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To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/μl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020–2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox. [ABSTRACT FROM AUTHOR]

ABSTRACTTobacco mosaic virus (TMV) causes a significant yield loss in solanaceous crops. Accurate and rapid diagnosis of TMV is an essential pre-requisite for proactive and tailored disease management strategies, filling the gap between more rigorous laws regarding pesticide use and maintaining crop production. Herein, we developed a time-resolved fluorescence immunoassay (TRFIA) to quantitatively detect TMV in tobacco. TMV virions were used as immunogens to develop monoclonal antibodies, which were then conjugated to Eu3+ nanospheres. The detection limit of the TRFIA platform was 0.24 ng/mL, with recovery rates in the range of 90.71–108.11% in spiked tobacco. The specificity was confirmed using ten species of tobacco-infecting phytopathogens. In-field applicability was implemented by screening for TMV in 10,000 tobacco leaf samples collected from five fields in Kunming, China. Overall, the proposed TRFIA platform is rapid, accurate, and end-user friendly, demonstrating its application in TMV onsite testing in tobacco.

Xylella fastidiosa is an aggressive plant pathogenic bacterium of significant quarantine concern. Accurate and reliable detection tools are essential to minimise the risk of the pathogen's spread and for outbreak control, as limited post-infection management strategies are possible. Here, we report the development of a specific and potentially field-deployable assay combining a pre-existing Loop-Mediated Isothermal Amplification (LAMP) assay and a Cas12a-based DNA Endonuclease-Targeted (DETECTR) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) trans reporter for X. fastidiosa detection. The LAMP-CRISPR-Cas12a integrated assay detected the amplified target region of the X. fastidiosa specific rimM gene at the low femto-molar range within 10 min of initiation. The assay detected varied X. fastidiosa sub-species in a range of naturally infected and economically relevant host material, with no non-target amplification recorded. The results show integration of LAMP with CRISPR-based detection is a specific, sensitive and a potentially field-adaptable strategy for the detection of X. fastidiosa and has the potential for further operationally focused improvements. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a quarantine priority pest in Europe and several other countries as it represents a major threat to many cultivated and wild plants. Early detection of the pathogen is needed to reduce potential losses and to stop the bacterial dissemination. Very few molecular methods are available for rapid field detection to identify the pathogen. Here, we evaluated a new rapid commercial detection test called AmplifyRP, based on molecular recombinase polymerase amplification (RPA). The AmplifyRP test was compared with the quantitative PCR (qPCR) Harper's assay, which represents the standard method used in most laboratories all over the world. Specificity was tested in vitro on a panel of target and nontarget strains. Additionally, the sensitivity of the RPA test was tested on X. fastidiosa genomic DNA, and the selectivity was compared between different host plant matrices spiked with X. fastidiosa cell suspensions. Although the qPCR assay showed a higher sensitivity than the RPA test, the latter appeared to be a very specific, rapid, and portable technique for which no skills are required to proceed. These findings should enable growers and inspectors to choose an appropriate diagnostic method. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Shrimp has become a highly traded global seafood product, with 8 million tons of shrimp produced annually. Acute hepatopancreatic necrosis disease (AHPND) is the most prevalent and severe disease affecting shrimp aquaculture, resulting in considerable economic losses. The AHPND incidence in shrimp farming was as high as 60%–80% in China, resulting in reduced farming capacity and unstable production. Vibrio parahaemolyticus has been identified as the main causative agent of AHPND. In addition, V. harveyi, V. cambelii, V. algolyticus, and V. owenii are capable of causing similar diseases, demonstrating a distinct pathogenic diversity. Previous studies have indicated that not all of the above-mentioned Vibrios species are capable of causing AHPND, and whole gene sequencing and knockout genes have revealed that pirA and pirB are the primary pathogenic factors responsible for AHPND in shrimp. Specifically, the causative agent for AHPND should be a specific strain of Vibrio carrying the binary toxins pirAVp and pirBVp on the extrachromosomal virulence plasmid pVA1. Among them, the pirB toxin mainly determines the pathogenicity of the bacterium, whereas the pirA virulence is relatively weak. Furthermore, it has been demonstrated that the virulence plasmids encoding the binary genes pirAVp and pirBVp are the main causative agents of AHPND. The virulence gene toxR is prevalent in Vibrio and plays an important role through the genetic diversity of 16S rRNA genes during shrimp infection. Real-time fluorescence quantitative PCR technology has less contamination, more accurate quantification, real-time monitoring, and greater automation than conventional PCR technology, which has been utilized in the fields of transgenic detection, environmental science, and medicine. However, this technique is time-consuming, involves multiple instruments and reagents, and requires personnel with extensive professional skills and experience. As a result of its small size, low sample and reagent consumption, rapid detection speed, miniaturization, and integration, microfluidic chip assay technology has emerged as a new focal point in assay technology. Therefore, in this study, we designed specific primers and established a microfluorescence quantitative PCR assay based on two genes, pirA and pirB, to address the genetic similarity of AHPND pathogens carrying a large plasmid encoding a binary toxin, pirA and pirB. The method was specific for the pathogenic pirA and pirB genes, and only when DNA from AHPND-infected samples was tested could the two genes be successfully amplified, while all other pathogenic bacteria were detected with negative results. The sensitivity was high, and the minimum detection limits for the pirA and pirB genes were 5.43×100 and 4.31×101 copies/μL, respectively. Standard curves for pirA and pirB were constructed and demonstrated good linearity in the concentration range of 5.43×109–5.43×104 copies/μL for pirA (y= –3.145x+6.63, R2=0.999) and 4.31×109–4.31×104 copies/μL for pirB (y= –3.015x+5.45, R2=0.999), with an average sample detection time of approximately 26 min. In order to evaluate the efficacy of the method in practice, artificial infection experiments with V. parahaemolyticus were performed. In this study, artificial infection experiments were induced by both injection and immersion, and samples were collected at different time periods to clinically validate the established method and compare its effectiveness in detecting different shrimp tissues, thereby facilitating a more thorough analysis of the pathogenic pathways of infection. The experimental group with injection as the mode of infection was found to be positive for all tissues in all time periods except the water test, which was negative. The experimental group that used immersion as the infection method showed different results for various time periods and with different genetic tests. In terms of the infection method, the tissues could be infiltrated within 2 h using the injection method, whereas the target genes were not detected in the hepatopancreas at 6 h using the immersion method. This indicated that the injection method infiltrated the tissues more rapidly than the immersion method. According to the comparison results of the three genes, pirB was only negative in the intestine at 2 h and positive in all tissues the rest of the time; pirA was negative in the hepatopancreas and intestine at 2 h, only the intestine was negative at 6 h, and all tissues were positive at 12 h; and toxR was negative in all tissues at 2 h. The rate of infestation from rapid to slow showed that pirB > pirA > toxR. Based on the rate of tissue infestation, pirA and pirB were detected in both cheek filaments and muscles at 2 h, making them the most rapid infiltration agents. Therefore, the strategy of using pirB as the primer and gill filament or muscle as the target tissue is more suitable for the rapid detection of AHPND in the field. In this study, we established a method for microfluidic fluorescent quantitative PCR that has the advantages of being rapid, sensitive, high throughput, less contaminated, on-site detectable, and integrated. The method is not only applicable to the laboratory but also meets the requirements of rapid field detection at hatcheries and farms, and can be used as a new technical method for shrimp fry quality detection and disease control.

Background: Long-term care (LTC) residents have been disproportionately affected by COVID-19 with higher morbidity and mortality. The use of canines for the detection of COVID-19 has been successful in controlled laboratory settings. They have the potential to provide rapid, non-invasive screening in congregate living settings. The ability to assess whether laboratory-trained canines can transfer their scent detection skills to the clinical settings has had limited evaluation. Methods: At Vancouver Coastal Health, two canines were previously trained and validated to differentiate COVID-19 positive and negative PCR samples from breath, sweat and gargle clinical samples from scent stands. Subsequently, they were taught to alert from pillowcases. Following successful validation and in collaboration with a local LTC, the canines performed weekly blind screening of residents' pillowcases during the Omicron wave. Results: A third-party, double-blind validation on pillowcases demonstrated an overall sensitivity of 100%. The specificity was 100% for the first canine and 82.6% for the second canine. Although no clinical cases occurred during the six-week pilot project, the agreement between the two canine teams was 98.4% on room alerts. In addition, the two teams were able to seamlessly transfer their laboratory skill sets to the LTC setting. Conclusion: Third-party evaluation determined that canines could successfully be trained to detect COVID-19 from pillowcases. The canine teams were then able to efficiently shift their skill set from the laboratory into an operational setting. This pilot project supports that the deployment of canine detection in a clinical setting is possible and could be considered in congregate living settings. [ABSTRACT FROM AUTHOR]

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography. [ABSTRACT FROM AUTHOR]

The invasive fungal pathogen Cronartium ribicola causes white pine blister rust which is considered one of the most destructive diseases of five‐needle (white) pines in North America. The disease has a life cycle that requires two hosts: white pines and Ribes spp., although other non‐Ribes species, including Castilleja and Pedicularis, have been demonstrated as alternate hosts as well. Detection of this disease can be difficult because of the ephemeral nature of sporulation on pine hosts with ambiguity in other symptoms, and the alternate hosts for C. ribicola can also be an alternate host for other pine rust species. We used the previously published C. ribicola genome and species‐specific real‐time PCR assay to develop a field‐ready loop‐mediated isothermal amplification (LAMP) specific colorimetric assay for this pathogen. Specificity results across regionally identified pine rust pathogens showed the assay is highly specific to C. ribicola and can detect as little as 40 pg of pathogen DNA. We also developed a simple DNA extraction method that works with several tissue types (bark/phloem, aeciospores, and urediniospores/telia) to prepare the DNA samples for the LAMP assay. The DNA extraction and LAMP assay take ~70 min to complete and require a relatively small investment in equipment. This tool enables quick and efficient detection of white pine blister rust. [ABSTRACT FROM AUTHOR]

Metagenomic next-generation sequencing (mNGS) is receiving increased attention for the detection of new viruses and infections occurring at the human–animal interface. The ability to actively transport and relocate this technology enables in situ virus identification, which could reduce response time and enhance disease management. In a previous study, we developed a straightforward mNGS procedure that greatly enhances the detection of RNA and DNA viruses in human clinical samples. In this study, we improved the mNGS protocol with transportable battery-driven equipment for the portable, non-targeted detection of RNA and DNA viruses in animals from a large zoological facility, to simulate a field setting for point-of-incidence virus detection. From the resulting metagenomic data, we detected 13 vertebrate viruses from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including avian leukosis virus in domestic chickens (Gallus gallus), enzootic nasal tumour virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA (CRESS DNA) viruses in several mammal species. More significantly, we demonstrate that the mNGS method is able to detect potentially lethal animal viruses, such as elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the newly described human-associated gemykibivirus 2, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for the first time. [ABSTRACT FROM AUTHOR]

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In order to reveal the influencing factors and causes of nitrate in karst groundwater in Baiquan spring area of Xingtai, a portable spectrophotometer was used to establish a rapid on-site quantitative detection method for nitrate-nitrogen in water. Based on hydrogeological structure and hydrochemical analysis, the distribution characteristics of nitrate-nitrogen in karst groundwater in Baiquan spring area and its relationship with conventional hydrochemical indexes were studied. The experimental results show that the linear relationship of nitrate-nitrogen is good in the range of 0. 60 ~30. 00 mg/ L, and the detection limit is 0. 2 mg/ L. The relative standard deviation between the results of field test and laboratory standard method is 0. 23% ~6. 70%, and the difference test result is t =0. 984, which indicates that the results of the two methods are identical. The concentration of nitrate-nitrogen in the runoff area is generally low, only in the karst exposed area in the northwest of Baiquan spring area and the karst water discharge area overlying Quaternary Upper Pleistocene sand gravel aquifer in the east with poor pollution prevention, the concentration is high. Nitrate-nitrogen concentration in the study area is between class Ⅰ and class Ⅲ water specified in the quality standard of groundwater. The hydrochemical types of karst groundwater in the study area are complex, with Ca and Ca·Mg type is the main cation, HCO3 and HCO3·SO4 type is dominant anion. Nitrate-nitrogen content in groundwater of HCO3·Cl-Ca·Mg and Cl·HCO3·SO4 -Ca type is relatively higher. The main source of nitrate is urban sewage, which is less affected by atmospheric precipitation and agricultural activities. The research results provide reference basis for establishing standard method of nitrate-nitrogen in groundwater on-site detection and water supply safety and ecological protection of karst water resources in Baiquan spring area. [ABSTRACT FROM AUTHOR]

Plant parasitic nematodes (PPNs) cause an important class of diseases that occur in almost all types of crops, seriously affecting yield and quality and causing great economic losses. Accurate and rapid diagnosis of nematodes is the basis for their control. PPNs often have interspecific overlays and large intraspecific variations in morphology, therefore identification is difficult based on morphological characters alone. Instead, molecular approaches have been developed to complement morphology-based approaches and/or avoid these issues with various degrees of achievement. A large number of PPNs species have been successfully detected by biochemical and molecular techniques. Newly developed isothermal amplification technologies and remote sensing methods have been recently introduced to diagnose PPNs directly in the field. These methods have been useful because they are fast, accurate, and cost-effective, but the use of integrative diagnosis, which combines remote sensing and molecular methods, is more appropriate in the field. In this paper, we review the latest research advances and the status of diagnostic approaches and techniques for PPNs, with the goal of improving PPNs identification and detection.

Wheat blast, caused by the fungus Magnaporthe oryzae Triticum (MoT) pathotype, is a devastating disease persistent in South America and Bangladesh. Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced, disease control by fungicide application solely based on the detection of visual symptoms is ineffective. To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control, we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments, MoT-6098 and MoT-6099, that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae (MoO) pathotype. Using polymerase chain reaction (PCR), we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh. To test the efficiency of the two markers, we first established a loop-mediated isothermal amplification (LAMP) method to detect MoT at isothermal conditions, without the use of a PCR machine. Following this, we used the Cas12a protein and guide RNAs (gRNAs) to target the MoT-6098 and MoT-6099 sequences. The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease (ssDNase) activity. We then combined target-dependent Cas12a ssDNase activation with recombinase polymerase amplification (RPA) and nucleic acid lateral flow immunoassay (NALFIA) to develop a method that accurately, sensitively, and cost-effectively detects MoT-specific DNA sequences in infected wheat plants. This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.

Metagenomic next-generation sequencing (mNGS) is receiving increased attention for the detection of new viruses and infections occurring at the human–animal interface. The ability to actively transport and relocate this technology enables in situ virus identification, which could reduce response time and enhance disease management. In a previous study, we developed a straightforward mNGS procedure that greatly enhances the detection of RNA and DNA viruses in human clinical samples. In this study, we improved the mNGS protocol with transportable battery-driven equipment for the portable, non-targeted detection of RNA and DNA viruses in animals from a large zoological facility, to simulate a field setting for point-of-incidence virus detection. From the resulting metagenomic data, we detected 13 vertebrate viruses from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including avian leukosis virus in domestic chickens (Gallus gallus), enzootic nasal tumour virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA (CRESS DNA) viruses in several mammal species. More significantly, we demonstrate that the mNGS method is able to detect potentially lethal animal viruses, such as elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the newly described human-associated gemykibivirus 2, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for the first time.

In order to solve the problem of early field inspection of porcine epidemic diarrhea, the experiment established a colloidal gold immunochromatographic method for the detection of porcine epidemic diarrhea virus (PEDV) in pig feces using a new kind of colloidal golds prepared via 3-hydroxymethyl aminomethane (Tris) assisted reduction. The preparation conditions including labeled pH and protein concentration was optimized. The sensitivity, specificity and accuracy of the established method were finally evaluated. The results showed that the established PEDV immunochromatography method could detect PEDV in pig feces within 15 min, and the minimum detection limit was 1×102 TCID50/mL. No cross reaction with porcine infectious gastroenteritis virus, rotavirus, Salmonella and other common pathogens was observed. In addition, there was no significant difference between intra-batch and inter-batch detection results. The coincidence rate of 107 clinical samples and RT-PCR was 100%. The study indicates that the established PEDV immunochromatographic strip based on the new colloidal gold marker had high sensitivity, strong specificity, good repeatability, with simple and quick operation, and is suitable for the rapid diagnosis of PED in primary farms. [ABSTRACT FROM AUTHOR]

Tomato yellow leaf curl disease which is caused by Tomato yellow leaf curl virus (TYLCV) is economically important and a widely spread tomato disease in China. Rapid and accurate detection methods are important in the control TYLCV. Here, a rapid method was developed to identify TYLCV on the basis of recombinase polymerase amplification (RPA) that can be visualized in 5 min using lateral flow dipsticks. The sensitivity and the specificity of this method were evaluated. This method can detect 0·5 pg DNA after 30 min at 37°C without any expensive instrumentation. In addition, it showed higher sensitivity than a PCR method when purified DNA was used. Moreover, the TYLCV was specifically detected, whereas other viruses infecting tomato produced negative results. The crude tomato extracts used in this assay has potential application in minimally equipped plant clinic laboratories. This method will facilitate the early and rapid detection of TYLCV for the timely application of control measures. [ABSTRACT FROM AUTHOR]

Rapid and accurate detection of genetically modified crops (GMC) is significant for crop supervision and genetically modified product labeling. For field detection of GMC, the rapidity and simplicity of the sample pretreatment and detection process are very important. Simplifying the extraction process of DNA in crops and speeding up the detection process are the technical problems that need to be solved. Therefore, this paper provides a field detection method for GMC. First, the selected universal LAMP primers of the chloroplast gene can be used to detect reference genes in nine crops with the most transgenic detection needs. The universal primers of reference genes can not only improve the accuracy of detection, but also omit the design and synthesis of reference gene primers in different crops. Second, a high concentration of DNA can be obtained using simple and fast DNA extraction methods within 5 min. The specific concentration of GuHCl in lysis buffer can accelerate the subsequent nucleic acid amplification reaction. Third, a new probe-LAMP method was established, with high reaction speed and specificity. Using the dual probe-LAMP for transgenic and reference genes in a handheld nucleic acid detector, the detection of GMC can be completed within 25 min. Two different fluorescence signals generated from the LAMP reaction are processed through a special logic gate, and finally three kinds of results of "Positive", "Negative" and "Invalid" are output on a smartphone. This method from sample processing to intelligent result output can achieve accurate, rapid, simple, low-cost, pollution-free field detection of GMC. • Universal LAMP primers for reference genes in nine transgenic crops were selected. • The DNA extraction method can obtain a high concentration of DNA within 5 min. • The concentration of GuHCl in lysis buffer can accelerate the LAMP reaction. • A new probe-LAMP method was established, with high reaction speed and specificity. • Test results of transgenic crops can be output on a smartphone within 25 min. [ABSTRACT FROM AUTHOR]

Abstract Bean common mosaic virus (BCMV) is one of the most widespread and damaging viruses of cultivated legumes in the world. In addition to serious yield reduction and germplasm decline, BCMV infection also makes legumes more vulnerable to other pathogens. Early diagnosis of the virus is particularly important in limiting its spread. Recombinase polymerase amplification (RPA) is a novel isothermic amplification technology. The whole reaction can be done outside the laboratory environment after the nucleic acid sample is obtained. In this study, we established a rapid and sensitive RPA combined with the lateral flow dipstick (LFD) assay for detection of BCMV, based on the conserved BCMV coat protein (CP) gene sequence. Specific primers and a probe were designed, which amplify ~ 150 bp CP fragments from BCMV-infected samples under a constant temperature of 37 °C for 20 min. The end-labeled amplification products were detected by high-affinity LFD within 5 min. Sensitivity of this RPA-LFD assay was 1000 times greater than that of the conventional polymerase chain reaction (PCR) assay. Furthermore, when the primers/probe were used against related potyviruses including soybean mosaic virus (SMV), bean yellow mosaic virus (BYMV), and turnip mosaic virus (TuMV), the three potyviruses were not detected, indicating that the assay was BCMV species-specific. The RPA-LFD assay was also successfully applied for the detection of seed-borne BCMV in beans. The RPA-LFD assay has great potential application in the rapid diagnosis of BCMV in the field.

In this study, the one-step switchable hydrophilic solvent (SHS)-based effervescence tablet microextraction (ETME) was coupled with smartphone digital image colorimetry (SDIC) for the field detection of nickel ion (Ni2+) for the first time. Both extractant and CO 2 were generated in situ when the novel SHS-based effervescence tablet was placed in the sample solution. The complexant 1-(2-pyridinylazo)-2-naphthaleno (PAN) dissolved from the effervescence tablet to form a stable complex with Ni2+, and the extractant was uniformly dispersed in the sample solution under the action of CO 2 and fully in contact with Ni-PAN, which enabled efficient extraction of Ni2+. The color changes of the extraction phase were captured by smartphone, then a quantitative relationship between the concentrations of Ni2+ and color intensity of images captured using a smartphone was established by customized applet WASDIC, which realized quantitative analysis of Ni2+ in different samples. Under optimal conditions, the enhancement factor (EF) of the proposed method was 65.1, the limit of detection (LOD) and limit of quantification (LOQ) were 1.69 and 5.64 μg L−1, respectively. The developed method was successfully applied to the detection of trace Ni2+ in the environmental samples and natural medicines. And the applicability of the method for use in field analysis was validated. The one-step effervescence tablet microextraction (ETME) based on switchable hydrophilic solvent (SHS) was coupled with smartphone digital image colorimetry (SDIC) for detection of nickel ion (Ni2+) in environmental samples (waters and soil) and the natural medicines (Salvia yunnanensis and Amomum villosum Lour.), and the applicability of the method for use in field analysis was validated. [Display omitted] • The novel SHS-ETME was firstly combined with SDIC. • The applicability of the combination for field analysis was firstly validated. • The coupling was firstly used to analyze environmental samples and medicinal plants. • Extractant and CO 2 were generated in situ and extraction was completed in one step. • Box-Behnken design was used for the optimization of experimental variables. [ABSTRACT FROM AUTHOR]

• Complex matrices with amounts of protein and fat do not affect RPA amplification. • Limiting template concentration contributes significantly to RPA specificity. • High sequence similarity causes higher LOD of cows in mixtures with goats. • A dual RPA assay of goats and sheep for further rapid special dairy identification. • Special dairy with whey protein could be at risk of error if defined as adulterated. The authentication of dairy species has great significance for food safety. This study focused on a more rapid method for identifying major dairy species, and specific recombinase polymerase amplification (RPA)-based assays for cattle, goat, sheep, camel and donkey were developed. Through the developed RPA-based assays, goats and sheep could be simultaneously identified and bovine families could be differentiated. The performances of the RPA assays were validated using 37 milk powder samples, of which 16.2% (6/37) were suspected of being adulterated and 24.3% (9/37) were potentially at risk of being wrongly identified as adulteration. The effectiveness of the developed assays for crude DNA detection was also validated by a rapid nucleic acid extraction kit, and results showed that the presence of large amounts of protein and fat did not affect the qualitative results. Therefore, these assays could combine with the rapid nucleic acids extraction methods for being used in field detection. [ABSTRACT FROM AUTHOR]

This paper is focused on a description of a new method of detecting continuities in a binary image. The detecting method is called “Binary Large Object” (BLOB). A new algorithm with other elementary parameters, as processing speed and memory capability, are described here. The developed BLOB method with described algorithms is implemented in MATLAB. The simulation of the algorithms is tested in different conditions, with the time dependences determination. The research results of the computing time or the BLOB memory demand during computation are presented as well. The developed BLOB method is usable as one of the elementary image processing methods of the field detection dependencies in the images, like pattern recognition or OCR algorithm.

Intensive monoculture production of agricultural plants, climate change, global trade and the evolutionary ability of pathogens to adapt rapidly are main source of current emergences. Incurable plant diseases caused by viroids can limit crop production and quality and can result in significant losses. An important factor in preventing the spread is represented by diagnostic methods that can rapidly and reliably detect viroid infections. The nature of viroids, which are naked RNA without any coding potential, restricts its detection based on antigen-antibody recognition. Therefore, only nucleic acid detection methods can be applied. Some of them can be timeconsuming, require the use of expensive equipment and technical skills, which may be an obstacle for field detection. An alternative way is the use of isothermal tests, such as the loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Isothermal tests are simple, easy to perform, and reliable and can be perform using lateral flow devices. This technology has the advantage to be implemented in field-based scenarios because tests require a minimal sample preparation, and are performed at constant low temperature (37-- 42°C) without the use of sophisticated equipment. Here we briefly describe isothermal assays (LAMP and RPA), that are suitable for onsite detection and their potential for viroid detection. [ABSTRACT FROM AUTHOR]

Pectobacterium carotovorum is the causal agent of bacterial soft rot in a wide range of vegetable host species. Once P. carotovorum infects the plant, the spread of the disease is difficult to control. In this study, a rapid and sensitive method based on loop-mediated isothermal amplification (LAMP) was developed for detecting P. carotovorum in celery with soft rot using a primer set designed from the pmrA conserved sequence of P. carotovorum. The specificity of the LAMP primer set for P. carotovorum was extensively validated on both P. carotovorum strains and non-target strains. The sensitivity was 1 pg of P. carotovorum genomic DNA, which demonstrated 10 times more sensitive than the conventional PCR assay. LAMP was also used to detect P. carotovorum in bacterial suspension. The lowest detection concentration was 104 CFU · mL−1. In addition, a LAMP assay, in conjunction with a crude DNA extraction method, was successfully performed on P. carotovorum-infected samples derived from both artificially and naturally infected plants. In summary, the LAMP assay established in this study constitutes a simple, sensitive, and rapid method for the detection of P. carotovorum, and has potential application in the control of celery soft rot disease through early detection.

The study of deformation characteristics of weak surrounding rock around fault zone is of great significance for roadway support optimization and preventing large deformation of the surrounding rock.Based on the engineering background of Chengjiao coal mine in Henan province, surface displacement, top-plate deformation and loose ring of surrounding rock are monitored in the excavation process of the second-level west-wing track roadway.The stress of fault structure gradually decreases under the continuous influence of the excavation of roadways, and the deformation speed of the tunnel increases first and then decreases with the advance of the roadway.Therefore, the force caused tunnel deformation in fault zone mainly comes from fault tectonic stress.The deep surrounding rock is affected by fault structure, the crack is well developed, and the roof is unstable, the shallow surrounding rock has smaller abscission layer due to the excavation of the roadway.The loose ring around the fault is greatly affected by fault structure and the roof forms a large loosen ring.In this paper, the support optimization scheme of U-type steel and anchorage cable is proposed according to the results of field monitoring and actual geological conditions.

The remote, timely and in-field detection of sulfured additives in natural gas pipelines is a challenge for environmental, commercial and safety reasons. Moreover, the constant control of the level of odorants in a pipeline is required by law to prevent explosions and accidents. Currently, the detection of the most common odorants (THT = tetrahydrothiophane; TBM = tertiary butyl mercaptan) added to natural gas streams in pipelines is made in situ by using portable gas chromatography apparatuses. In this study, we report the analysis of the ultraviolet spectra obtained by a customized ultraviolet spectrophotometer, named Spectra, for the in-field detection of thiophane and tertiary butyl mercaptan. Spectra were conceived to accomplish the remote analysis of odorants in the pipelines of the natural gas stream through the adoption of technical solutions aimed to adapt a basic bench ultraviolet spectrophotometer to the in-field analysis of gases. The remotely controlled system acquires spectra continuously, performing the quantitative determination of odorants and catching systemic or accidental variations of the gaseous mixture in different sites of the pipeline. The analysis of the experimental spectra was carried out also through theoretical quantum mechanical approaches aimed to detect and to correctly assign the nature of the intrinsic electronic transitions of the two odorants, thiophane and tertiary butyl mercaptan, that cause the ultraviolet absorptions. So far, these theoretical aspects have never been studied before. The absorption maxima of thiophane and tertiary butyl mercaptan spectra were computationally simulated through the usage of selected molecular models with satisfactory results. The good matches between the experimental and theoretical datasets corroborate the reliability of the collected data. During the tests, unexpected pollutants and accidental malfunctions have been detected and also identified by Spectra, making this instrument suitable for many purposes. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a widespread blood‐borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma, particularly in south‐east Asian and African countries where HBV is highly endemic and the budget and resources are limited. Therefore, simple, rapid, and portable field detection methods are crucial to efficiently control HBV infection. In this study, using heat‐treated DNA, we developed two‐field applicable detection assays for HBV based on recombinase‐aided amplification (RAA). One was an internal controlled duplex RAA assay using a portable real‐time fluorescence detection device, another was an instrument‐free visual observation assay using lateral flow dipsticks. The entire experimental time was greatly shortened to less than 40 minutes at 39.0°C. The sensitivities, specificities, and clinical performance of both assays were evaluated. Compared with quantitative polymerase chain reaction assay as a reference, our results demonstrated that the two RAA‐based assay obtained 97.18% and 95.77% of sensitivity, respectively, and the specificity was 100%, by testing a total of 157 serum samples with HBsAg positive. We conclude that the advantages of rapidity, simplicity, portability, and visualization of proposed two assays make them great potentials in point‐of‐care testing of HBV infection by untrained people in resource‐limited situations. Research Highlights: HBV is a widespread blood‐borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma. We present a rapid RAA assay to detect HBV without DNA extraction. This assay can be performed in a single closed tube at 39℃ within 30 min. The sensitivity of the internally controlled RAA assay was 102 IU/ml. The introduction of IAC effectively reduced the occurrence of false negatives. [ABSTRACT FROM AUTHOR]

Banana bract mosaic virus (BBrMV) causes one of the major viral diseases of the fiber crop abaca at a great economic loss in the Philippines. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect BBrMV. A set of six primer pairs was designed based on the conserved coat protein gene sequences of BBrMV. The RT-LAMP can be completed in 30 min and was 100,000-fold more sensitive than RT-PCR. The RT-LAMP assay is suitable for field detection of BBrMV using crude RNA extracts and fluorescent DNA intercalating dyes that produce easily visualized results. [ABSTRACT FROM AUTHOR]

This paper is focused on a description of a new method of detecting continuities in a binary image. The detecting method is called "Binary Large Object" (BLOB). A new algorithm with other elementary parameters, as processing speed and memory capability, are described here. The developed BLOB method with described algorithms is implemented in MATLAB. The simulation of the algorithms is tested in different conditions, with the time dependences determination. The research results of the computing time or the BLOB memory demand during computation are presented as well. The developed BLOB method is usable as one of the elementary image processing methods of the field detection dependencies in the images, like pattern recognition or OCR algorithm. [ABSTRACT FROM AUTHOR]

Sorghum mosaic virus (SrMV) is a virus that causes mosaic symptom on sugarcane worldwide. In recent years, SrMV is becoming the most predominant pathogen for sugarcane mosaic disease (SMD) in Guangxi province, the major sugarcane-growing region in China. To facilitate the detection of SrMV, high throughput and sensitive serology-based detection techniques were developed and compared with RT-PCR assay. A high titer polyclonal antibody against bacterial-expressed recombinant coat protein of SrMV was first generated. The specificity and sensitivity of the resultant antibodies were evaluated by Western blotting, indirect-ELISA and Dot-ELISA. SrMV could be detected in crude sap at dilution 1:100 by Dot-ELISA, 1:1000 by indirect-ELISA, and at 1:100,000 by IC-RT-PCR. Meanwhile, a non-reactive result was found in SrMV-free extracts. In a randomly collected field sample pool, 60% of the samples showed SrMV-positive by IC-RT-PCR, similar to that of RT-PCR assay, compared with 46% by indirect-ELISA or 48% by Dot-ELISA. Besides, IC-RT-PCR, Dot-ELISA and indirect-ELISA methods achieved an agreement with RT-PCR procedure of 90, 86 and 78% for the detection of SrMV, respectively. Taken altogether, our findings indicate that IC-RT-PCR is a simple, sensitive and reliable method for SrMV detection, which holds a great potential for SMD control, quarantine programs and the diagnosis of SrMV in asymptomatic infected plant samples. [ABSTRACT FROM AUTHOR]

Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site. [ABSTRACT FROM AUTHOR]

Copyright of Natural Gas Industry is the property of Natural Gas Industry Journal Agency and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Nonoriented coupling of nanobodies (Nb) to nanomaterials can considerably reduce epitope recognition or induce other dysfunctions, thereby restricting the application of Nb in field detection. Herein, using Avi-tag/streptavidin (Avi/SA)-oriented coupling strategy, we developed a lateral-flow immunochromatographic assay (LFIA) based on Nb-Avi/SA@quantum dots (QDs) probes (Nb-LFIA) for Aflatoxin B 1 (AFB 1) detection. Compared with nonoriented coupled Nb@QD probes, the detection sensitivity and stability of Nb-Avi/SA@QD probes were greatly retained. Furthermore, Nb showed higher tolerance to the extreme detection environments compared with monoclonal antibodies in traditional enzyme-linked immunosorbent assay and LFIA. After optimization, the limit of detection of Nb-LFIA was 0.095 ng mL−1, half-maximal inhibitory concentration was 0.85 ng mL−1, and the visual cut-off level was 1.25 ng mL−1. Nb-Avi/SA@gold nanoparticle probes were assessed for the wide applicability of the Avi/SA strategy, and they showed excellent performance (coefficient of determination = 0.999). Finally, the analysis performance of probes was assessed in oat samples, revealing a recovery rate of 88.8–116.7%, which was consistent with the results of high-pressure liquid chromatography coupled with tandem mass spectrometry. Altogether, our study provides a convenient and sensitive tool for AFB 1 detection, and presents a viable path for efficient Nb application. ● An effective oriented coupling strategy for the development of nanobody and nanomaterials. ● A nanobody-based lateral flow immunochromatographic assay was developed for the rapid, convenient field detection of AFB 1. ● A nanobody-based lateral flow immunochromatographic assay showed the tolerance in extreme detection environments. ● Avi/SA oriented coupling strategy would greatly retain the advantageous functions of nanobody. [ABSTRACT FROM AUTHOR]

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography.

Several tobamoviruses cause substantial economic losses to tomato and pepper crops globally, especially the pepper mild mosaic virus (PMMoV), tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV). A fast and accurate detection method is essential for virus identification. An all-in-one reaction method combining a one-step reverse-transcription recombinase-aided amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one mixture was developed to rapidly screen and accurately differentiate among these four tobamoviruses for field detection in tomato and pepper plants. With a generic RT-RAA primer set and a mix of four specific crRNAs, along with a portable metal incubator and the use of a crude extraction method, this method screened for PMMoV, ToBRFV, ToMV, and ToMMV concurrently in less than 1 h, enabling field workers to take action immediately. The accurate differentiation of these four viruses could be achieved by later adding a single specific crRNA.

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The use of portable gas chromatography–mass spectrometry (GC-MS) is an important capability that has been available commercially for almost 25 years. These systems have been used within a variety of different industries, including their extensive use by environmental scientists for the analysis of hazardous air pollutants. Recently, these systems were deployed to conventional military forces for use in theater to detect and identify toxic chemicals including chemical warfare agents (CWAs). The challenges of deploying such complex analytical instruments to these military users are unique. Among other things, these organizations have considerable and variable mission strains, complex and difficult logistics and coordination needs, and variability in user backgrounds. This review outlines the value portable GC-MS systems offer to these warfighters in theater, discusses some important aspects of the design of portable systems that makes their deployment to this type of end user possible, and proposes methods that can be used to overcome challenges to successful deployment of portable GC-MS to non-scientists working within hostile environments. [ABSTRACT FROM AUTHOR]

Illegal drugs are among the multitude of goods examined by customs officers at the border. With the emergence of fentanyl and fentanyl analogues, the goal of field detection of drugs becomes two-fold: to interdict illegal drug movement and to assist in the protection of the health and safety of frontline officers. This work reports a study of nine fentanyl-related substances (acetylfentanyl, benzoylfentanyl, beta-hydroxythiofentanyl, butyrylfentanyl, carfentanil, despropionylfentanyl, fentanyl, furanylfentanyl and parafluoroisobutyrylfentanyl) using a commercial off-the-shelf ion mobility spectrometer (COTS IMS) and the results from analyzing 84 seized samples containing fentanyl-related substances. The results showed that the COTS IMS was capable of detecting the presence of programmed fentanyl-related substances. Unprogrammed fentanyl-related substances were detected, however no alarm was generated and the information was accessible via the advanced user mode only. The COTS IMS was not able to distinguish between several pairs of substances with similar reduced mobility values. During this study, six fentanyl analogues were intercepted by our laboratory for the first time (2-isopropylfuranylfentanyl, 2-methoxyfuranylfentanyl, 2-methfuranylfentanyl, 4-chlorofuranylfentanyl, cyclopropylfentanyl and methoxyacetylfentanyl) and their reduced mobility values are presented herein. [ABSTRACT FROM AUTHOR]

Commercial off-the-shelf (COTS) ion mobility spectrometers (IMS) are widely used for the trace detection of drugs and explosives in security applications and are designed to be operated by non-technical users. In non-technical user mode, the detection algorithm of the COTS IMS evaluates the sample data against the programmed parameters for a specific and limited list of target substances, and reports if any of the programmed target substances are detected. The simplicity in operation is offset by the loss of analytical information available from the spectral details. In this work, we present for the first time a COTS IMS being operated in technical user mode in the field, analyzing packages selected for customs examination. The COTS IMS was used in combination with a Fourier transform infrared (FTIR) spectrometer to evaluate the effectiveness of presumptive field testing. Three case studies are presented to demonstrate the usefulness of operating COTS IMS in technical user mode: to corroborate results obtained from the FTIR, to indicate the presence of unprogrammed substances, and to indicate the presence of substances in minor to trace amounts that were not identified by the FTIR. This work also demonstrates the use of IMS in negative mode to identify the citrate salt form of a drug. [ABSTRACT FROM AUTHOR]

The rapid and accurate detection of the presence of microorganisms, such as viruses, has been an important issue in the fields of public health, as well as agriculture. A PCR‐based detection method has been developed and applied in these fields to determine the presence of specific pathogens. Although the major advantage of real‐time PCR is the monitoring of amplification and ability to quantify the template genes, the method described here should solve the problem of nonspecific product synthesis. We obtained viral RNA from infected samples by freezing and thawing; we rapidly synthesized cDNA from RNA, and then amplified the cDNA by rapid PCR in 10 Min. Finally, the PCR products were hybridized and quickly confirmed to be the target analyte on a DNA chip. Our newly proposed methods overcome the drawbacks of PCR‐based detection and provide three additional advantages, namely, rapidly obtaining large amounts of RNA from samples, quickly detecting infective or pathogenic genes, and speedily confirming the detected exogenous genes. This application might be useful for detecting viral RNA and for the diagnosis of RNA virus‐mediated diseases. [ABSTRACT FROM AUTHOR]

H9N2 avian influenza viruses (AIVs) have been detected from wild birds and domestic poultry worldwide. Serious diseases combined with secondary infection have caused high mortality and great economic losses to poultry industry. Therefore, simple, rapid, sensitive and accurate methods suitable for field detection of H9N2 AIVs are crucial to efficiently control virus infection and spread in time. In this study, an isothermal reverse transcription recombinase polymerase amplification with lateral‐flow dipstick (RT‐RPA‐LFD) assay for detection of hemagglutinin (HA) gene of H9 subtype influenza viruses was developed. The optimal forward and reverse primers targeting HA gene of H9 subtype influenza viruses were labeled with fluorescein isothiocyanate (FITC) and biotin at the 5′‐end, respectively. The amplification reaction could be finished in 20 min at a wide temperature range of 30–42°C, and then the products could be visualized with naked eyes. The developed H9 RT‐RPA‐LFD was able to detect 0.15 pg of H9N2 AIV RNA, which was 10 times more sensitive than that of conventional RT‐PCR. The H9 RT‐RPA‐LFD assay did not detect nucleic acids extracted from H9 negative samples or from other poultry respiratory pathogens. The clinical performance of H9 RT‐RPA‐LFD was determined by testing 120 cloacal samples collected from chickens with respiratory syndromes. The coincidence rate of the detection results between RT‐RPA‐LFD and conventional RT‐PCR was 95.8%. Therefore, the developed RT‐RPA‐LFD assay provides a rapid, reliable and sensitive method for field diagnosis of H9 subtype AIVs. [ABSTRACT FROM AUTHOR]

Detecting, monitoring, and mapping transgenic sequences in the environment has been a long-standing challenge in ecology. Given the level of social, economic, and scientific interest in them, transgenic (GM) DNA sequences stand as paradigmatic; such sequences further have the unique property of functioning as unequivocal markers in the environment, clearly showing the paths of gene flow between crops and their wild relatives, and across industrial and traditional forms of agriculture. True mapping of transgenic DNA at the landscape level requires a method of DNA amplification that is robust and isothermal in order to be cost effective and field-based. We present here a method with these characteristics. Our multiplex fluorescent method is for the detection of the p35s CaMV promoter frequently used in transgenic plants as well as the alpha-zein protein specific to maize, but can be applicable to any other DNA sequence. The method uses loop-mediated isothermal amplification (LAMP) with visualization achieved using a fluorophore-quencher system of DNA hybridizing probes. We demonstrate the applicability of this tri-color method to transgenic corn, non-transgenic corn, and non-transgenic/non-corn species. [ABSTRACT FROM AUTHOR]