Establishment of High-sensitivity Rapid Fluorescence Quantitative Detection Method for Antibody against Peste des Petits Ruminants Virus

[Objectives]This study was conducted to establish a rapid quantitative method for detecting antibody against Peste des Petits Ruminants Virus(PPR V)in sheep serum.[Methods]Soluble N protein and NH fusion protein were obtained in Escherichia coli prokaryotic expression system by optimizing codons and expression conditions of E.coli.Furthermore,based on the purified soluble N protein and NH fusion protein,a high-sensitivity fluorescence immunoassay kit for detecting the antibody against PPR V was established.[Results]The method could quickly and quantitatively detect PPR V antibody in sheep serum,with high sensitivity and specificity,without any cross reaction to other related sheep pathogens.The intra-batch and inter-batch coefficients of variation were less than 10%and 15%,respectively,and the method had good repeatability.Through detection on 292 clinical serum samples,it was compared with the French IDVET competitive ELISA kit,and the coincidence rate of the two methods reached 93.84%.Compared with the serum neutralization test,the detected titer value of the high-sensitivity rapid fluorescence quantitative detection method was basically consistent with the tilter value obtained by the neutralization test on the standard positive serum(provided by the WOAH Brucellosis Reference Laboratory of France).[Conclusions]This method can realize rapid quantitative detection of PPR V antibody on site,and has high practical value and popularization value.

Quantitative Damage Detection for Planetary Gear Sets Based on Physical Models

Planetary gear set is the critical component in helicopter transmission train, and an important problem in condition monitoring and health management of planetary gear set is quantitative damage detection. In order to resolve this problem, an approach based on physical models is presented to detect damage quantitatively in planetary gear set. A particular emphasis is put on a feature generation and selection method, which is used for sun gear tooth breakage damage detection quantitatively in planetary gear box of helicopter transmission system. In this feature generation procedure, the pure torsional dynamical models of 2K-H planetary gear set is established for healthy case and sun gear tooth-breakage case. Then, a feature based on the spectrum of simulation signals of the dynamical models is generated. Aiming at selecting the best feature suitable for quantitative damage detection, a two-sample Z-test procedure is used to analyze the performance of features on damage evolution tracing. A feature named SR, which had better performance in tracking damage, is proposed to detect damage in planetary gear set. Meanwhile, the sun gear tooth-chipped seeded experiments with different severity are designed to validate the method above, and then the test vibration signal is picked up and used for damage detection. With the results of several experiments for quantitative damage detection, the feasibility and the effect of this approach are verified. The proposed method can supply an effective tool for degradation state identification in condition monitoring and health management of helicopter transmission system.

Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.

Utility of Droplet Digital PCR Assay for Quantitative Detection of Norovirus in Shellfish, from Production to Consumption in Guangxi, China

Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.

Study on the Simultaneously Quantitative Detection for β-Lactoglobulin and Lactoferrin of Cow Milk by Using Protein Chip Technique

Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.

Lateral Flow Immunoassay for Quantitative Detection of Ractopamine in Swine Urine

A strip reader based lateral flow immunoassay （LFIA） was established for the rapid and quantitative detection of ractopamine （RAC） in swine urine. The ratio of the optical densities （ODs） of the test line （AT） to that of the control line （Ac） was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration （IC50） of 0.59＋0.06 ng/mL. The limit of detection （LOD） of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.

Quantitative and sensitive detection of prohibited fish drugs by surface-enhanced Raman scattering

Rapid and simple detections of two kinds of prohibited fish drugs, crystal violet （CV） and malachite green （MG）, were accomplished by surface-enhanced Raman scattering （SERS）. Based on the optimized Au/cicada wing, the detectable concentration of CV/MG can reach 10-7 M, and the linear logarithmic quantitative relationship curves between log/and logC allows for the determination of the unknown concentration of CV/MG solution. The detection of these two analytes in real environment was also achieved, demonstrating the application potential of SERS in the fast screening of the prohibited fish drugs, which is of great benefit for food safety and environmental monitoring.

Mesoporous SiO_2 Nanoparticles:A Unique Platform Enabling Sensitive Detection of Rare Earth Ions with Smartphone Camera

Fast and sensitive detection of dilute rare earth species still represents a challenge for an on-site survey of new resources and evaluation of the economic value. In this work, a robust and low-cost protocol has been developed to analyze the concentration of rare earth ions using a smartphone camera. The success of this protocol relies on mesoporous silica nanoparticles(MSNs) with large-area negatively charged surfaces, on which the rare earth cations(e.g., Eu^(3+)) are efficiently adsorbed through electrostatic attraction to enable a ‘‘concentrating effect''. The initial adsorption rate is as fast as 4025 mg(g min)^(-1), and the adsorption capacity of Eu^(3+)ions in the MSNs is as high as 4730 mg g^(-1)(equivalent to ～41.2 M) at 70 °C. The concentrated Eu^(3+)ions in the MSNs can form a complex with a light sensitizer of 1,10-phenanthroline to significantly enhance the characteristic red emission of Eu^(3+)ions due to an ‘‘antenna effect'' that relies on the efficient energy transfer from the light sensitizer to the Eu^(3+)ions.The positive synergy of ‘‘concentrating effect'' and ‘‘antenna effect'' in the MSNs enables the analysis of rare earth ions in a wide dynamic range and with a detection limit down to ～80 nM even using a smartphone camera. Our results highlight the promise of the protocol in fieldwork for exploring valuable rare earth resources.

A Real-time Fluorescent Quantitative PCR Method for Detection of Genetically Modified Maize MON88017

In order to improve the standardized technical system of quantitative analyses for genetically modified organisms( GMOs) and protect China's bio-safety and reduce ecological risk,we establish a quantitative detection method for the genetically modified( GM) maize MON88017 using real-time fluorescent quantitative PCR. Meanwhile,the method is evaluated by several methodological indicators such as specificity,sensitivity,accuracy and uncertainty of measurement. The results show that the method has strong specificity in analysis of genetically modified maize MON88017. The mean value(1. 54%) repeatedly measured for 29 times with the relative deviation of 2. 7% was close to the real value(1. 50%) and the variation coefficient of the measured value was 0. 1. The tested recovery rate is 100% and the uncertainty of measurement is 0. 096. 5 copies of the MON88017 molecular fragment can be detected at 97. 5% confidence level. Consequently,the quantitative detection method established in this paper for the GM maize MON88017 has fairly high specificity,accuracy and sensitivity and this technology established in this paper can provide good technical support for the safety supervision of genetically modified organisms in China.

Critical Considerations in the Immunochemical Detection and Quantitation of Antigenic Biomarkers

The formation of covalent adducts as a result of the interaction of metabolically activated chemicals with host macromolecules is a common critical event in mutagenic, carcinogenic, and immunologic phenomena. Because of their antigenicity and their immunogenicity, covalent adducts may be detected using sensitive immunochemical techniques. The immunochemical approaches to biomonitoring and molecular dosimetry of DNA damage are particularly attractive because they allow sensitive quantitation of specific DNA adducts present in small samples and do not rely on the use of radiolabeled adducts. Two examples of biomarker immunoassay development are presented: an avidin/biotin-amplified ELISA for the major DNA adduct of the human bladder carcinogen 4-aminobiphenyl (ABP), and a particle concentration fluorescent immunoassay (PCFIA) for the major protein adduct associated with toxicity by the prototype hepatotoxin acetaminophen. The examples illustrate critical steps in the development of biomarker immunoassays which include selection of the relevant adduct, preparation of an appropriate immunogen, immunization, characterization of antisera, and development of application-specific sample processing techniques for biomarker quantitation. Immunochemical procedures may be combined with other analytical techniques to form hybrid systems which take advantage of both the antigenicity and the physical or chemical properties of a biomarker to achieve greater specificity and/or sensitivity. The future usefulness of these new tools of molecular epidemiology will depend on a compound-by-compound validation of methods and critical evaluation of the biologic importance of the particular antigenic biomarker as an indicator of exposure and as an indicator of risk.

DETECTION AND QUANTITATION OF TRACE ETHYL CARBAMATE IN ALCOLLOLIC BEVERAGES BY GC/GC AND GC/GC/MS

Analytical difficulties encountered in the determination of ethyl carbamate, a known cancinogen, in a wide variety of wines and spirits have been overcome by spe- cific, sensitive GC/GC and CC/CC/MS methods with a relatively shorter extraction procedure. The lowest detection limits were estimated to be 0. 1 and 0. 01μg/L for GC/GC and GC/GC/MS respectively. The RSD of the GC/GC method was 2. 5%.

Quantitative detection and comparison of sulfate glycosaminoglycans content in extracellular matrix of in vitro cultured epiphyseal, articular and rib chondrocytes

Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage

Gear Pitting Measurement by Multi-Scale Splicing Attention U-Net

The judgment of gear failure is based on the pitting area ratio of gear.Traditional gear pitting calculation method mainly rely on manual visual inspection.This method is greatly affected by human factors,and is greatly affected by the working experience,training degree and fatigue degree of the detection personnel,so the detection results may be biased.The non-contact computer vision measurement can carry out non-destructive testing and monitoring under the working condition of the machine,and has high detection accuracy.To improve the measurement accuracy of gear pitting,a novel multi-scale splicing attention U-Net(MSSA U-Net)is explored in this study.An image splicing module is first proposed for concatenating the output feature maps of multiple convolutional layers into a splicing feature map with more semantic information.Then,an attention module is applied to select the key features of the splicing feature map.Given that MSSA U-Net adequately uses multi-scale semantic features,it has better segmentation performance on irregular small objects than U-Net and attention U-Net.On the basis of the designed visual detection platform and MSSA U-Net,a methodology for measuring the area ratio of gear pitting is proposed.With three datasets,experimental results show that MSSA U-Net is superior to existing typical image segmentation methods and can accurately segment different levels of pitting due to its strong segmentation ability.Therefore,the proposed methodology can be effectively applied in measuring the pitting area ratio and determining the level of gear pitting.

Establishment and Preliminary Application of One-step Reverse Transcriptase Droplet Digital PCR Assay for Bovine Viral Diarrhea Virus

[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.

TOFD detection of shallow subsurface defects in aluminum alloy thin plates by half-skip mode-converted wave

Aluminum alloy is widely applied to the aerospace field.However,the inspection of thin plates using Time-of-Flight Diffraction(TOFD)technique is restricted by the near-surface dead zone because of the coupling between diffracted longitudinal wave and lateral wave.The halfskip mode-converted wave is introduced to decrease dead zone and detect defects in aluminum alloy thin plates by increasing ray path and propagation time.The quantitative correlation for the diffracted shear wave from longitudinal back-wall wave is deduced in combination with the acoustic path,realizing the accurate location of shallow subsurface defects.Simulated and experimental results indicate that the dead zone is decreased by 38% by the half-skip mode-converted wave,and the location errors are within 5% for the aluminum alloy plate with a thickness of 7.0 mm.Compared to other alternative TOFD techniques,half-skip mode-converted wave has better response amplitude and positioning accuracy,demonstrating strong applicability in TOFD inspection of thin plates.

Self-cleaning SERS sensor based on flexible Ni_(3)S_(2)/MoS_(2)@Ag@PDMS composites for label-free multiplex volatile organic compounds detection

Flexible self-cleaning surface-enhanced Raman scattering(SERS)sensors are highly desirable for the detection of various environmental pollutants,including volatile organic compounds(VOCs).However,achieving sensitive detection without labeling and ensuring efficient cyclic use remain significant challenges.Herein,we introduce a direct approach to create a versatile Ni_(3)S_(2)/MoS_(2)@Ag@PDMS(PDMS=polydimethylsiloxane)composite SERS substrate using chemical vapor deposition technology.The produced substrate shows outstanding performance,offering extremely low detection sensitivity(1.0×10^(−12)M 4-aminobenzenethiol)and high enhancement factors(approximately 107).The interactions between the rod-shaped Ni_(3)S_(2)/MoS_(2)@Ag heterostructure and the molecules facilitate the transfer of charge,resulting in an increased electric field enhancement of the exciton resonance.This has the dual benefit of providing a self-cleaning effect and enhancing SERS efficiency.Importantly,the substrate enables sensitive detection of VOCs gas molecules without the need for labels,achieving a minimum detectable concentration as low as 1 ppm for o-dichlorobenzene,due to the preconcentration effect of PDMS.Theoretical calculations further explain the combined effect of electromagnetic and chemical enhancement in this composite substrate.By utilizing the developed visual SERS barcode,quick multiple detection and analysis of mixtures can be accomplished.This flexible and versatile SERS technique has significant potential for on-site detection and analysis of environmental pollutants,opening the doors for the development of a wearable in-situ SERS sensing platform.

Defect Detection in c-Si Photovoltaic Modules via Transient Thermography and Deconvolution Optimization

Defects may occur in photovoltaic(PV)modules during production and long-term use,thereby threatening the safe operation of PV power stations.Transient thermography is a promising defect detection technology;however,its detection is limited by transverse thermal diffusion.This phenomenon is particularly noteworthy in the panel glasses of PV modules.A dynamic thermography testing method via transient thermography and Wiener filtering deconvolution optimization is proposed.Based on the time-varying characteristics of the point spread function,the selection rules of the first-order difference image for deconvolution are given.Samples with a broken grid and artificial cracks were tested to validate the performance of the optimization method.Compared with the feature images generated by traditional methods,the proposed method significantly improved the visual quality.Quantitative defect size detection can be realized by combining the deconvolution optimization method with adaptive threshold segmentation.For the same batch of PV products,the detection error could be controlled to within 10%.

On-treatment quantitative hepatitis B e antigen predicted response to nucleos(t)ide analogues in chronic hepatitis B

AIMTo investigate potential predictors for treatment response to nucleos(t)ide analogues (NAs) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. METHODSSeventy-six HBeAg-positive CHB patients received 96-wk NAs optimized therapy (lamivudine and adefovir dipivoxil) were studied retrospectively. Serum hepatitis B surface antigen, HBeAg, hepatitis B core antibody, hepatitis B virus (HBV) DNA and alanine aminotransferase levels were quantitatively measured before and during the treatment at 12 and 24 wk. Stepwise logistic regression analyses were performed to identify predictors for treatment response, and areas under the receiver operating characteristic curves (AUROC) of the independent predictors were calculated. RESULTSForty-three CHB patients (56.6%) achieved virological response (VR: HBV DNA ≤ 300 copies/mL) and 15 patients (19.7%) developed HBeAg seroconversion (SC) after the 96-wk NAs treatment. The HBeAg level (OR = 0.45, P = 0.003) as well as its declined value (OR = 2.03, P = 0.024) at 24-wk independently predicted VR, with the AUROC of 0.788 and 0.736, respectively. The combination of HBeAg titer 1.6 lg PEIU/mL at 24-wk predicted VR with a sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of 85%, 100%, 100% and 83%, respectively, and the AUROC increased to 0.923. The HBeAg level (OR = 0.37, P = 0.013) as well as its declined value (OR = 2.02, P = 0.012) at 24-wk also independently predicted HBeAg SC, with the AUROC of 0.828 and 0.814, respectively. The HBeAg titer 2.2 lg PEIU/mL at 24-wk predicted HBeAg SC with a sensitivity, specificity, PPV, NPV of 88%, 98%, 88% and 98%, respectively, and the AUROC reached 0.928. CONCLUSIONThe combination of HBeAg level and its declined value at 24-wk may be used as a reference parameter to optimize NAs therapy.

An effective surface-enhanced Raman scattering template based on gold nanoparticle/silicon nanowire arrays

A large-scale Si nanowire array （SiNWA） is fabricated with gold （Au） nanoparticles by simple metal-assisted chemical etching and metal reduction processes. The three-dimensional nanostructured Au/SiNWA is evaluated as an active substrate for surface-enhanced Raman scattering （SERS）. The results show that the detection limit for rhodamine 6G is as low as 10-7 M, and the Raman enhancement factor is as large as 105 with a relative standard deviation of less than 25%. After the calibration of the Raman peak intensifies of rhodamine 6G and thiram, organic molecules could be quantitatively detected. These results indicate that Au/SiNWA is a promising SERS-active substrate for the detection of biomolecules present in low concentrations. Our findings are an important advance in SERS substrates to allow fast and quantitative detection of trace organic contaminants.

Production of monoclonal antibodies against AFLM(Ver-1),a middle key protein involved in aflatoxin biosynthesis

Aflatoxins are potent carcinogens,mutagens and teratogens,and are harmful to both humans and animals.As many as 30 genes are involved in aflatoxin biosynthesis.Among them,aflM(ver-1)gene was predicted to encode a 28-kDa NADPH-dependent ketoreductase(AFLM),which catalyzed middle enzymatic steps in aflatoxin biosynthetic pathway.AFLM(Ver-1)was proved to be necessary for conversion of versicolorin A(VERA)to demethylsterigmatocystin(DMST)in aflatoxin B1(AFB1)biosynthesis.For these reasons,aflM gene was cloned and specific monoclonal antibodies for AFLM was developed to better define potential pathways of AFLM involved in AFB1 biosynthesis.Monoclonal antibodies 11B2-1D7 and 3G5-4E7 were successfully screened out by immunizing mouse.Immunoblot analysis revealed that both had high sensitivity and specificity to identify native AFLM protein in A.flavus with detection limit of 11 ng/mL and 8 ng/mL respectively.These results showed that it was suitable for quantitative detection of AFLM in A.flavus isolate.Further investigation revealed that aflatoxin accumulations of various A.flavus were not dependent on AFLM biosynthesis.Overall,this is the first report for development for AFLM monoclonal antibody development and application in A.flavus quantitative detection.