Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential i...Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential in the development,registration,and quality control of rhGH pharmaceutical products[2].However,the existing in vivo bioassay procedure based on somatropin-induced weight gain in rats is complicated,and the use of a rat cell line-based approach(Nb2-11 bioassay),which measures the production of adenosine triphosphate(ATP)as a direct indicator of cell growth,has a low mechanism of action(MOA)relevance.Therefore,novel rhGH bioassays are still needed.To this end,we developed a reporter gene assay(RGA)based on the GH/insulin-like growth factor-1(IGF-1)axis.展开更多
Ostreopsis cf.ovata is a marine benthic dinoflagellate in tropical and temperate seas and can produce potent toxic compounds.The existence of O.cf.ovata has been found in the Chinese coastal areas,but studies on its t...Ostreopsis cf.ovata is a marine benthic dinoflagellate in tropical and temperate seas and can produce potent toxic compounds.The existence of O.cf.ovata has been found in the Chinese coastal areas,but studies on its toxicity are very few.This study investigated the toxicity of the O.cf.ovata(TIO991)isolated from Weizhou Island in the South China Sea by using methanol and chloroform to extract toxic compounds from the algal cells cultured indoor.Experiments on mouse acute toxicity showed that the crude methanol extract(CME)of O.cf.ovata caused the death of mice in 16–18 min.Furthermore,CME inhibited the cell reproduction of human neuroblastoma cells(BE(2)-M17 cells)by Cell Counting Kit-8 with a dose-and time-effect relationship and caused cell death in the form of cell necrosis.We found that CME had strong hemolytic activity and was significantly inhibited by ouabain,indicating that CME might contain palytoxins.By contrast,the crude chloroform extract of O.cf.ovata was relatively weak in toxicity as obtained in our experiments on mouse acute toxicity,cytotoxicity,and hemolytic activity.This suggests that the algae may raise the potential threat to marine ecosystems and public health.展开更多
Succinylcholine(SC)is a widely used depolarizing muscle relaxant,but improper use can lead to arrhythmias and,in severe cases,pose a life-threatening risk.Additionally,some criminals exploit SC for illicit activities....Succinylcholine(SC)is a widely used depolarizing muscle relaxant,but improper use can lead to arrhythmias and,in severe cases,pose a life-threatening risk.Additionally,some criminals exploit SC for illicit activities.Therefore,rapid SC detection is paramount for clinical practice and public safety.Currently,however,limited methods are available for the rapid detection of SC.A fluorescent indicator displacement assay sensor based on molecular recognition of an amide naphthotube was developed.This sensor enabled the rapid fluorescent detection of SC through competitive binding between SC and methylene blue with the amide naphthotube.The sensor exhibited exceptional sensitivity with a detection limit as low as 1.1μmol/L and a detection range of 1.1~60μmol/L,coupled with outstanding selectivity and robust stability.Furthermore,this sensor accurately determined SC levels in biological samples such as serum.In summary,this research provides a new solution for the rapid and accurate sensing of SC in complex matrices and offers new insights for the swift identification and detection of toxins.展开更多
African swine fever(ASF),caused by the African swine fever virus(ASFV),has brought enormous economic loss and represents a major threat to the global pig industry(Vergne et al.2017).ASFVs are divided into 24 genotypes...African swine fever(ASF),caused by the African swine fever virus(ASFV),has brought enormous economic loss and represents a major threat to the global pig industry(Vergne et al.2017).ASFVs are divided into 24 genotypes based on their B646L gene,with only genotypes Ⅰ and Ⅱ circulating globally(Dixon et al.2019).展开更多
Dear Editor,The frequent emergence of life-threatening infectious diseases has posed a constant global threat in recent decades.Since the first identification of Ebola Virus Disease(EVD)in the Democratic Republic of C...Dear Editor,The frequent emergence of life-threatening infectious diseases has posed a constant global threat in recent decades.Since the first identification of Ebola Virus Disease(EVD)in the Democratic Republic of Congo(then Zaire)in 1976,there have been approximately 40 outbreaks.展开更多
Background:Organ transplantation recipients encounter significant risks from acute or chronic infections that threaten graft survival.BK virus(BKV)and JC virus(JCV)are 2 prominent opportunistic infection viruses,and t...Background:Organ transplantation recipients encounter significant risks from acute or chronic infections that threaten graft survival.BK virus(BKV)and JC virus(JCV)are 2 prominent opportunistic infection viruses,and they may cause polyomavirus-associated nephropathy and graft kidney loss in patients who are in an immunosuppressed state after kidney transplantation.Hence,timely detection and sustained monitoring of the viral load are indispensable.However,the current diagnostic methods remain limited,and the development of new molecular detection technology is extremely urgent.Methods:The sequences and concentrations of clustered regularly interspaced short palindromic repeats(CRISPR)RNA(crRNA),the concentration of Cas13a,and the primers for recombinase polymerase amplification(RPA)were optimized for BKV and JCV detection.Next,a novel microfluidic dual-droplet chip was designed and fabricated,and it was integrated with CRISPR(ddCRISPR)to simultaneously qualitatively detect BKV and JCV.Subsequently,the ddCRISPR assay was verified using clinical samples.Then,a lateral flow strip combined with CRISPR(LFCRISPR)was developed for the detection of BKV and JCV in resource-limited settings.Results:A one-pot RPA-CRISPR reaction system was established and optimized for BKV and JCV detection.ddCRISPR can simultaneously and rapidly detect BKV and JCV with high sensitivity(10 copies/ml for BKV and 1 copy/ml for JCV),and provide absolute quantification,which is suitable for viral load detection and conducive to personalized and precise treatment for organ transplant recipients.LFCRISPR simplified the operational process through a simple visual readout,facilitating virus screening after organ transplantation.Conclusion:These platforms incorporate molecular testing into the transplantation treatment model,thereby reducing costs,prolonging the survival time of the graft,improving the clinical outcomes of postoperative management in kidney transplantation,and enhancing the patients’quality of life.展开更多
A novel coronavirus of zoonotic origin(SARSCoV-2)has recently been recognized in patients with acute respiratory disease.COVID-19 causative agent is structurally and genetically similar to SARS and bat SARS-like coron...A novel coronavirus of zoonotic origin(SARSCoV-2)has recently been recognized in patients with acute respiratory disease.COVID-19 causative agent is structurally and genetically similar to SARS and bat SARS-like coronaviruses.The drastic increase in the number of coronavirus and its genome sequence have given us an unprecedented opportunity to perform bioinformatics and genomics analysis on this class of viruses.Clinical tests like PCR and ELISA for rapid detection of this virus are urgently needed for early identification of infected patients.However,these techniques are expensive and not readily available for point-of-care(POC)applications.Currently,lack of any rapid,available,and reliable POC detection method gives rise to the progression of COVID-19 as a horrible global problem.To solve the negative features of clinical investigation,we provide a brief introduction of the general features of coronaviruses and describe various amplification assays,sensing,biosensing,immunosensing,and aptasensing for the determination of various groups of coronaviruses applied as a template for the detection of SARS-CoV-2.All sensing and biosensing techniques developed for the determination of various classes of coronaviruses are useful to recognize the newly immerged coronavirus,i.e.,SARS-CoV-2.Also,the introduction of sensing and biosensing methods sheds light on the way of designing a proper screening system to detect the virus at the early stage of infection to tranquilize the speed and vastity of spreading.Among other approaches investigated among molecular approaches and PCR or recognition of viral diseases,LAMP-based methods and LFAs are of great importance for their numerous benefits,which can be helpful to design a universal platform for detection of future emerging pathogenic viruses.展开更多
Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient c...Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies.Therefore,a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101.In this study,a sensitive bioanalytical method was developed and validated,using a Quanterix single molecular array(Simoa)assay.Moreover,to thoroughly assess the platform,enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed,and their performance was compared with that of this novel technology platform.The assay was validated in compliance with the current guidelines.Measurements with the Simoa assay were precise and accurate,presenting a valid assay range from 6.55 to 4000 pg/mL.The intra-and inter-run accuracy and precision were within-19.3%to 15.3%and 5.5%to 17.0%,respectively.S3101 was stable in human serum for 280 days at-20℃and-70℃,for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature(22℃-28℃),respectively,and after five and two freeze-thaw cycles at-70℃and-20oC,respectively.The Simoa assay also demonstrated sufficient dilution linearity,assay sensitivity,and parallelism for quantifying S3101 in human serum.The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.展开更多
Objective To perform the modulation of an assay system for the sensory integration of 2 sensory stimuli that inhibit each other.Methods The assay system for assessing the integrative response to 2 reciprocally-inhibit...Objective To perform the modulation of an assay system for the sensory integration of 2 sensory stimuli that inhibit each other.Methods The assay system for assessing the integrative response to 2 reciprocally-inhibitory sensory stimuli was modulated by changing the metal ion barrier.Moreover,the hen-1,ttx-3 and casy-1 mutants having known defects in integrative response were used to evaluate the modulated assay systems.Based on the examined assay systems,new genes possibly involved in the sensory integration control were identified.Results In the presence of different metal ion barriers and diacetyl,locomotion behaviors,basic movements,pan-neuronal,cholinergic and GABAergic neuronal GFP expressions,neuronal development,structures of sensory neurons and interneurons,and stress response of nematodes in different regions of examined assay systems were normal,and chemotaxis toward different concentrations of diacetyl and avoidance of different concentrations of metal ions were inhibited.In the first group,most of the nematodes moved to diacetyl by crossing the barrier of Fe2＋,Zn2＋,or Mn2＋.In the second group,almost half of the nematodes moved to diacetyl by crossing the barrier of Ag＋,Cu2＋,Cr2＋,or Cd2＋.In the third group,only a small number of nematodes moved to diacetyl by crossing the barrier of Pb2＋ or Hg2＋.Moreover,when nematodes encountered different metal ion barriers during migration toward diacetyl,the percentage of nematodes moving back and then turning and that of nematodes moving straight to diacetyl were very different.With the aid of examined assay systems,it was found that mutations of fsn-1 that encodes a F-box protein,and its target scd-2 that encodes a receptor tyrosine kinase,caused severe defects in integrative response,and the sensory integration defects of fsn-1 mutants were obviously inhibited by scd-2 mutation.Conclusion Based on the nematode behaviors in examined assay systems,3 groups of assay systems were obtained.The first group may be helpful in evaluating or identifying the very subtle deficits in sensory integration,and the third group may be useful for the final confirmation of sensory integration defects of mutants identified in the first or the second group of assay systems.Furthermore,the important association of sensory integration regulation with stabilization or destabilization of synaptic differentiation may exist in C.elegans.展开更多
[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD...[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.展开更多
A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELI...A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).展开更多
Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofl...Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.展开更多
A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an...A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.展开更多
Objective To detect the response of lymphocytes to radiation in untreated breast cancer patients with three different genetic assays. Methods Blood samples were collected from 25 untreated patients and 25 controls. Ea...Objective To detect the response of lymphocytes to radiation in untreated breast cancer patients with three different genetic assays. Methods Blood samples were collected from 25 untreated patients and 25 controls. Each blood sample was divided into two parts： one was irradiated by 3-Gy X-ray （irradiated sample）, the other was not irradiated （non-irradiated sample）. The radiosensitivity of lymphocytes was assessed by comet assay, cytokinesis-block micronucleus （CBMN） assay and 6-TG-resistant cells scored （TG） assay. Results The baseline values of micronucleated cell frequency （MCF） and micronucleus frequency （MNF） in the patients were significantly higher than those in the controls （P〈0.01）, and 3-Gy X-ray induced genetic damage to lymphocytes in the patients increased significantly as compared with that in the controls as detected with the three genetic assays （P〈0.01）. The proportion of radiosensitive cases in the patient group was 48% for the mean tail length （MTL）, 40% for the mean tall moment （MTM）, 40% for MCF, 44% for MNF, and 48% for mutation frequencies of the hprt gene （Mfs-hprt）, respectively, whereas the proportion of radiosensitive cases in the control group was only 8% for all the parameters. Conclusion The difference in the lymphocyte radiosensitivity between the breast cancer patients and the controls is significant. Moreover, there are wide individual variations in lymphocyte radiosensitivity of patients with breast cancer. In some cases, the radiosensitivity of the same patient may be different as detected with the different assays. It is suggested that multiple assays should be used to assess the radiosensitivity of patients with breast cancer before therapy.展开更多
BACKGROUND Current diagnosis of hepatitis C virus(HCV)infection requires two sequential steps:testing for anti-HCV followed by HCV RNA PCR to confirm viremia.We have developed a highly sensitive and specific HCV-antig...BACKGROUND Current diagnosis of hepatitis C virus(HCV)infection requires two sequential steps:testing for anti-HCV followed by HCV RNA PCR to confirm viremia.We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA)for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera:10 without HCV or HIV infection;54 with viremic HCV monoinfection;38 with viremic HCV/HIV coinfection;and 45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding,it was 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test.In five sera with HCV infection,HCV RNA was as low as 50-59 IU/mL,and four out of five tested positive for HCV-Ags EIA.Likewise,it was also 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection,regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL.It is highly sensitive and specific in the setting of HIV coinfection,regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.展开更多
Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania an...Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stressinducible protein-1 with/without adjuvant.After three vaccinations,mice were challenged by Leishmania tropica promastigotes.Two months after challenge,the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods.Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium.For real-time PCR,DNA of the lymph nodes was extracted,equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers.The data of the two methods were compared by appropriate statistical methods.Results:Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice.In addition,wherever parasite load of a group was estimated high(or low)by one method,the estimated parasite load by another method was the same,although statistically significant differences were found between some groups.Conclusions:Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups.However,due to the lower errors and faster process,the real-time PCR method is preferred.展开更多
In order to compare sensitivity of EIA and RIA assay kits for hepatitis B and C virus (HBV and HCV, respectively) infection markers, 100 serum samples in total were collected form 50 adult women each in urban and rura...In order to compare sensitivity of EIA and RIA assay kits for hepatitis B and C virus (HBV and HCV, respectively) infection markers, 100 serum samples in total were collected form 50 adult women each in urban and rural areas in northeast China. The number of positive cases to the three infection markers on HBV (i.e., HBsAg +, anti HBs +, and anti HBc +) and the one on HCV (anti HCV +) were examined in two laboratories, i.e., in Laboratory A with EIA kits produced in China and in Laboratory B with RIA kits. HCV infection positivity (anti HCV +) was examined by EIA kits in both laboratories, but from different sources in and outside of China, respectively. The assay in Laboratory A gave 2 HBsAg + cases out of the 100 cases examined, whereas there were 9 positive cases in Laboratory B. In contrast, 19 cases were positive to anti HCV when examined in Laboratory A, and there were 3 cases in Laboratory B. Thus, the kits used in Laboratory A gave fewer HBsAg + and more anti HCV + cases than the kits used in Laboratory B. The prevalence of anti HBs + or anti HBc + and cases did not differ when assayed in the two laboratories with EIA and RIA kits, respectively. The agreement of positive and negative findings between the two sets of testing were 93%, 93%, 93%, 86% and 82% for HBsAg, anti HBs, anti HBc, HBV (i.e., either positive to anyone of the three markers or negative to all three markers), and anti HCV, respectively. The implication of the observation on epidemiology on HBV and HCV infection prevalence was discussed.展开更多
基金supported by the first batch of grants from the State Key Laboratory of Drug Regulatory Science,China(Grant No.:2023SKLDRS0108).
文摘Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential in the development,registration,and quality control of rhGH pharmaceutical products[2].However,the existing in vivo bioassay procedure based on somatropin-induced weight gain in rats is complicated,and the use of a rat cell line-based approach(Nb2-11 bioassay),which measures the production of adenosine triphosphate(ATP)as a direct indicator of cell growth,has a low mechanism of action(MOA)relevance.Therefore,novel rhGH bioassays are still needed.To this end,we developed a reporter gene assay(RGA)based on the GH/insulin-like growth factor-1(IGF-1)axis.
基金Supported by the Special Research for the Science and Technology Basic Resources Investigation Program of China(No.2018FY100200)the National Natural Science Foundation of China(No.42176206)the Natural Science Foundation of Shandong Province(No.ZR2021MD071)。
文摘Ostreopsis cf.ovata is a marine benthic dinoflagellate in tropical and temperate seas and can produce potent toxic compounds.The existence of O.cf.ovata has been found in the Chinese coastal areas,but studies on its toxicity are very few.This study investigated the toxicity of the O.cf.ovata(TIO991)isolated from Weizhou Island in the South China Sea by using methanol and chloroform to extract toxic compounds from the algal cells cultured indoor.Experiments on mouse acute toxicity showed that the crude methanol extract(CME)of O.cf.ovata caused the death of mice in 16–18 min.Furthermore,CME inhibited the cell reproduction of human neuroblastoma cells(BE(2)-M17 cells)by Cell Counting Kit-8 with a dose-and time-effect relationship and caused cell death in the form of cell necrosis.We found that CME had strong hemolytic activity and was significantly inhibited by ouabain,indicating that CME might contain palytoxins.By contrast,the crude chloroform extract of O.cf.ovata was relatively weak in toxicity as obtained in our experiments on mouse acute toxicity,cytotoxicity,and hemolytic activity.This suggests that the algae may raise the potential threat to marine ecosystems and public health.
文摘Succinylcholine(SC)is a widely used depolarizing muscle relaxant,but improper use can lead to arrhythmias and,in severe cases,pose a life-threatening risk.Additionally,some criminals exploit SC for illicit activities.Therefore,rapid SC detection is paramount for clinical practice and public safety.Currently,however,limited methods are available for the rapid detection of SC.A fluorescent indicator displacement assay sensor based on molecular recognition of an amide naphthotube was developed.This sensor enabled the rapid fluorescent detection of SC through competitive binding between SC and methylene blue with the amide naphthotube.The sensor exhibited exceptional sensitivity with a detection limit as low as 1.1μmol/L and a detection range of 1.1~60μmol/L,coupled with outstanding selectivity and robust stability.Furthermore,this sensor accurately determined SC levels in biological samples such as serum.In summary,this research provides a new solution for the rapid and accurate sensing of SC in complex matrices and offers new insights for the swift identification and detection of toxins.
基金supported by the National Natural Science Fund for Distinguished Young Scholars of China(31925036 and 32025034)the Shandong Provincial Key R&D Program,China(2021LZGC001)+3 种基金the Biological Breeding-Major Projects,Institute of Zoology,Chinese Academy of Sciences and Institute of Animal Science,Chinese Academy of Agricultural Sciences(2023ZD04074 and 2023ZD0404604)the Special Project of Longhu Laboratory,China(LHLab_ZD20230012)the National Natural Science Foundation of China(32230100,32330099 and 32201257)the National Key Research and Development Program of China(2020YFA0509503,2022YFF0710703,2021YFA0805902 and 2022XAGG0121,2022YFF1002803).
文摘African swine fever(ASF),caused by the African swine fever virus(ASFV),has brought enormous economic loss and represents a major threat to the global pig industry(Vergne et al.2017).ASFVs are divided into 24 genotypes based on their B646L gene,with only genotypes Ⅰ and Ⅱ circulating globally(Dixon et al.2019).
基金financially supported by the National Key Research and Development Program of China(grant No.2021YFF0703600)the Science and Technology Development Program of Jilin Province(Grant20250601001RC).
文摘Dear Editor,The frequent emergence of life-threatening infectious diseases has posed a constant global threat in recent decades.Since the first identification of Ebola Virus Disease(EVD)in the Democratic Republic of Congo(then Zaire)in 1976,there have been approximately 40 outbreaks.
基金supported by the National Key Research and Development Program(2023YFC2306200)the National Natural Science Foundation of China(82472374)+1 种基金the Cross-Research Fund of Biomedical Engineering of Shanghai Jiaotong University(YG2024LC02)the Clinical Excellence project of Shanghai Key Laboratory of Nucleic Acid Chemistry and Nanomedicine(2024ZY008).
文摘Background:Organ transplantation recipients encounter significant risks from acute or chronic infections that threaten graft survival.BK virus(BKV)and JC virus(JCV)are 2 prominent opportunistic infection viruses,and they may cause polyomavirus-associated nephropathy and graft kidney loss in patients who are in an immunosuppressed state after kidney transplantation.Hence,timely detection and sustained monitoring of the viral load are indispensable.However,the current diagnostic methods remain limited,and the development of new molecular detection technology is extremely urgent.Methods:The sequences and concentrations of clustered regularly interspaced short palindromic repeats(CRISPR)RNA(crRNA),the concentration of Cas13a,and the primers for recombinase polymerase amplification(RPA)were optimized for BKV and JCV detection.Next,a novel microfluidic dual-droplet chip was designed and fabricated,and it was integrated with CRISPR(ddCRISPR)to simultaneously qualitatively detect BKV and JCV.Subsequently,the ddCRISPR assay was verified using clinical samples.Then,a lateral flow strip combined with CRISPR(LFCRISPR)was developed for the detection of BKV and JCV in resource-limited settings.Results:A one-pot RPA-CRISPR reaction system was established and optimized for BKV and JCV detection.ddCRISPR can simultaneously and rapidly detect BKV and JCV with high sensitivity(10 copies/ml for BKV and 1 copy/ml for JCV),and provide absolute quantification,which is suitable for viral load detection and conducive to personalized and precise treatment for organ transplant recipients.LFCRISPR simplified the operational process through a simple visual readout,facilitating virus screening after organ transplantation.Conclusion:These platforms incorporate molecular testing into the transplantation treatment model,thereby reducing costs,prolonging the survival time of the graft,improving the clinical outcomes of postoperative management in kidney transplantation,and enhancing the patients’quality of life.
基金Nanjing Forestry University[Grant Nos.163020139,164020818,163020217 and 16302023]National Natural Science Foundation of China(5201101466).
文摘A novel coronavirus of zoonotic origin(SARSCoV-2)has recently been recognized in patients with acute respiratory disease.COVID-19 causative agent is structurally and genetically similar to SARS and bat SARS-like coronaviruses.The drastic increase in the number of coronavirus and its genome sequence have given us an unprecedented opportunity to perform bioinformatics and genomics analysis on this class of viruses.Clinical tests like PCR and ELISA for rapid detection of this virus are urgently needed for early identification of infected patients.However,these techniques are expensive and not readily available for point-of-care(POC)applications.Currently,lack of any rapid,available,and reliable POC detection method gives rise to the progression of COVID-19 as a horrible global problem.To solve the negative features of clinical investigation,we provide a brief introduction of the general features of coronaviruses and describe various amplification assays,sensing,biosensing,immunosensing,and aptasensing for the determination of various groups of coronaviruses applied as a template for the detection of SARS-CoV-2.All sensing and biosensing techniques developed for the determination of various classes of coronaviruses are useful to recognize the newly immerged coronavirus,i.e.,SARS-CoV-2.Also,the introduction of sensing and biosensing methods sheds light on the way of designing a proper screening system to detect the virus at the early stage of infection to tranquilize the speed and vastity of spreading.Among other approaches investigated among molecular approaches and PCR or recognition of viral diseases,LAMP-based methods and LFAs are of great importance for their numerous benefits,which can be helpful to design a universal platform for detection of future emerging pathogenic viruses.
文摘Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies.Therefore,a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101.In this study,a sensitive bioanalytical method was developed and validated,using a Quanterix single molecular array(Simoa)assay.Moreover,to thoroughly assess the platform,enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed,and their performance was compared with that of this novel technology platform.The assay was validated in compliance with the current guidelines.Measurements with the Simoa assay were precise and accurate,presenting a valid assay range from 6.55 to 4000 pg/mL.The intra-and inter-run accuracy and precision were within-19.3%to 15.3%and 5.5%to 17.0%,respectively.S3101 was stable in human serum for 280 days at-20℃and-70℃,for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature(22℃-28℃),respectively,and after five and two freeze-thaw cycles at-70℃and-20oC,respectively.The Simoa assay also demonstrated sufficient dilution linearity,assay sensitivity,and parallelism for quantifying S3101 in human serum.The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.
基金supported by the National Natural Science Foundation of China (No. 30870810)National Basic Research Program of China (No. 2011CB933404)
文摘Objective To perform the modulation of an assay system for the sensory integration of 2 sensory stimuli that inhibit each other.Methods The assay system for assessing the integrative response to 2 reciprocally-inhibitory sensory stimuli was modulated by changing the metal ion barrier.Moreover,the hen-1,ttx-3 and casy-1 mutants having known defects in integrative response were used to evaluate the modulated assay systems.Based on the examined assay systems,new genes possibly involved in the sensory integration control were identified.Results In the presence of different metal ion barriers and diacetyl,locomotion behaviors,basic movements,pan-neuronal,cholinergic and GABAergic neuronal GFP expressions,neuronal development,structures of sensory neurons and interneurons,and stress response of nematodes in different regions of examined assay systems were normal,and chemotaxis toward different concentrations of diacetyl and avoidance of different concentrations of metal ions were inhibited.In the first group,most of the nematodes moved to diacetyl by crossing the barrier of Fe2＋,Zn2＋,or Mn2＋.In the second group,almost half of the nematodes moved to diacetyl by crossing the barrier of Ag＋,Cu2＋,Cr2＋,or Cd2＋.In the third group,only a small number of nematodes moved to diacetyl by crossing the barrier of Pb2＋ or Hg2＋.Moreover,when nematodes encountered different metal ion barriers during migration toward diacetyl,the percentage of nematodes moving back and then turning and that of nematodes moving straight to diacetyl were very different.With the aid of examined assay systems,it was found that mutations of fsn-1 that encodes a F-box protein,and its target scd-2 that encodes a receptor tyrosine kinase,caused severe defects in integrative response,and the sensory integration defects of fsn-1 mutants were obviously inhibited by scd-2 mutation.Conclusion Based on the nematode behaviors in examined assay systems,3 groups of assay systems were obtained.The first group may be helpful in evaluating or identifying the very subtle deficits in sensory integration,and the third group may be useful for the final confirmation of sensory integration defects of mutants identified in the first or the second group of assay systems.Furthermore,the important association of sensory integration regulation with stabilization or destabilization of synaptic differentiation may exist in C.elegans.
文摘[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.
基金supported by the National Basic Research Program of China (973 Program,no. 2007CB714507)National Nature Science Foundation of China (no. 90813015)
文摘A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).
文摘Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.
基金the grants from the Ministry of Sciences and Technology of China, No. 2006AA02A408, 2008ZX09312-014
文摘A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.
基金supported by the Ministry of Science and Technology of the People’s Republic of China (No. 2000-0120)the Science and Technology Department of Zhejiang Province (No. 012104)+1 种基金the Science and Technology Department of Jiaxing City (No. 2005AY3042)the Education Department of Zhejiang Province (No. 25000964).
文摘Objective To detect the response of lymphocytes to radiation in untreated breast cancer patients with three different genetic assays. Methods Blood samples were collected from 25 untreated patients and 25 controls. Each blood sample was divided into two parts： one was irradiated by 3-Gy X-ray （irradiated sample）, the other was not irradiated （non-irradiated sample）. The radiosensitivity of lymphocytes was assessed by comet assay, cytokinesis-block micronucleus （CBMN） assay and 6-TG-resistant cells scored （TG） assay. Results The baseline values of micronucleated cell frequency （MCF） and micronucleus frequency （MNF） in the patients were significantly higher than those in the controls （P〈0.01）, and 3-Gy X-ray induced genetic damage to lymphocytes in the patients increased significantly as compared with that in the controls as detected with the three genetic assays （P〈0.01）. The proportion of radiosensitive cases in the patient group was 48% for the mean tail length （MTL）, 40% for the mean tall moment （MTM）, 40% for MCF, 44% for MNF, and 48% for mutation frequencies of the hprt gene （Mfs-hprt）, respectively, whereas the proportion of radiosensitive cases in the control group was only 8% for all the parameters. Conclusion The difference in the lymphocyte radiosensitivity between the breast cancer patients and the controls is significant. Moreover, there are wide individual variations in lymphocyte radiosensitivity of patients with breast cancer. In some cases, the radiosensitivity of the same patient may be different as detected with the different assays. It is suggested that multiple assays should be used to assess the radiosensitivity of patients with breast cancer before therapy.
文摘BACKGROUND Current diagnosis of hepatitis C virus(HCV)infection requires two sequential steps:testing for anti-HCV followed by HCV RNA PCR to confirm viremia.We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA)for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera:10 without HCV or HIV infection;54 with viremic HCV monoinfection;38 with viremic HCV/HIV coinfection;and 45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding,it was 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test.In five sera with HCV infection,HCV RNA was as low as 50-59 IU/mL,and four out of five tested positive for HCV-Ags EIA.Likewise,it was also 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection,regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL.It is highly sensitive and specific in the setting of HIV coinfection,regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.
基金supported by Pasteur Institute of Iran(funding No 754)Kermanshah University of Medical Sciences(funding No 980467).
文摘Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stressinducible protein-1 with/without adjuvant.After three vaccinations,mice were challenged by Leishmania tropica promastigotes.Two months after challenge,the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods.Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium.For real-time PCR,DNA of the lymph nodes was extracted,equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers.The data of the two methods were compared by appropriate statistical methods.Results:Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice.In addition,wherever parasite load of a group was estimated high(or low)by one method,the estimated parasite load by another method was the same,although statistically significant differences were found between some groups.Conclusions:Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups.However,due to the lower errors and faster process,the real-time PCR method is preferred.
文摘In order to compare sensitivity of EIA and RIA assay kits for hepatitis B and C virus (HBV and HCV, respectively) infection markers, 100 serum samples in total were collected form 50 adult women each in urban and rural areas in northeast China. The number of positive cases to the three infection markers on HBV (i.e., HBsAg +, anti HBs +, and anti HBc +) and the one on HCV (anti HCV +) were examined in two laboratories, i.e., in Laboratory A with EIA kits produced in China and in Laboratory B with RIA kits. HCV infection positivity (anti HCV +) was examined by EIA kits in both laboratories, but from different sources in and outside of China, respectively. The assay in Laboratory A gave 2 HBsAg + cases out of the 100 cases examined, whereas there were 9 positive cases in Laboratory B. In contrast, 19 cases were positive to anti HCV when examined in Laboratory A, and there were 3 cases in Laboratory B. Thus, the kits used in Laboratory A gave fewer HBsAg + and more anti HCV + cases than the kits used in Laboratory B. The prevalence of anti HBs + or anti HBc + and cases did not differ when assayed in the two laboratories with EIA and RIA kits, respectively. The agreement of positive and negative findings between the two sets of testing were 93%, 93%, 93%, 86% and 82% for HBsAg, anti HBs, anti HBc, HBV (i.e., either positive to anyone of the three markers or negative to all three markers), and anti HCV, respectively. The implication of the observation on epidemiology on HBV and HCV infection prevalence was discussed.
