Reference Protocol Research Articles

Evaluation of plasma ACTH stability using the Roche Elecsys immunoassay

BackgroundAdrenocorticotropic hormone (ACTH) has been reported to be labile in blood due to proteolytic degradation and stringent procedures are followed to prevent in vitro degradation after sample collection. ObjectiveThe purpose of this study was to examine the effect of time and temperature before and after separation of plasma from cells in the quantitation of plasma ACTH. MethodsOur current protocol includes sample collection in a pre-chilled tube, transport on ice and immediate centrifugation at 4 °C. These reference conditions were compared against sample processing in tubes and centrifuge set at room-temperature; using delayed centrifugation at 4 °C. ACTH stability was evaluated at ambient and refrigerated temperatures after collection and plasma separation using the reference protocol. Plasma samples were analyzed using the Roche Elecsys ACTH immunoassay. ResultsQuantification of ACTH was not impacted by the use of non-chilled tubes and centrifuge and up to a 4 h delay in separation of plasma from cells. Average percent differences in plasma ACTH concentration from time 0 was <10% up to 12 h at ambient temperature. Refrigeration of plasma did not preserve ACTH stability at 12 h and longer storage resulted in significant ACTH degradation at both ambient and refrigerated temperatures. ConclusionsAs supported by these data, previously recommended strict specimen collection and processing requirements are not necessary for measuring ACTH with the Roche Elecsys immunoassay.

Securing Delay-Sensitive CR-IoT Networking Under Jamming Attacks: Parallel Transmission and Batching Perspective

Cognitive radio (CR) is envisioned as an enabling technology for the Internet-of-Things (IoT) networking by providing spectrum opportunities to the huge number of communicating IoT devices (the so-called CR-IoT networking). Due to the nature of its wireless operating media, CR-IoT networks are vulnerable to jamming attacks. In CR-IoT networks with delay-sensitive applications, such attacks can incur longer packet transmission delay, resulting in received obsolete packets and service unavailability. Therefore, an important challenge in this domain is how to perform a secured channel assignment that can provide soft guarantees on the timeline of the packet delivery while satisfying a predefined Quality-of-Service (QoS) requirements. In this article, we propose a security-aware batch-based MAC protocol for delay-sensitive CR-IoT networks that attempts at improving the network performance by exploiting the parallel transmission capabilities of the CR-IoT devices while being jamming aware. Our protocol performs simultaneous assignment decisions for contending CR-IoT devices. Specifically, we formulate the channel assignment problem that attempts at maximizing the number of served CR-IoT users subject to user-rate demand, number of transceivers per CR-IoT device, and delay (packet invalidity) constraints as an optimization problem. This problem is shown to be a binary linear programming problem, which is, in general, NP-hard. Hence, we use a sequential fixing linear programming technique, which provides a near-optimal solution. To implement our channel assignment in a distributed manner, we adopt an access window (AW)-based access protocol that allows the users to exchange their control information and conduct the spectrum assignment. Compared to reference protocols, simulation results show that our protocol significantly improves spectrum efficiency and CR-IoT network performance.

Accelerated MP2RAGE imaging using Cartesian phyllotaxis readout and compressed sensing reconstruction.

MP2RAGE T1 -weighted imaging has been shown to be beneficial for various applications, mainly because of its good grey-white matter contrast, its B1 -robustness and ability to derive T1 maps. Even using parallel imaging, the method requires long acquisition times, especially at high resolution. This work aims at accelerating MP2RAGE imaging using compressed sensing. A pseudo-phyllotactic Cartesian MP2RAGE readout was implemented allowing for flexible reordering and undersampling factors. The sampling pattern was first optimized based on fully sampled data and a compressed sensing reconstruction. Changes in contrast ratios, automated brain segmentation results, and quantitative T1 values were used for benchmarking. In vivo undersampled data from eleven healthy subjects were then acquired using a 4-fold acceleration with the optimized sampling pattern. The resulting images were compared to the standard parallel imaging MP2RAGE protocol by visual inspection and using the above quality metrics. The application of incoherent undersampling and iterative compressed sensing reconstruction on MP2RAGE acquisitions allows for a 57% time reduction (corresponding to 4-fold undersampling with maintained reference lines, TA = 3:35 minutes) compared to the reference protocol using parallel imaging (GRAPPAx3 acceleration, TA = 8:22 minutes) while obtaining images with similar image quality, morphometric (volume differences = [0.07 ± 1.2-3.8 ± 1.9]%) and T1 -mapping outcomes (T1 error = [6 ± 5.1-37 ± 12.3] ms depending on the different structures). A whole-brain MP2RAGE acquisition is feasible with compressed sensing in less than 4 minutes without appreciably compromising image quality.

Sampling and PCR method for detecting pathogenic Fusarium oxysporum strains in onion harvest.

Fusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onion-pathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTATM filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTATM technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the post-harvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion.

Multidisciplinary health care in cases of childhood suicidal ideation: operational and organizational limits.

to know the health care process performed by the multidisciplinary team in cases of suicidal ideation for children and adolescents in Primary and Secondary Care. a descriptive study with a qualitative approach carried out with 12 professionals from ESFs, EACS and CAPS II in the city of Santarém. The software IRAMUTEQ 0.7 alpha 2 was used to build the similarity tree and analyze speech content. the word "no" is present prominently in the interviewees' speeches about experiences and strategies for coping with suicide, revealing an absence of individual preparation and structure in the segments of SUS Primary and Secondary Care. Final considerations: health team assistance finds operational and organizational limits during the implantation of intervention strategies and coping with the factors that trigger child and youth suicide. It is important to make specific resources feasible, to organize reference protocols and support programs for patients and families.

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.

Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.ml−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

Three-dimensional simultaneous brain T1 , T2 , and ADC mapping with MR Multitasking.

To develop a simultaneous T1 , T2 , and ADC mapping method that provides co-registered, distortion-free images and enables multiparametric quantification of 3D brain coverage in a clinically feasible scan time with the MR Multitasking framework. The T1 /T2 /diffusionweighting was generated by a series of T2 preparations and diffusion preparations. The underlying multidimensional image containing 3 spatial dimensions, 1 T1 weighting dimension, 1 T2 -preparation duration dimension, 1 b-value dimension, and 1 diffusion direction dimension was modeled as a 5-way low-rank tensor. A separate real-time low-rank model incorporating time-resolved phase correction was also used to compensate for both inter-shot and intra-shot phase inconsistency induced by physiological motion. The proposed method was validated on both phantom and 16 healthy subjects. The quantification of T1 /T2 /ADC was evaluated for each case. Three post-surgery brain tumor patients were scanned for demonstration of clinical feasibility. Multitasking T1 /T2 /ADC maps were perfectly co-registered and free from image distortion. Phantom studies showed substantial quantitative agreement ( ) with reference protocols for T1 /T2 /ADC. In vivo studies showed nonsignificant T1 (P = .248), T2 (P = .97), ADC (P = .328) differences among the frontal, parietal, and occipital regions. Although Multitasking showed significant differences of T1 (P = .03), T2 (P < .001), and ADC (P = .001) biases against the references, the mean bias estimates were small (ΔT1 % < 5%, ΔT2 % < 7%, ΔADC% < 5%), with all intraclass correlation coefficients greater than 0.82 indicating "excellent" agreement. Patient studies showed that Multitasking T1 /T2 /ADC maps were consistent with the clinical qualitative images. The Multitasking approach simultaneously quantifies T1 /T2 /ADC with substantial agreement with the references and is promising for clinical applications.

Impact of b-Value Sampling Scheme on Brain IVIM Parameter Estimation in Healthy Subjects.

Purpose:Intravoxel incoherent motion (IVIM) analysis has attracted the interest of the clinical community due to its close relationship with microperfusion. Nevertheless, there is no clear reference protocol for its implementation; one of the questions being which b-value distribution to use. This study aimed to stress the importance of the sampling scheme and to show that an optimized b-value distribution decreases the variance associated with IVIM parameters in the brain with respect to a regular distribution in healthy volunteers.Methods:Ten volunteers were included in this study; images were acquired on a 1.5T MR scanner. Two distributions of 16 b-values were used: one considered ‘regular’ due to its close association with that used in other studies, and the other considered ‘optimized’ according to previous studies. IVIM parameters were adjusted according to the bi-exponential model, using two-step method. Analysis was undertaken in ROI defined using in the Automated Anatomical Labeling atlas, and parameters distributions were compared in a total of 832 ROI.Results:Maps with fewer speckles were obtained with the ‘optimized’ distribution. Coefficients of variation did not change significantly for the estimation of the diffusion coefficient D but decreased by approximately 39% for the pseudo-diffusion coefficient estimation and by 21% for the perfusion fraction. Distributions of adjusted parameters were found significantly different in 50% of the cases for the perfusion fraction, in 80% of the cases for the pseudo-diffusion coefficient and 17% of the cases for the diffusion coefficient. Observations across brain areas show that the range of average values for IVIM parameters is smaller in the ‘optimized’ case.Conclusion:Using an optimized distribution, data are sampled in a way that the IVIM signal decay is better described and less variance is obtained in the fitted parameters. The increased precision gained could help to detect small variations in IVIM parameters.

Assessment of potential reduction in multidetector computed tomography doses using FBP and SAFIRE for detection and measurement of the position of the inferior alveolar canal.

The objective was to identify the lowest doses required to detect and measure the position of the inferior alveolar canal (IAC) on multidetector computed tomography (MDCT) images using filtered backprojection (FBP) and sinogram-affirmed iterative reconstructions (SAFIRE) 3 and SAFIRE 5. Four cadaveric mandibles were imaged using a reference protocol with standard dose and FBP and 3 ultra-low-dose protocols (LD1-LD3), using an MDCT scanner. All test examinations were reconstructed with FBP, SAFIRE 3, and SAFIRE 5. Subjective visibility of the IAC in the images and digital measurements of the height of the ridge above the IAC were recorded from test images and compared with those from the reference image using one-sample t tests, Bland-Altman plots, and linear regression. Subjective visibility comparable to the standard protocol was obtained with an 84.6% dose reduction using the LD2 protocol. No statistically significant difference was found between the height measurements from the reference protocol and any of the LD1 and LD2 protocols. The t tests indicated a significant difference between the measurements from the reference and all LD3 test protocols. SAFIRE did not have an advantage over FBP images. Significant dose reduction from the reference dose can allow adequate detection and measurements of the IAC.

Development of highly efficient protocols for extraction and amplification of cytomegalovirus DNA from dried blood spots for detection and genotyping of polymorphic immunomodulatory genes.

Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection.

A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples

Documenting the extent of soil contamination by Toxoplasma gondii oocysts is a key issue to prevent the worldwide infection caused by this protozoan. Our aim was to improve the practicability and sensitivity of a low-cost method to detect T. gondii DNA in soil samples developed a few years ago. Various parameters of the reference protocol were modified to determine their effect on the detection of T. gondii DNA in soil samples (“natural soil” and “sand”) spiked with oocysts. We tested i) filtration using stomacher bags, ii) Tween 80, Tween 20, SDS and Triton X100 as dispersion solutions, iii) sucrose solution, zinc chloride solution, Optiprep and Percoll as density gradients, iv) freeze/thaw versus mechanical grinding as lysis methods, and v) Qiagen versus Fastprep as extraction kits The optimized protocol is quicker and easier to use than the previous one, and includes the following items: 0.1% Tween80/PBS for dispersion, sucrose solution for flotation, mechanical grinding, and FastDNA spin kit for extraction. It accurately detects T. gondii DNA in both fresh and frozen soil samples and displays a detection limit below 1 oocyst/g of fresh soil.

Abstract 4116: Immune cell checkpoint profiling of solid tumors by multiplex immunofluorescence

Abstract Immune checkpoint proteins are important regulators in self-tolerance. However, these same molecules also allow cancer cells to evade immune destruction. Checkpoint inhibitor (CKI) blockade therapies that aim at restoring antitumoral immunity have resulted in long-lasting remission in patients with cancers that were previously seen as incurable. However, only a fraction of the patients respond to these treatments. Research is currently focusing on identifying biomarkers that can predict a response to CKI therapies and stratify patient populations. As it is likely that several CKI need to be targeted at once to restore immune responses, numerous clinical trials associating more than one CKI are under evaluation. We have developed a multiplex immunohistochemistry panel, validated by in-depth image analysis, for immune cell checkpoint profiling. The base of the panel consists of CD3/CD8/PD-1/PD-L1 with the capability of adding additional targets including other immune checkpoint (e.g. TIGIT, SIRPα, CTLA-4) or tumor markers. Here we present our validation assay which uses the base panel and the ability to interchange the additional targets. Assay conditions for the individual biomarkers, including antigen retrieval, antibody concentration, panel position, and efficiency of stripping, are optimized. Simplex protocols are analyzed to ensure accuracy and specificity of the staining, success of stripping, and a balance of signal intensities. Validation includes the comparison of staining (% positive cells or % positive area) between a reference protocol and the position of interest. Multiplex optimization consists of staining serial, single stained slides of the individual biomarkers and the multiplex slide in parallel. The staining concordance (% positive cells) is evaluated with imaging software. Repeatability and robustness of the panels are validated by image analysis. This technique gives clinicians the potential to determine candidate patients for CKI single or double therapies and their progress during treatment. The established, rigorous workflow that we have developed allows our customers to adapt the panels to their needs and to have the highest confidence in the consequent images and results related to the profiling of immune cell checkpoints in solid tumors. Citation Format: Amanda Finan, Nicolas Goulange, Manon Motte, Maroua Tliba, Alexandra Mace, Jean-Philippe Coton, Domenico Lazzaro, Renaud Burrer. Immune cell checkpoint profiling of solid tumors by multiplex immunofluorescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4116.

First experiences of a low-dose protocol for CT-guided musculoskeletal biopsies combining different radiation dose reduction techniques.

The use of computed tomography (CT) for image guidance during biopsies is a powerful approach. The method is, however, often associated with a significant level of radiation exposure to the patient and operator. To investigate if a low-dose protocol for CT-guided musculoskeletal (MSK) biopsies, including a combination of different radiation dose (RD) techniques, is feasible in a clinical setting. Fifty-seven patients underwent CT-guided fine-needle aspiration cytology (FNAC) utilizing the low-dose protocol (group A). A similar number of patients underwent CT-guided FNAC using the reference protocol (group B). Between-group comparisons comprised radiation dose, success rate, image quality parameters, and workflow. In group A, the mean total dose-length product (DLP) was 41.2 ± 2.9 mGy*cm, which was statistically significantly lower than of group B (257.4 ± 22.0 mGy*cm), corresponding to a mean dose reduction of 84% ( P<0.001). The mean CTDIvol for the control scans were 1.88 ± 0.09 mGy and 13.16 ± 0.40 mGy for groups A and B, respectively ( P < 0.001). The success rate in group A was 91.2% and 87.9% in group B ( P = 0.56). No negative effect on image-quality parameters, time of FNAC, and number of control scans were found. We successfully developed a low-dose protocol for CT-guided MSK biopsies that maintains diagnostic accuracy and image quality at a fraction of the RD compared to the reference biopsy protocol at our clinic.

Isomeric and Conformational Analysis of Small Drug and Drug-Like Molecules by Ion Mobility-Mass Spectrometry (IM-MS).

This chapter provides a broad overview of ion mobility-mass spectrometry (IM-MS) and its applications in separation science, with a focus on pharmaceutical applications. A general overview of fundamental ion mobility (IM) theory is provided with descriptions of several contemporary instrument platforms which are available commercially (i.e., drift tube and traveling wave IM). Recent applications of IM-MS toward the evaluation of structural isomers are highlighted and placed in the context of both a separation and characterization perspective. We conclude this chapter with a guided reference protocol for obtaining routine IM-MS spectra on a commercially available uniform-field IM-MS.

Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL-1 Cas9 mRNA and 25ngμL-1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.

Development and Standardization of Narayana Churna—A Polyherbal Ayurvedic Formulation

Aim: The present study aims to develop pharmacognostical standards, standard operating procedure (SOP), and analytical profiling including physicochemical analysis of Narayana Churna, a polyherbal Ayurvedic formulation. Materials and methods: The pharmacognostical (macroscopy, microscopy and powder drug analysis), thin layer chromatography (TLC), and quantitative physicochemical analysis including loss on drying, alcohol and water-soluble extractive values, total and acid-insoluble ash, and pH value were performed as per the standard procedures described in the Ayurvedic Pharmacopoeia of India (API). The microbial limit, aflatoxins, heavy metals and pesticide residues were also analyzed. Results and discussion: Narayana Churna is of grayish-brown color with a slightly pungent taste. The powder microscopy revealed the presence of pentagonal/hexagonal/polygonal cork cells, polygonal pale green endosperm, oval/polygonal/irregular shaped stone cells, elongated and flat-shaped bordered pitted vessels, pitted tracheids, annular vessels, unicellular covering trichomes, prismatic crystals of calcium oxalate, starch grains and oil globules. The TLC fingerprint was developed using toluene:ethyl acetate:formic acid (6:4:1) as the solvent system. The standardized limits of the physicochemical parameters, microbial count, aflatoxins, heavy metals and pesticide residues were also laid down. Conclusion: This is the first ever attempt made in order to develop the SOP and standardized parameters of Narayana Churna. Thus, the present study would be useful as the standardized reference protocol for the identification and standardization of this formulation.

Development and Standardization of Chaturbhadra Kvath Churna

Aim: The present study aims to develop pharmacognostical standards, the standard operating procedure (SOP), and analytical profiling including the physicochemical analysis of Chaturbhadra Kvath Churna, an Ayurvedic formulation. Materials and methods: The pharmacognostical (macroscopy, microscopy, and powder drug analysis), thin-layer chromatography (TLC), quantitative physicochemical analysis including loss on drying, alcohol- and water-soluble extractive values, total acid-insoluble ash and pH value were performed as per the standard procedures described in the Ayurvedic Pharmacopoeia of India (API). The microbial limit, aflatoxins, heavy metals, and pesticide residues were also determined. Results and discussion: Chaturbhadra Kvath Churna is greyish-brown color with slightly pungent taste. The powder microscopy revealed the presence of hexagonal thin-walled/polygonal/wavy-walled polygonal/stratified cork cells, crystalloid fiber of phloem, unicellular warty trichomes, bordered-pitted/annular/spiral vessels, nonlignified spindle-shaped fibers, stone cells, brown matter, prism-shaped calcium oxalate crystals, volatile oils, and free and compound types of starch grains. The TLC fingerprint was developed using hexane: ethyl acetate: formic acid (4:5:1) as a solvent system. The standardized limits of the physicochemical parameters, microbial count, aflatoxins, heavy metals, and pesticide residues were also laid down. Conclusion: This is the first-ever attempt made in order to develop the SOP and standardized specifications of Chaturbhadra Kvath Churna. Thus, the present study would be useful as the standardized reference protocol for the identification and standardization of this formulation.

Towards By-Product Utilisation of Pea Hulls: Isolation and Quantification of Galacturonic Acid.

In order to evaluate by-products from food processing as alternative raw materials for pectin extraction, their amount of galacturonic acid (GalA) has to be analysed as a marker for pectin content. In the present study, significant differences in GalA release using different digestion methods are shown for pea hulls, as an example of by-products with a high content of cellulose. Complete digestion of the fibre matrix was assumed for Saeman hydrolysis as a reference protocol. Significantly lower GalA release was achieved by a treatment with trifluoracetic acid (TFA). An alternative treatment with ethylenediaminetetraacetic acid (EDTA) at pH 11 followed by an enzymatic digestion at pH 4.5 using a combination of polygalacturonase (Vegazyme M) and cellulase (Celluclast 1.5L) resulted in a similar release of GalA compared to Seaman hydolysis. Pea hull samples, analysed by this alternative protocol, showed on average a GalA content of 11.2%. Therefore, pea hulls may serve as new raw material for pectin extraction.

Effects of extraction method and storage of dry tissue on marine lipids and fatty acids

Various protocols are currently used to study marine lipids, but there is a growing interest in working on dry samples that are easier to transport. However, reference protocols are still lacking for dry samples. In order to make recommendations on this use, lipid classes and fatty acids (FA) obtained from six analytical protocols using two different tissue states (dry vs wet) and three extraction methods (automat vs manual potter vs leaving the solvent to work on tissue) were compared. Three dry storage modes of tissue (freezer vs gas nitrogen vs dry room) during one and three months were also compared. These comparisons were made on seven marine species with different lipid profiles, including fishes, crustaceans and mollusks. Lipid classes and FA obtained from wet and dry tissues were similar, but they were affected by the extraction methods. Regardless of tissue state, “Leave to work” methods obtained the highest lipid quantities, followed by manual potter and automat methods (ca. 90% and 80% of “Leave to work” methods, respectively). Linear relationships allowed correction for lipid classes and FA concentrations obtained from different protocols. The repeatability of all protocols still needs to be improved, especially for fish species. Increasing the replicate number for each sample might be an indirect way to improve lipid quantification. Our results show that storing dry tissues in the freezer for more than one month was associated with a decrease in lipids, which is also observed for other storage methods. For qualitative studies of FA (expressed in %), a three-month storage of dry tissue in freezer did not affect the relative composition of species/tissues with a lipid content below 20% of dry weight.

Development of a method for direct extraction of viral RNA from bivalve molluscs.

A direct extraction procedure was developed to recover viral RNA from shellfish with improved efficiency in comparison to reference extraction method (ISO 15216). Without the need for specific equipment, this procedure offers an alternative for performing food safety controls and for risk assessment studies. Given the inclusion in this extraction method of several steps for the efficient removal of food components inhibiting PCR reaction, this approach could serve as a general scheme for the extraction of nucleic acids of other enteric viruses and/or from other food categories.