method that took one hour. In addition, the collected DNA was subjected to spectrophotometry, agarose gel electrophoresis, and PCR for both quantitative and qualitative examination. The results presented in this work show that boiling method pretreatment facilitates efficient cell lysis and DNA extraction, leading to increased yields of isolated bacterial DNA. In addition, DNA suitability for PCR-based identification of mecA,16SrRNA, VanB to Gram positive S. aureus strains was analyzed. Our findings demonstrated that the DNA generated by this simple approach is low-cost, quick, and safe, and that the process may be utilized in PCR methods on a wide range of gram-positive and gram negative species, as well as in laboratories without the necessary materials, tools, or technology

Bacteriophage-assisted lysis and eluted genomic DNA-based detection of pathogenic bacterial contamination in food

Foodborne diseases represent a significant global health concern, with a substantial number of illnesses and fatalities reported annually owing to the consumption of contaminated food. Current methods for identifying foodborne pathogens cannot effectively distinguish live from dead bacteria or efficiently detect multiple targets. Therefore, we developed a selective detection method for three pathogenic bacteria in food samples that leverages bacteriophage-assisted live-cell lysis and exposed genomic DNA amplification. The antimicrobial activity and host specificity of these three bacteriophages were employed to confirm the target bacteria selectivity. Bacterial DNA purification using bacteriophage-assisted lysis demonstrated the superior activity of the selected phages in extracting DNA from Escherichia coli O157:H7 and Salmonella Typhimurium. The results from reactions involving the three strains, including Bacillus cereus, highlighted that a 1 h incubation period yielded the most efficient DNA extraction and purification. The polymerase chain reaction (PCR) products obtained were comparable in terms of concentration and purity with those generated using bacterial DNA prepared using a commercial kit. In the practical food sample application experiment, bacterial strains were successfully recovered and concentrated using a polyethersulfone filter, resulting in a concentration factor >3 for all samples. Furthermore, the concentration of purified bacterial DNA from contaminated kimchi cabbage exceeded 10 ng/μL. Notably, we could detect E. coli O157:H7, S. Typhimurium, and B. cereus spiked at 103 colony-forming units/mL using multiplex PCR. This innovative bacteriophage-based approach holds substantial promise for the selective and practical detection of live foodborne pathogens, thereby significantly advancing food safety standards.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1pg/µl while root gDNA remained amplifiable at 10pg/µl. Genomic DNA obtained was also suitable for sequencing. This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Magnetic composite microspheres with a controlled mesoporous shell for highly efficient DNA extraction and fragment screening.

Rapid extraction and screening of high-purity DNA fragments is an indispensable technology in advanced molecular biology. In this article, mesoporous magnetic composite microspheres (MSP@mTiO2) with tunable pore sizes were successfully fabricated for high-purity DNA extraction and fragment screening. Owing to the strong complexation ability of Ti ions with DNA phosphate groups and the high specific surface area of mesoporous microspheres, the MSP@mTiO2 microspheres possess excellent adsorption performance, where the saturated loading capacity of MSP@mTiO2 with a specific surface area of 122 m2 g-1 is as high as 575 μg mg-1 for a salmon sperm specimen. ITC experiments demonstrated that DNA adsorption on MSP@mTiO2 microspheres is mainly driven by entropy, which gives us more potential ways to regulate the balance of adsorption and desorption. Meanwhile, the mesoporous MSP@mTiO2 microspheres exhibit a much higher extraction efficiency compared with non-porous MSP@TiO2 for whole genome DNA from Arabidopsis thaliana plants. Interestingly, DNA fragments with different lengths could be screened by simply regulating the pore size of MSP@mTiO2 or the concentration of Na3PO4 in the eluent. A small pore size and low phosphate concentration are advantageous for the extraction of short-stranded DNA fragments, and DNA fragments (≤1000 bp) can be efficiently extracted when the mesopore size of MSP@mTiO2 is lower than 7.6 nm. The extraction results from the mesoporous composite microspheres provide new promising insights into the purification and screening of DNA from complex biological samples.

Spore-DNA localization and extraction efficiencies of Bacillus subtilis for accurate results in quantitative real-time polymerase chain reaction.

Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reaction（qPCR）. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNA（tDNA）extracted from spore cells and intracellular（iDNA）and extracellular DNA（eDNA）extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xx（PMAxx）before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following: 1）amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2）lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.

Rapid and efficient DNA extraction method from high oily content seeds:

Ricinus communis seeds harbor high oil and polyphenolics contents that hinder DNA extraction. Here, a rapid and efficient protocol for isolating total DNA from R. communis seeds was developed. The current method implies the use of repeated cycles of freeze/heat shock for the seed tissue to lyse the cells. DNA isolated with this protocol was successfully used as a template for PCR amplification of the internal transcribed spacer (ITS) region of the rRNA encoding gene that widely used for molecular identification of different plant species. As far to our knowledge, this study is the first one that report the efficient use of freeze/heat shock repeated cycles for isolation of a high-quality DNA from plant cells. The current protocol would support the subsequent analysis for seed lot purity analysis.

A portable, integrated microfluidics for rapid and sensitive diagnosis of Streptococcus agalactiae in resource-limited environments

Streptococcus agalactiae (Group B Streptococcus, GBS) has been the leading cause of infections in newborns. Rapid and accurate diagnosis of GBS in pregnant women is a deterministic strategy to prevent newborn infection. Conventional detection methods based on nucleic acid amplification assay have been applied in GBS diagnosis in central laboratories, with demonstrated high sensitivity. However, their heavy dependence on instrumentation and trained technicians forms remarkable obstacles to GBS detection in wide scenarios, including self-testing, and bedside-/community-screening. Furthermore, the structures of GBS bring about extra challenges to the nucleic acid extraction and purification. Novel GBS diagnosis platforms integrating sample processing, amplification, and read-out, are highly desired in clinical. Here, we report a portable, integrated microfluidics that enables rapid extraction of DNA from sampling swabs (<10 min), power-free DNA amplification (<30 min), and simple read-out in GBS detection. The platform works without an external pump, achieving rapid and highly efficient DNA extraction from clinical samples, with a significantly reduced time from 6 h to less than 50 min. Systematic clinical tests based on 47 patient samples validated the high performance of the platform, highlighted with a low limit of detection (LOD, 103 copies/ml), high sensitivity (100%), and specificity (100%). Head-to-head comparisons showed that the device improved the LOD by an order of magnitude than the traditional PCR method, showing a simple yet powerful POCT platform for home-/community-based testing towards GBS (and other pathogens) prevention in remote areas.

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

Comparative Study of Genomic DNA Extraction Methods for Common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is one of the significant grains used in the human diet, accounting for half of all grain legumes consumed globally. To enhance production, conventional breeding and molecular approaches have been used so far. An efficient and rapid genomic DNA extraction method is required for these molecular approaches. The aim of this study was to compare and optimize an efficient and rapid DNA extraction protocol for common bean. Modified cetyl trimethylammonium bromide (CTAB) and potassium chloride (KCL) extraction methods were used. The mean DNA yield per nanoliter was 209 µg from modified CTAB and 150.3 µg from the KCL method. The concentration of gDNA was significantly (P&lt; 0.05) higher for the KCL method, which was 5.01 µg/µl and 2.09 µg/µl for the CTAB method. The obtained DNA was also pure, with an absorbance ratio at 260 nm to an absorbance of 280 nm (A260/280) of 1.75-2.23 for the KCL method and 1.86-2.09 for the modified CTAB method. Gel electrophoresis separation was used to evaluate the quality of the total DNA extracted by the present protocols. The results showed that intense bands close to the gel wells were obtained from both extraction methods. DNA isolated with the two methods was successfully used for PCR-based downstream analysis, which includes random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. In this study, it took approximately 150 minutes for KCL and 240 minutes for the CTAB for whole process. In contrast to the CTAB method, the KCL method uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. Therefore, it is more convenient and economical than the traditional technique.

Immobilization of Multivalent Titanium Cations on Magnetic Composite Microspheres for Highly Efficient DNA Extraction and Amplification.

Magnetic-assisted DNA testing technology has attracted much attention in genetics, clinical diagnostics, environmental microbiology, and molecular biology. However, achieving satisfying DNA adsorption and desorption efficiency in real samples is still a big challenge. In this paper, a new kind of high-quality magnetic composite microsphere of MM@PGMA-PA-Ti4+ was designed and prepared for DNA extraction and detection based on the strong interaction of Ti4+ and phosphate groups. By taking the advantages of high magnetic susceptibility and high Ti4+ content, the MM@PGMA-PA-Ti4+ microspheres possessed remarkable extraction capacity for mimic biological samples (salmon sperm specimens) with saturated loadings up to 533.0 mg/g. When the DNA feeding amount was 100 μg and the MM@PGMA-PA-Ti4+ dosage was 1 mg, the adsorption and desorption efficiencies were 80 and 90%, respectively. The kinetic and equilibrium extraction data were found to fit well with the pseudo-second-order model and Freundlich isotherm model. Furthermore, the MM@PGMA-PA-Ti4+ microspheres were successfully employed for DNA extraction from mouse epithelial-like fibroblasts. The extraction ability (84 ± 4 μg/mg) and DNA purity were superior to the comparative commercial spin kits, as evaluated by electrophoresis assays and qPCR analysis. The experimental results suggest that the MM@PGMA-PA-Ti4+ microspheres possess great potential as an adsorbent for DNA purification from complex biological samples.

An efficient extraction method for short single-stranded DNA from agarose gels in aptamer screening

With the rapid advancements in aptamer screening, the efficient extraction of short single-stranded DNA (ssDNA) from agarose gel has become a new requirement. However, the currently available products are primarily designed for double-stranded DNA (dsDNA) and exhibit limited efficacy when applied to the extraction of short ssDNA. In this study, we successfully developed a novel method based on amino-modified silica-coated magnetic particles (ASMPs) for the extraction of short ssDNA from agarose gel. The gel slices containing short ssDNA were subjected to centrifugation in a spin column/centrifugation tube assembly with silica wool, followed by the adsorption using ASMPs. Subsequently, reagents containing phosphate groups were employed to desorb ssDNA from the surface of ASMPs. Through optimization of each step, we realized remarkable efficiency in the extraction of short ssDNA. To assess the efficacy of our method, we utilized it in aptamer screening. The results demonstrated that our method outperformed three commercially available DNA gel extraction products (Q-kit, S-kit, and V-kit). The relative recovery rates of all methods were as follows: M-dNTP (100.00 %) > M-BB (63.38 %) > Q-kit (46.64 %) > S-kit (15.98 %) > V-kit (0.38 %). The results strongly suggest that the developed method holds promise for short ssDNA extraction from agarose gel.

Miniaturized Microscale Solid-Phase Extraction-Based Module for Highly Efficient DNA Extraction and Purification from Grapevine Samples.

Several developments over the last few years are being directed toward improving DNA-based analysis to simplify, miniaturize, and reduce the time and cost of analysis, with the objective to allow its use in decentralized settings. One of the most interesting fields is DNA extraction and purification, a key step for ensuring good analytical performance. In this sense, microscale solid phase extraction (μSPE) offers paramount advantages for an improved DNA yield. In this work, we have developed a miniaturized module for DNA purification based on μSPE using a borosilicate glass microfiber filter as the solid phase. We also established a protocol for highly efficient DNA purification from vegetable samples, including leaves and grapes from four different varieties from the PDO Douro and two varieties from the Minho wine regions. The protocol demonstrated excellent performance when compared with a commercial kit with a DNA recovery yield of around 50%.

An Improved Method for Efficient DNA Extraction from Grapevine

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops worldwide which has been the subject of extensive genetic studies including gene mapping, genetic transformation, and DNA fingerprinting. Grapevines are rich in polysaccharides, polyphenolic compounds, and various secondary metabolites, many of which have significant importance in food, agrochemical, and pharmaceutical industries. While metabolites are one of the indicators of quality of grapevines, the presence of them makes grapevine one of the most difficult plants to extract DNA from. These metabolites not only affect DNA extraction procedures but also downstream reactions such as restriction digestion and PCR. Development of new genotyping techniques based on sequencing such as genotyping by sequencing (GBS) requires high-quality DNA for digestion and sequencing. To date, several protocols have been developed for DNA extraction from grapevine. In this study, three different protocols with modifications were compared for DNA extraction performance from grapevine leaves from four different cultivars. Efficiencies of these methods were determined by extracted DNA’s quantity and quality. To confirm the suitability for GBS, extracted DNA was digested with restriction enzymes. Although all protocols were based on the traditional CTAB method, they resulted in different DNA yield and restriction digestion efficiency. The modified protocol including PVP-40 and ß-mercaptoethanol was found to be the most efficient method to obtain high quality and quantity grapevine DNA that is amenable to restriction digestion.

Quantitative Real-Time PCR for the Detection of Allergenic Species in Foods.

Food allergy is an increasing challenge to public health, with widespread global distribution. With no cure for this pathology, the food-allergic individuals are forced to adopt food eviction measurements, relying on label information to avoid consuming the offending foods. To safeguard these individuals, the analytical methods based on real-time PCR approaches are currently faced as excellent tools to verify labeling compliance, aiding industry and regulatory agencies to efficiently manage food allergen control programs. Therefore, this chapter intends to describe a protocol of real-time PCR to analyze allergenic food species. For method development, the main steps to be considered are (i) in silico sequence analysis and primer/hydrolysis probe design, (ii) preparation of calibrators (model foods containing the allergenic ingredient), (iii) efficient DNA extraction from complex food matrices, (iv) amplification by real-time PCR with hydrolysis probe (90-200bp) targeting a highly specific DNA region (allergen-encoding gene), (v) sequencing PCR products for identity confirmation, and (vi) validation and application to commercial foods. Herein, a real-time PCR approach for the detection and quantification of cashew nut as an allergenic food is described as an example protocol, including all the steps for method development and validation.

Identification of Heliothinae species (Lepidoptera: Noctuidae) in chickpeas in central Brazil

ABSTRACT Chickpea is a commercially exploited crop on a relatively new scale in Brazil, that is why, little information on the main pests is available. Mostly caterpillars showing Heliothinae morphological characteristics have emerged in production areas. In order to point out the species of these insects in chickpea fields produced in the Federal District and the surrounding areas, this study aimed to determine the morphological and molecular identification of these invertebrates in order to assist producers in pest management in a more sustainable way. Caterpillars were hand-collected directly from the plants in six sites and the moth collection was collected using light traps in four sites, monthly, in 2017 and 2018. The morphological identification was made through the adult insect genitalia and the subsequent molecular identification was performed using the cytochrome oxidase I enzyme with the quantitative PCR technique, which resulted in the development of an efficient and low-cost DNA extraction protocol to be used in samples under unfavorable storage conditions. Morphological and molecular studies showed the presence of Helicoverpa armigera, Helicoverpa zea, Chloridea virescens and Chloridea subflexa, with a predominance of Chloridea virescens in chickpea plants. Chloridea subflexa was verified for the first time feeding on chickpeas in Brazil.