Development Of Test Methods Research Articles

Evaluation of early African swine fever virus detection using CP204L gene encoding the p30 protein using quantitative polymerase chain reaction.

The African swine fever virus (ASFV), spanning 170-193 kb, contains over 200 proteins, including p72 and p30, which play crucial roles in the virus's entry and expression. This study investigated the capability of detecting ASFV early through the analysis of genes B646L and CP204L, encoding p72 and p30 antigen proteins, by employing ASFV, diagnosis, immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR), and IHC techniques. Samples were taken from both experimentally and field-infected pigs to evaluate the effectiveness of qPCR and IHC in detecting ASFV. Twenty-two infected pigs were necropsied at 3-, 5-, 7-, and 9-day post-infection to obtain the first set of samples, collecting anticoagulated blood and tissues each time. The thymus, spleen, and lymph nodes were processed by fixing in 10% formalin, paraffin-blocking, and undergoing IHC staining. Forty anticoagulated blood samples were collected from clinically infected sows at a pig farm for the second batch of samples. Based on the lowest Ct values, three blood samples were diluted fivefold for qPCR DNA testing, and their tissues were used for both qPCR and IHC analyses. At 1-day post-infection, p30-qPCR identified more ASFV-positive pigs and measured lower Ct values compared to p72-qPCR. At later time points, both methods showed similar levels of detection. ASFV was detected earlier and with lower Ct values in lymphoid tissues using p30-qPCR compared to p72-qPCR, particularly in the spleen and lymph nodes. In a field outbreak study, p30-qPCR demonstrated superior sensitivity and lower Ct values when detecting ASFV in blood samples compared to p72-qPCR. The early detection of the CP204L gene encoding p30 and its corresponding antigenic protein in ASFV diagnosis compared to the gene encoding p72 suggests that CP204L and p30 are promising candidates for the development of more effective antigen and antibody testing methods.

Guidelines for Manufacturing and Application of Organoids: Lung.

The objective of standard guideline for utilization of human lung organoids is to provide the basic guidelines required for the manufacture, culture, and quality control of the lung organoids for use in non-clinical efficacy and inhalation toxicity assessments of the respiratory system. As a first step towards the utilization of human lung organoids, the current guideline provides basic, minimal standards that can promote development of alternative testing methods, and can be referenced not only for research, clinical, or commercial uses, but also by experts and researchers at regulatory institutions when assessing safety and efficacy.

Development and evaluation of test methods for commercial spatial repellent products using the common fruit flies in laboratory and simulated-use scenarios

Fruit flies and other miniscule flying insects often cause a nuisance in the kitchen areas of our dwellings. To mitigate the invasion of indoor insect pests, behavioral modifications using spatial repellents containing natural products are emerging as alternatives to chemical insecticides especially around the kitchens and food storage areas. However, the absence of standardized testing methods for spatial repellency has undermined the reliability of these products. We introduced three distinct laboratory-scale repellency assays and their relevancy to simulated-use conditions. Out of eleven commercial products tested, five showed effectiveness in laboratory assays, and three of these were further proven effective in simulated-use tests as well. Another issue limiting the utility of commercially available spatial repellents was the lack of clear and comprehensive use instructions on product labels, potentially leading to ineffective applications. We observed that increasing the applied quantity improved the efficacy of one product that was initially determined to be ineffective in a simulated-use test. Based on our findings, we propose three strategies for regulations of spatial repellents: 1) consideration of mixture effects beyond single active ingredients especially using the natural products as their active ingredients, 2) the use of multiple testing systems, while acknowledging the limitations of laboratory assays in predicting real-world efficacy, and 3) ensuring product labels provide detailed and clear instructions for effective use.

Characterizing MEMS Switch Reliability for Cryogenic Applications such as Quantum Computing

Micro Electromechanical Systems (MEMS) switches offer many advantages over conventional larger switches. One potential application we are exploring is the use of commercial radio frequency (RF) MEMS switches for quantum computing applications. However, it is well-documented that cryogenic temperatures can cause mechanical reliability issues for MEMS. Furthermore, commercial RF MEMS switches are designed for room temperature operation, thus their reliability at cryogenic temperatures is unknown. Commercial MEMS switches are also packaged inside sealed housings, which prevent the use of optical methods for characterization and inspection. We are therefore developing test methods to evaluate the reliability of commercial RF MEMS switches at cryogenic temperatures. We describe our test methods and preliminary reliability test data from room temperature down to 55 K.

Development and evaluation of test methods for the detection and enumeration of opportunistic waterborne pathogens from the hospital environment

Many Gram-negative bacteria other than Pseudomonas aeruginosa have been implicated in waterborne outbreaks, but standardized laboratory detection methods for these organisms have not been established. This study aimed to establish laboratory testing methodologies for six waterborne pathogens: Acinetobacter spp., Burkholderia spp., Cupriavidus spp., Delftia acidovorans, Elizabethkingia spp. and Stenotrophomonas maltophilia. Water samples were spiked by UK Health Security Agency laboratories and sent to the Glasgow Royal Infirmary laboratory for analysis. Water samples were spiked with either a pure culture of target organism or the target organism in water containing normal background flora, to ensure that the methodology could identify organisms from a mixed culture. Volumes of 100mL were filtered under negative pressure on to culture media and incubated at 30°C and 37°C. The incubation time was 7 days, with plates read on days 2, 5 and 7. Further identification of colonies was undertaken using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar, chocolate bacitracin agar and pseudomonas selective agar. The optimal temperature for isolation was 30°C. The optimal incubation time was 5 days, and MALDI-TOF MS identified all test species reliably. The methodology described was able to detect the six tested waterborne pathogens reliably, and can be utilized by laboratories involved in testing water samples during outbreak investigations.

KoCVAM-led development of phototoxicity alternative test method using reconstructed human epidermis model (KeraSkin™)

Phototoxicity is an acute toxic reaction induced by topical skin exposure to photoreactive chemicals followed by exposure to environmental light and thus chemicals that absorb UV are recommended to be evaluated for phototoxic potential. There are currently three internationally harmonized alternative test methods for phototoxicity. One of them is the in vitro Phototoxicity: RhE Phototoxicity test method (OECD TG498).Korean center for the Validation of Alternative Methods (KoCVAM) developed an in vitro phototoxicity test method using a KeraSkin™ reconstructed human epidermis model (KeraSkin™ Phototoxicity Assay) as a ‘me-too’ test method of OECD TG498.For the development and optimization of KeraSkin™ Phototoxicity Assay, the following test chemicals were used: 6 proficiency chemicals in OECD TG498 (3 phototoxic and 3 non-phototoxic), 6 reference chemicals in OECD Performance Standard No. 356 (excluding the proficiency test chemicals, 3 phototoxic and 3 non-phototoxic) and 13 additional chemicals (7 phototoxic and 6 non-phototoxic). Based on the test results generated from the test chemicals above, the overall predictive capacity of KeraSkin™ Phototoxicity Assay was calculated. In particular, the assay exhibited 100 % accuracy, 100 % sensitivity, and 100 % specificity. Therefore, it fulfills the requirements to be included as a ‘me-too’ test method in OECD TG498.

A point-of-care testing assay for clonorchiasis using a EuNPs-CsTR1 fluorescent probe-based immunoassay.

Clonorchis sinensis is one of the most important fish-borne zoonotic parasitic worms in humans, and is distributed in several countries with more than 15 million people infected globally. However, the lack of a point-of-care testing (POCT) method is still the critical barrier to effectively prevent clonorchiasis. With the application of novel fluorescent nanomaterials, the development of on-site testing methods with high signal enhancement can provide a simple, precise and inexpensive tool for disease detection. In this study, Eu-(III) nanoparticles (EuNPs) were used as indicative probes, combined with C. sinensis tandem repeat sequence 1 (CSTR1) antigen to capture specific antibodies. Afterward, the complex binds to mouse anti-human IgG immobilized on the test line (T-line) producing a fluorescent signal under UV light. The EuNPs-fluorescent immunoassay (EuNPs-FIA) was successfully constructed, allowing sample detection within 10 min. It enabled both qualitative determination with the naked eye under UV light and quantitative detection by scanning the fluorescence intensity on the test line and control line (C-line). A total of 133 clinical human sera (74 negative, 59 clonorchiasis, confirmed by conventional Kato-Katz (KK) methods and PCR via testing fecal samples corresponding to each serum sample) were used in this study. For qualitative analysis, the cut-off value of fluorescence for positive serum was 31.57 by testing 74 known negative human samples. The assay had no cross-reaction with other 9 parasite-infected sera, and could recognize the mixed infection sera of C. sinensis and other parasites. The sensitivity and specificity of EuNPs-FIA were both 100% compared with KK smear method. Taking advantage of its high precision and user-friendly procedure, the established EuNPs-FIA provides a powerful tool for the diagnosis and epidemiological survey of clonorchiasis.

The Accomplishments of KoCVAM in the Development and Implementation of Alternative Methods in Korea.

The Korean Center for the Validation of Alternative Methods (KoCVAM), which promotes the Three Rs principles and the use of alternative methods in Korea, has been operating within the Toxicological Screening and Testing Division of the Ministry of Food and Drug Safety (MFDS) since 2009. KoCVAM has exchanged opinions and information on the development and validation of non-animal alternative test methods as part of the International Cooperation on Alternative Test Methods (ICATM), and provided input into draft OECD Test Guidelines (TGs). Several Korean laws (e.g. the Cosmetics Act) encourage the use of alternative test methods for chemical testing and assessment. To promote and support the use of alternative test methods in the country, KoCVAM has published information and provided training on the national guidelines, which are based on the OECD TGs. In addition, KoCVAM has held annual training workshops on alternative test methods, to help Korean research institutions (including GLP test facilities) to implement them. In addition, by helping to develop and validate alternative test methods that were adopted in OECD TG 442B, TG 492 and TG 439, KoCVAM has contributed to the enhanced competitiveness of Korean industry on the worldwide stage.

Examination of Analytical Method for Tris(2,3-dibromopropyl)phosphate and Bis(2,3-dibromopropyl)phosphate to Revise the Official Methods Based on "Act on the Control of Household Products Containing Harmful Substances"

In Japan, the use of frame retardants [tris(2,3-dibromopropyl)phosphate: TDBPP and bis(2,3-dibromopropyl)phosphate: BDBPP] in several household textile products is banned under the "Act on the Control of Household Products Containing Harmful Substances." As the official analytical methods for testing these substances have not been revised for over 42 years, several issues such as the using of harmful reagents, have been pointed out. Therefore, we developed a new method to revise the official method in our previous study. In this study, the validity of the developed test method is evaluated at six laboratories using two types of textile samples spiked with TDBPP and BDBPP at three concentrations (4, 8, and 20 µg/g). TDBPP and BDBPP are extracted under reflux using methanol containing hydrochloric acid. TDBPP is analyzed using GC-MS, and BDBPP is also analyzed using GC-MS after methylation with trimethylsilyl diazomethane. Although the accuracy (70-120%), repeatability (<10%), and reproducibility (<15%) of a few samples, mainly low concentration samples, are out of range, overall, the concentration level of detection limits of TDBPP and BDBPP (8 and 10 µg/g) in official analytical methods are quantifiable with sufficient precision using the proposed method. Furthermore, harmful reagents are not used in this method. Thus, the method validated in this study is effective as a revised method for the testing of TDBPP and BDBPP in household textile products.

Development of test methods for fire-retardant coatings of steel engineering structures during operation

Introduction. Prediction of the durability of flame-retardant coatings of steel engineering structures and preservation of their performance during operation remain important research directions. There is a lack of normative documents in the field of fire protection, regulating the process of testing of flame-retardant coatings during operation, as well as determination of their durability (service life).Aim. To develop test methods for determining the resistance of flame-retardant coatings of steel engineering structures exposed to climatic factors, preservation of their fireproof and anti-corrosion properties during operation.Materials and methods. Test specimens included 600 x 600 x 5 mm plates made of 08kp and 08ps sheet steel according to State Standard 16523-97 and State Standard 9045-93 with a flame-retardant agent applied on the front side.Results. Methods for testing thin-layer intumescent and structural flame-retardant coatings during operation are proposed. The methodology of accelerated climatic testing of specimens coated with thin-layer intumescent flame-retardant coatings (flame-retardant paints) corresponds to State Standard 9.401-2018. These coatings are inherently high-solid paint materials. A new methodology, sequence, and modes of testing are developed for structural flame-retardant coatings. The subsequent assessment of fireproof properties of coatings and their preservation is carried out by the methods of fire protection efficiency according to State Standard R 53295-99 and the methods of thermal analysis. These methods imply comparison of the characteristics of the initial flame-retardant coating and those obtained after sample aging. The preservation of fireproof properties by thin-layer intumescent and structural coatings is additionally evaluated by the intumescence coefficient and the change in thermal conductivity, respectively.Conclusions. Test methods for flame-retardant coatings of steel engineering structures during operation are developed. Threshold levels of changes in their properties are established. After accelerated climatic tests, fire protection efficiency should not decrease by no more than 20 %. For structural fire protection, an increase in thermal conductivity by no more than 5 % is permitted. For thin-layer coatings, the arithmetic mean value of the intumescence coefficient should not decrease by no more than 30 % of the initial value.Implications. The developed methods were used in the preparation of a draft national standard of the Russian Federation “Steel engineering structures with fireproof coatings. Test methods for anticorrosion properties and resistance to climatic factors during operation” to ensure regulatory fire safety requirements for these structures.

Development of Self-Passivating, High Strength Ferritic Alloys for CSP and TES Application

The addition of aluminum to ferritic stainless steels can result in self-passivation by the formation of a compact Al2O3 top layer, which exhibits significantly higher corrosion resistance to solar salt compared to a Cr2O3 surface layer. The development and qualification of realistic experimental methods for fatigue testing under superimposed salt corrosion attack will enable safe component design. Salt corrosion experiments were carried out at 600 °C with and without mechanical fatigue loading at a novel, self-passivating trial steel, using “solar salt” (60 wt.% NaNO3 and 40 wt.% KNO3). Cyclic salt corrosion tests at 600 °C under flowing synthetic air (without mechanical loading) showed that self-passivation to molten salt attack and mechanical strengthening by precipitation of fine Laves phase particles is possible in novel ferritic HiperFerSCR (Salt Corrosion Resistant) steel. A compact, continuous Al2O3 layer was formed on the surface of the model alloys with Al contents of 5 wt.% and higher. A distribution of fine, strengthening Laves phase precipitates was achieved in the metal matrix.

Weld Cracking Susceptibility of High-Cr Ni-Base Fe Alloys and Its Improvement—Development of Novel Test Method for Ductility-Dip Cracking and New Alloy

Weld Cracking Susceptibility of High-Cr Ni-Base Fe Alloys and Its Improvement—Development of Novel Test Method for Ductility-Dip Cracking and New Alloy

A Review of the Development of Glucocorticoid Testing Methods in Doping Control

A Review of the Development of Glucocorticoid Testing Methods in Doping Control

Development of optical-digital methods for non-destructive testing of aerospace thin-walled shell structures (Review)

Development of optical-digital methods for non-destructive testing of aerospace thin-walled shell structures (Review)

Newborn screening for lipid disorders.

Newborn screening is one of the most successful public health programs of the last century and offers unparalleled access to universal screening for a variety of metabolic and other disorders. Interest in development of newborn screening for lipid disorders has intensified in recent years. Screening newborns for lipid disorders has important implications for the health of the newborn as well as their relatives, and in the case of more common lipid disorders like familial hypercholesterolemia, could have important public health implications. Recent studies have demonstrated feasibility of measuring biomarkers for heterozygous familial hypercholesterolemia from newborn screening dried blood spot specimens. Another lipid disorder, cerebrotendinous xanthomatosis, is currently amenable to newborn screening utilizing currently available assays. New research in next-generation sequencing as a primary screen in newborns will also identify both common and rare lipid disorders in newborns. Historically, newborn screening for lipid disorders was not done for many reasons, but new research has developed testing methods that may successfully identify common and rare lipid disorders. This will impact the health of the newborn but could also impact family members and public health.

Experimental assessment of appropriate time for aggregate exposure at the surface of cement concrete pavement

ABSTRACT Exposed aggregate concrete (EAC) pavement is a commonly employed technology in Europe for the construction of highways. The technical challenges associated with pavement construction include achieving both a comfortable ride and the desired skid resistance, while ensuring the long-term concrete durability. Maintaining uniformity of concrete mix, precise dosing of retarding agents, optimal selection of brushing time, and ensuring adequate curing conditions are identified as critical factors for achieving the designed ride performance. This study is focused on determining the appropriate time for the brushing operation, conducted to expose aggregate grains at the surface of the pavement. Laboratory tests were performed on concrete mixes designed to replicate job mixes for the upper layer of a two-layer concrete pavement. Measurements of the mass of evaporated water from the cement paste, isothermal calorimetry tests, and modified Vicat tests were employed to predict the appropriate brushing time. The texture depth was determined using a laser profiler as a function of brushing time. Compressive strength, the rate of chloride ion migration, and scaling resistance were determined through tests conducted on specimens cut from exposed aggregate slabs. Results revealed the suitability of the developed test method for determining the appropriate time for brushing EAC pavements.

Etiological diagnostic methods and research progress of forest encephalitis

Forest encephalitis is a natural focal disease transmitted through the bite of hard ticks, and its pathogen is the tick-borne encephalitis virus from the Flaviviridae family. The mortality rate of forest encephalitis is relatively high, making laboratory testing significant in diagnosing this disease. This article elaborates on the etiological diagnostic methods and recent research progress in forest encephalitis. Laboratory tests for forest encephalitis mainly include routine examinations, serological tests, virus isolation, and molecular biological testing. The detection of serum-specific IgM antibodies against the forest encephalitis virus is of great importance for early diagnosis, and specific IgG antibodies serve as a "gold standard" for differentiation from other diseases. Techniques such as enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay for detecting specific IgM antibodies in serum and/or cerebrospinal fluid, the serum hemagglutination inhibition test or serum complement fixation test, and the double serum hemagglutination inhibition test or complement fixation test all contribute to the early diagnosis. The development of molecular testing methods is rapid, and techniques such as metabolomics, digital PCR, and matrix metalloproteinases are also applied in the early diagnosis of forest encephalitis.

Evaluation of the cardiotoxicity potential of bisphenol analogues in human induced pluripotent stem cells derived cardiomyocytes

The importance of evaluating the cardiotoxicity potential of common chemicals as well as new drugs is increasing as a result of the development of animal alternative test methods using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Bisphenol A (BPA), which is used as a main material in plastics, is known as an endocrine-disrupting chemical, and recently reported to cause cardiotoxicity through inhibition of ion channels in CMs even with acute exposure. Accordingly, the need for the development of alternatives to BPA has been highlighted, and structural analogues including bisphenol AF, C, E, F, and S have been developed. However, cardiotoxicity data for analogues of bisphenol are not well known. In this study, in order to evaluate the cardiotoxicity potential of analogues, including BPA, a survival test of hiPSC-CMs and a dual-cardiotoxicity evaluation based on a multi-electrode array were performed. Acute exposure to all bisphenol analogues did not affect survival rate, but spike amplitude, beat period, and field potential duration were decreased in a dose-dependent manner in most of the bisphenols except bisphenol S. In addition, bisphenols, except for bisphenol S, reduced the contractile force of hiPSC-CMs and resulted in beating arrest at high doses. Taken together, it can be suggested that the developed bisphenol analogues could cause cardiotoxicity even with acute exposure, and it is considered that the application of the MEA-based dual-cardiotoxicity evaluation method can be an effective help in the development of safe alternatives.

Experimental analysis on flow characteristics of particulate materials conveyed by airslides

Increasing demands on efficient airslides conveying in the fields of construction, agriculture, pharmacy and power require deep understanding of the flow characteristics of materials being conveyed. This study designs a full-scaled airslide test rig, in conjunction with an experimental method, to investigate the flow behaviour of two different materials, namely sand and flyash under vent and non-vent conditions. The flow modes of the sand and flyash are predicted based on the measured material properties and fluidisation. Furthermore, tests are conducted using the designed airslide, from which the flow is characterised by analysing the variations of the key parameters, including the mass flow rate, the superficial air velocity, the pressure inside the airslide and the flowing material bed height. The work shows the developed test rig and method are capable of characterising fluidised flow for airslide systems.

Development of Chemical and Mechanical Acceleration Stress Test Method for PEMFC Polymer Membranes

Development of Chemical and Mechanical Acceleration Stress Test Method for PEMFC Polymer Membranes