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Development of a Multiplex Real-Time Quantitative PCR assay for Detecting Vaginal Microbiota in Chinese Women-China,2021-2022

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China CDC weekly 2024年第31期6卷 793-798,I0001,I0002页

作者： Xiaolei Wang Yuanyuan Zhang Tingting Liu Chuan Song Xiaoyu Xue Jun Liu Hongxin Zhao Clinical Center for HIV/AIDS Beijing Ditan HospitalCapital Medical UniversityBeijingChina Beijing Institute of Infectious Diseases BeijingChina Beijing Key Laboratory of Emerging Infectious Diseases Institute of Infectious DiseasesBeijing Ditan HospitalCapital Medical UniversityBeijingChina National Center for Infectious Diseases Beijing Ditan HospitalCapital Medical UniversityBeijingChina Beijing Ditan Hospital Peking UniversityBeijingChina Department of Gynecology Beijing Ditan HospitalCapital Medical UniversityBeijingChina

Introduction:The Nugent score,limited by subjectivity and personnel requirements,lacks *** a precise and simple molecular test is therefore essential for detecting vaginal microbiota compositions and evaluating vaginal ***:We evaluated the vaginal health of Chinese women using quantitative polymerase chain reaction(qPCR)to target Lactobacillus crispatus(***),***,Gardnerella vaginalis(***),Atopobium vaginae(***),and Megasphaera *** vaginosis(BV)-related bacteria shared a fluorescent *** 16S rDNA sequencing as a reference standard,we evaluated and validated the diagnostic accuracy of the qPCR ***:Both qPCR and 16S rDNA sequencing demonstrated 90.5%concordance in segregating vaginal community state type(CST),as visualized through heatmaps and ***’s correlation analysis revealed strong correlations between the two methods in calculating the RA of ***(CST I),***(CST III),and BV-related bacteria(CST IV),with coefficients of 0.865,0.837,and 0.827,*** operating characteristic analysis showed that qPCR had significant diagnostic accuracy for CST I,CST III,and CST IV(molecular BV),with area under the curve values of 0.967,0.815,and 0.950,respectively,indicating strong predictive ***:Vaginal health can be evaluated using a single qPCR amplification experiment,making the multiplex qPCR assay a highly accurate tool for this purpose.

关键词： Vaginal assay precise

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Reversed-Phase-HPLC assay Method for Simultaneous Estimation of Sorbitol, Sodium Lactate, and Sodium Chlorides in Pharmaceutical Formulations and Drug Solution for Infusion

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American Journal of Analytical Chemistry 2024年第2期15卷 57-71页

作者： Sreenivas Pippalla Venugopal Komreddy Srinivasulu Kasa Vaishnavi Chintala Poluri Venkata Reddy Department of Chemistry Sikkim Professional University Gangtok India Analytical Research & Development Hikma Pharmaceuticals USA Inc. Bedford USA Department of Chemistry Osmania University Hyderabad India Analytical Research & Development Cambrex High Point High Point USA Department of Analytical Chemistry Annamalai University Annamalai Nagar India

A rapid, straightforward, sensitive, efficient, and cost-effective reverse-phase high-performance liquid chromatographic method was employed for the simultaneous determination of Sorbitol, Sodium Lactate, and Chlorides in a drug solution for infusion. Sorbitol, Sodium lactate, and Chloride are all officially recognized in the USP monograph. assay methods are provided through various techniques, with titrations being ineffective for trace-level quantification. Alternatively, IC, AAS, and ICP-MS, though highly accurate, are costly and often unavailable to most testing facilities. When considering methods, it’s important to prioritize both quality control requirements and user-friendly techniques. A simple HPLC simultaneous method was developed for the quantification of Chlorides, Sorbitol, and Sodium Lactate with a shorter run time. The separation utilized a Shimpack SCR-102(H) ion exclusion analytical column (7.9 mm × 300 mm, 7 μm), with a flow rate of 0.6 mL per min. The column compartment temperature was maintained at 40°C, and the injection volume was set at 10 μL, with detection at 200 nm. All measurements were conducted in a 0.1% solution of phosphoric acid. The analytical curves demonstrated linearity (r > 0.9999) in the concentration range of 0.79 to 3.8 mg per mL for Sodium Lactate (SL), 0.16 to 0.79 mg per mL for Sodium Chloride (SC), and 1.5 to 7.2 mg per mL for Sorbitol. Validation of the developed method followed the guidelines of the International Conference on Harmonization (ICH Q2B) and USP. The method exhibited precision, robustness, accuracy, and selectivity. In accelerated stability testing over 6 months, no significant variations were observed in organoleptic analysis and pH. Consequently, the developed method is deemed suitable for routine quality control analyses, enabling the simultaneous determination of Sodium Lactate, Sodium Chloride, and Sorbitol in pharmaceutical formulations and infusions.

关键词： Sorbitol Sodium Lactate and Chloride assay Analytical Validation HPLC

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A peptide-based assay discriminates individual antibody response to SARS-CoV-2

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Genes & Diseases 2022年第1期9卷 275-281页

作者： Immacolata Polvere Serena Voccola Gaetano Cardinale Maurizio Fumi Francesca Aquila Alfredina Parrell Jessica Raffaella Madera Romania Stilo Pasquale Vito Tiziana Zotti Dipartimento di Scienze e Tecnologie Universitàdegli Studi del SannioVia dei Mulini snc82100 BeneventoItaly Genus Biotech Universitàdegli Studi del SannioVia Appia s.n.c.82030 Apollosa(BN)Italy Azienda Ospedaliera San Pio Via Pacevecchia5382100 Benevento(BN)Italy

SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. Understanding the antibody response to SARS-CoV-2 is crucial for the development of vaccines, therapeutics and public health interventions. However, lack of consistency in methods used to monitor antibody response to SARS-CoV-2 leaves some uncertainty in our fine understanding of the human antibody response mounted following SARS-CoV-2 infection. We developed a peptide-based enzyme-linked immunosorbent assay (ELISA) by selecting 7 synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of SARS-CoV-2, which effectively detects the antibody response mounted by all COVID-19 convalescent tested. Strikingly, the assay shows a profound difference in antibody response among individual subjects, which may have a significant impact on disease severity. Together, our results define an efficient and specific serological assay to consistently measure the antibody response following SARS-CoV-2 infection, as well as help the design of vaccine and therapeuticals for prevention and treatment of COVID-19.

关键词： Antibodies assay COVID-19 ELISA Peptides SARS-Cov-2

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A homogeneous DNA assay by recovering inhibited emission of rare earth ions-doped upconversion nanoparticles

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Journal of Rare Earths 2019年第1期37卷 11-18,M0002页

作者： Yingzhu Zhou Yinghui Chen Hao He iayan Liao Hien T.T.Duong Maryam Parviz Dayong Jin Institute for Biomedical Materials and Devices School of Mathematical and Physical Sciences Faculty of Science University of Technology Sydney ARC Research Hub for Integrated Device for End-User Analysis at Low-levels Faculty of Science University of Technology Sydney The Faculty of Pharmacy The University of Sydney

Robust and easy-to-use kits specific for a particular DNA sequence are desirable for early detection of diseases. However, the major challenge with these tests is often the background fluorescence artifacts arising from biological species due to employing UV and visible range of light. Here, we have reported a near-infrared (NIR) fluorescence "turn-on" kit based on rare earth ions doped nanoparticles, upconversion nanoparticles (UCNPs), and gold nanoparticles (AuNPs), which forms a fluorescence-quencher pair,brought together by a hairpin structure through the formation of double-stranded DNA (dsDNA), with quenched upconversion luminescence. In the presence of analytes, the molecular beacon opens to push AuNPs away from UCNPs, with a distance longer than the efficient quenching distance, so that the inhibited upconversion emission will be restored. We demonstrated that this assay provides a homogeneous, facile, simple and highly selective HIV-1 based DNA detection system with restore efficiency up to 85%, and the detection limit of 5 nm.

关键词： Upconversion nanoparticles Gold nanoparticles LRET Homogenous assay DNA assay Fluorescence recovery

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Development of a homogenous assay based on fluorescent imprinted nanoparticles for analysis of nitroaromatic compounds

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Nano Research 2019年第12期12卷 3044-3050页

作者： Asma Elbelazi Francesco Canfarotta Joanna Czulak Michael J.Whitcombe Sergey Piletsky Elena Piletska Chemistry Department University of LeicesterLEI 7RHLeicesterUK Chemistry Department University of TripoliTripoliLibya MIP Diagnostics Ltd. Colworth Science ParkShambrookBedfordMK441LQUK

Herein we describe the development of a homogeneous assay for the detection of 4-nitroaniline(4-NA)and 2,4-dinitroaniline(2,4-diNA).Thisassay relies on fluorescent molecularly imprinted nanoparticles(nano MIPs)which,upon interaction with the target analytes,generate areduction in fluorescence emission intensity(quenching).This is due to a resp on sive fluorescent monomer(N-2-propenyl-(5-dimethylamino)-1-naphthalene sulphonamide)employed in the manufacture of the nanoMIPs which,by virtue of the imprinting process,is capable of selectiveinteraction with the target an alyte,thus giving rise to a quenching *** experiments showed excellent recognition propertiestoward the target *** optimal conditions,the fluorescence intensity of these nano MIPs decreased as the concentration of theimprinted analyte increased from 10 nM to 2.71 pM.A linear relation between the negative logarithm of 4-NA or 2,4-diNA concentrations and the fluorescence intensity for both nanosystems was found(R^2=0.991 and R^2=0.9895),with excellent sensitivity(limit of detection(LOD)=7 and 6 nM,respectively).Furthermore,both nanosystems have been successfully applied for detection of 4-NA or 2,4-diNA in tap water,withrecoveries between 90%to 100.6%and 92%to 100.3%,*** to the versatility of the imprinting process,this nanosystemholds the potential for further development of several optical sensors for many other compounds.

关键词： molecularly imprinted polymers quenching homogeneous assay nitroaromatic compounds

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Amine-rich carbon nitride nanoparticles:Synthesis,covalent functionalization with proteins and application in a fluorescence quenching assay

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Nano Research 2019年第8期12卷 1862-1870页

作者： Gabriele Capilli Simone Cavalera Laura Anfossi Cristina Giovannoli Marco Minella Claudio Baggiani Claudio Minero Department of Chemistry and NIS Center of Excellence University of Torino10125 TorinoItaly

Carbon nitride nanoparticles (CNNPs) have been employed as fluorescent sensing tools owing to their unique features, e.g. low cost production, high stability in water and high photoluminescence quantum yield. Here, an easy and versatile synthetic approach was exploited to design fluorescent nanoparticles with surface functionalities suitable for covalent binding to bioligands. High hydrophilic, brightly fluorescent CNNPs, rich of superficial amines, were obtained from the thermal condensation of urea and lysine (CNNP^Lys) and by tuning the precursor ratio and the heating time. Structure and size of the functionalized nanoparticles were characterized through infrared (IR) spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). Their optical properties were studied by ultraviolet-visible (UV-Vis) and fluorescence spectroscopy. The superficial primary amino groups, furnished by the lysine co-precursor, enabled for covalently linking CNNP^Lys to model proteins. The CNNP^Lys-protein conjugates excited under UV irradiation emit in the 400-450 nm visible range (quantum yield 24%). The applicability of CNNP^Lys as novel fluorescent probes was demonstrated by a fluorescence quenching assay, in which gold nanoparticles (GNPs) were attached to Staphylococcal protein A and employed to quench the CNNP^Lys fluorescence by Forster resonant energy transfer (FRET). The quenching occurred upon formation of the specific binding between the GNP-linked protein A and CNNP^Lys-tagged immunoglobulins, while the inhibition of the binding resulted in the recovery of CNNP^Lys luminescence. The synthetic strategy, based on combining a "conjugated polymer"-forming unit (urea) and a co-precursor able to provide the desired functional group (lysine), allows designing innovative materials for the development of new generation fluorescence biosensors in which easily functionalized fluorophores are needed.

关键词： fluorescent probe carbon nitride nanoparticles quenching assay fluorescent materials g-C3N4 Forster resonant energy transfer (FRET)

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Effects of ultrasonic treatment on dithiothreitol(DTT) assay measurements for carbon materials

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Journal of Environmental Sciences 2019年第10期31卷 51-58页

作者： Haotian Jiang Yun Xie Yanli Ge Hong He Yongchun Liu State Key Joint Laboratory of Environment Simulation and Pollution Control Research Center forEco-Environmental Sciences Chinese Academy of Sciences Beijing 100085 China Aerosol and Haze Laboratory Advanced Innovation Center for Soft Matter Science and Engineering Beijing University of Chemical Technology Beijing 100029 China University of Chinese Academy of Sciences Beijing 100049 China Key Laboratory of Environmental Biotechnology Research Center for Eco-Environmental Sciences Chinese Academy of Sciences Beijing 100085 China Center for Excellence in Urban Atmospheric Environment Institute of Urban Environment Chinese Academy of Sciences Xiamen 361021China

The dithiothreitol(DTT)assay is the most commonly used method to quantify the oxidative potential of fine ***,the reported DTT decay rates of carbon black(CB)materials vary greatly among different *** might have resulted from either the intrinsic toxicity of CB or the unsuitability of the DTT assay protocol for CB *** the current study,the protocol of the DTT assay for CB materials has been carefully *** was found that the dispersion degree of CB particles in water has a great influence on the DTT decay rate of CB *** CB particles(special black 4A(SB4A)and Printex U)and single-walled carbon nanotube tube(SWCNT),the DTT decay rate after sonication for 10 min became 4.2,4.6 and 1.7 times higher than that without *** rate continued to grow as a function of ultrasound time up to 30 min of *** the concentration of soluble transition metals and surface oxygen-containing species such as carbonyls increased slightly with sonication,they had no significant effects on the measured DTT activity,while the increase in the dispersion degree of aggregates was found to play a vital role in the observed enhancement of the DTT decay rates for different CB *** on our results,30 min of sonication is recommended for sample dispersion when measuring the DTT decay rate of CB materials.

关键词： Sonication Dispersion Dithiothreitol(DTT) assay Carbon nanomaterials

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A novel non-radioactive assay for HIV-RT(RdDp)based on pyrosequencing for high-throughput drug screening

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Protein & Cell 2010年第3期1卷 284-290页

作者： Chang Zhang Yang Wu Yuna Sun Chuan Hong Kehui Xiang Yu Guo Mark Bartlam Zhiyong Lou Tsinghua-Nankai-IBP Joint Research Group for Structural Biology Tsinghua UniversityBeijing 100084China National Laboratory of Macromolecules Institute of BiophysicsChinese Academy of SciencesBeijing 100101China College of Life Sciences and Tianjin State Laboratory of Protein Sciences Nankai UniversityTianjin 300071China

Current in vitro assays for the activity of HIV-RT(reverse transcriptase)require radio-labeled or chemically modified nucleotides to detect reaction ***,these assays are inherently end-point measurements and labor *** we describe a novel non-radioactive assay based on the principle of pyrosequencing coupledenzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate(PPi),which is generated during nascent strand *** results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid highthroughput drug screening targeting anti-HIV therapies due to its high speed and ***,this assay can be used to measure primase activity in an easy and sensitive manner,which suggests that this novel approach could be wildly used to analyze the activity of PPi-generated and ATP-free enzyme reactions.

关键词： HIV-RT(RdDp) assay non-radioactive high throughput drug screening

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Development and Evaluation of a Universal and Supersensitive NS1-Based Luciferase Immunosorbent assay to Detect Zika Virus-Specific IgG

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Virologica Sinica 2020年第1期35卷 93-102页

作者： Tianyu Wang Ying Zhan De Wu Zhihai Chen Wei Wu Yao Deng Wenling Wang Wenjie Tan Shixing Tang Guangdong Provincial Key Laboratory of Tropical Disease Research Department of EpidemiologySchool of Public HealthSouthern Medical UniversityGuangzhou 510515China NHC Key Laboratory of Biosafety National Institute for Viral Disease Control and PreventionChina CDCBeijing 102206China Key Laboratory for Repository and Application of Pathogenic Microbiology Research Center for Pathogens Detection Technology of Emerging Infectious DiseasesGuangdong Provincial Center for Disease Control and PreventionGuangzhou 511430China The National Clinical Key Department of Infectious Disease Beijing Ditan HospitalCapital Medical UniversityBeijing 100015China

Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay(LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase(NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization(MN) *** and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus(DENV), Japanese encephalitis virus(JEV), and hepatitis C virus(HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172–352 amino acids(aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV Ig G detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV Ig G in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.

关键词： Zika virus(ZIKV) NS1 Luciferase immunosorbent assay IgG Detection

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Silver nanoparticle labeled immunoresonance scattering spectral assay for trace fibrinogen

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Science China Chemistry 2007年第3期50卷 345-350页

作者： JIANG ZhiLiang CHEN YuanYuan LIANG AiHui TAO HuiLin TANG NingLi ZHONG FuXin School of Environment and Resource Guangxi Normal UniversityGuilin 541004China Department of Material and Chemical Engineering Guilin University of TechnologyGuilin 541004China

Silver nanoparticles in size of 8.0 nm was prepared by the trisodium citrate and used to label goat anti-human fibrinogen. In the pH 5.8 Na2HPO4-NaH2O4 buffer solution (PBS) and in the presence of polyethylene glycol (PEG) and KCl, the immune reaction between silver-labeled goat anti-human fibrinogen and fibrinogen took place and led the resonance scattering intensity at 465 nm (I465) to decreasing. The I465 decreased intensity was linear to the fibrinogen concentration in the range from 0.067 to 1.67 μg/mL, with a detection limit of 0.024 μg/mL. This method was applied to determination of fibrinogen in human plasma, with satisfactory results.

关键词： silver-nanoparticle fibrinogen goat anti-human immune fibrinogen serum resonance scattering spectral assay

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