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.

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.

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%.

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.

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.

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

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.

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.

Preliminary study on qualitative and quantitative detection of norfloxacin based on terahertz spectroscopy

Norfloxacin is a kind of quinolone bactericide which is widely used in food industry,livestock feed and medicine area.At present,how to develop a rapid,convenient and accurate technique for qualitative and quantitative detection of norfloxacin is still a challenge.In this study,Terahertz Time-domain Spectroscopy technology(THz-TDS)was used to explore qualitative and quantitative analysis of norfloxacin.Forty-five norfloxacin samples with different levels were prepared.The pure norfloxacin powder was mixed with polyethylene(PE)powder together and made into tablets by tablet-making machine.The minimum detection concentration was 10%and the maximum detection concentration evaluated in the paper was 90%.Then,terahertz(THz)spectra of each sample were measured on K15 THz-TDS equipment.The multiple linear regressions and the partial least squares regression algorithms were used to establish a model to make quantitative analysis.From the results,two typical fingerprint peaks of norfloxacin in terahertz band were observed at 0.944 THz and 1.206 THz,and the correlation coefficient and the root mean square error of the established model reached 0.9908 and 0.0481,respectively,which showed that the model performed well for the prediction.This preliminary study indicates that THz spectroscopy has great potential for future screening applications to detect the presence of norfloxacin residues in food,livestock feed and medicine industries.

Quantitative detection of Cymbidium mosaic virus by real time PCR

The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.

Immune fluorescence test strips based on quantum dots for rapid and quantitative detection of carcino-embryonic antigen

At present,many researchers focused on the point-of-care testing（POCT）,a method of disease markers detection without large-scale instruments and specialized persons.However,most POCT diagnostic methods were suffered from poor detection sensitivity or inefficiency in quantitative detection.Herein,we developed a newly QD-immune fluorescence test strips（QD-IFTS） based on quantum dots（QDs） as the fluorescence nanocarrier to prepare the immune fluorescence probes in the classical immunochromatography detection system for sensing carcino-embryonic antigen（CEA）,a kind of glycoprotein produced by intestinal tissue and a broad spectrum of tumor marker for cancer diagnosis.And we designed a homemade strips fluorescence reader for detection of fluorescence intensity of QDs on the QD-IFTS.Under the optimized reaction conditions,chromatographic time of the newly QD-IFTS was only25 min,sample volume of the newly QD-IFTS was only 40 m L and the LOD of the newly QD-IFTS was 0.72 ng/m L.In addition,the efficiency and robustness of the newly QD-IFTS were confirmed by successfully application in 300 clinical serum samples,and the results revealed great potential in clinical POCT of other biomarkers.

QUANTITATIVE DETECTION OF AMPLIFICATION OF PROTO-ONCOGENES IN BREAST CANCER

In the present study, dot-blot hybridization, serial dilution analysis and densitomctric scanning were used to detect amplification of proto- oncogenes including c-erbB2, c-myc, int-2 and c-Ha-ras in 104 paraffin-embedded breast cancers. Expression of c-erbB2 was also examined by immunohistochemistry. Amplification of c-erbB2. c-myc and int-2 genes was found in 34.7%, 17.8% and 11.9% of breast cancers respectively. However amplification of c-Ha-ras was not detected in all cases. In 11.9% of cases co-amplification of two or more oncogenes was observed. Positive immunostain-ing of c-erbB2 was seen in 23.8% of the cases and it was significantly associated, but not always corresponding to the amplification of the gene. There was no difference between primary and metastatic breast cancer in the alterations of proto-oncogenes examined in this study, which suggested that the amplification and overexpression of these proto-oncogenes occured prior to and maintained in the process of metastasis of breast cancer. Statistical analysis showed that high-scale of immunopositive staining of c-erbB2 and high-fold co-amplification of proto-oncogenes were significantly correlated with large size of the tumour and the number of involved lymph nodes. Our results indicate that the alterations of multiple oncogenes are involved in the development of breats cancer and some of them may have prognostic importance for breast cancer patients.

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.

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.

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.

Separation and quantitative detection of fermentationγ-polyglutamic acid

γ-Polyglutamic acid(γ-PGA)is a kind of polymer material with good biocompatibility and degradability,which is widely used in food,medicine,environmental protection,cosmetics and agriculture.Asγ-PGA is produced by microbial fermentation,its separation,purification and quantitative detection are very important.In this study,the separation methods such as organic solvent precipitation,copper salt precipitation and membrane separation precipitation were mainly discussed,and the quantitative determination methods such as spectrophotometer,enzyme method and viscosity method ofγ-PGA were discussed and compared.Furthermore,Molecular imprinting as a new method for the separation ofγ-PGA has also been analyzed and discussed in order to provide a reference for the separation and purification ofγ-PGA.

Development of an Event-specific Quantitative PCR for Genetically Modified Maize (Zea mays) Event NK603

[ Objective ] The aim of this study was to develop a quantitative PCR detection method for genetically modified maize event NK603, so as to provide ba- sis for quantitative analysis of event NK603. [ Methods ] A quantitative PCR detection method for genetically modified maize event NK603 was developed using primers and Taqman probe designed according to the flanking sequence of event NK603, which was then adopted to detect the samples containing 2% NK603 stand- ard （with uncertain quantity of 10% ）. [ Results ] The slope of standard curve ranged between -3.6 and -3.1, and the correlation coefficient was higher than 0. 99. The amplification efficiency of this method reached 100.2%, fallen between 90% and 110%. The detected quantity of the experimental sample was 1.9%, closer to the true quantity （2%）. [ Conclusion] This quantitative PCR detection method for genetically modified maize event NK603 is very precise and can be a- dopted in routine testing analysis.

A Preliminary Study of The Value of Quantitative Testing of TP-DNA In The Treatment of Patients with Syphilis

Objectives: To explore the relationship betweenquantitative Treponema pallidum DNA (TP-DNA) PCR testingand the Toludine Red Unheated Serum Test (TRUST) inpatients with syphilis before and after treatment, and evaluatethe clinical value of quantitative TP-DNA testing in thediagnosis and treatment evaluation of syphilis. Methods: 29 patients with primary (12 cases) or secondary(17 cases) syphilis, who met the criteria set for this study wererecruited as subjects. All patients were treated with 2.4 millionunits benzathine penicillin IM weekly for 3 weeks.Quantitative tests of TP-DNA in the patients' plasma wereperformed using FQ-PCR before and after the treatment.Serologic tests including TRUST and TPPA were alsoperformed. Results: Before the treatment, 9 out of 12 primary syphilispatients (75%) and all secondary syphilis patients (17/17)tested positive for Treponema pallidum (TP) by TP-DNAtesting. The average quantitative test values of TP-DNA inprimary and secondary syphilis patients were (3.38±2.34)×10~4and (5.73±1.33)×10~6 copies/ml, respectively. After threemonths of treatment, 1 of the 9 primary and 5 out of 17secondary syphilis patients were positive upon TP-DNAtesting, respectively. The average quantities of TP-DNA were2.01×10~2 copies/ml in primary and 5.87×10~2 copies/ml insecondary syphilis patients with positive TRUST and TP-DNAtests, and 3.09×10~2 copies/ml for those with negative TRUSTrespectively. After nine months of treatment, all the primaryand secondary syphilis patients were negative upon TP-DNAtesting, while all primary and 14 of 17 (82.35%) secondarysyphilis patients showed negative TRUST results. Conclusion: That the results of TP-DNA tests are notconsistent with those or TRUST before and after treatmentindicates that quantitative TP-DNA testing may have valuableclinical significance in the early diagnosis and evaluation oftreatment regimens for syphilis.

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%.