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@#Objective To develop a colloidal gold immunochromatographic test strip for rapid and accurate detection of Pseudomonas aeruginosa(P.aeruginosa,Pa).Methods After bioinformatics analysis of Pa outer membrane protein OprF,the gene sequence with abundant antigenic determinants and high intraspecific homology was chemically synthesized,and then connected to pET-28a(+)vector to construct the expression vector pET-28a-OprF,which was transformed into E.coli BL21(DE3)and induced by IPTG. The recombinant OprF protein was purified by Ni Sepharose~(TM)6 Fast Flow and used to immunize two female BALB/c mice for 3～4 times by multi-point subcutaneous injection in the back at the first immunization and intraperitoneal injection at subsequent immunizations. The monoclonal antibodies were screened by animal cell fusion technique,and the colloidal gold immunochromatographic test strip for rapid detection of Pa was prepared by using monoclonal antibody and double antibody sandwich immunochromatography technique. The specificity,sensitivity and stability of the test strip were evaluated.Results Two monoclonal antibodies,Pa-1# and Pa-2#,were obtained with the titer of 1∶409 600,and both of them recognized OprF specifically. The prepared colloidal gold immunochromatographic test strip showed a sensitivity of 1. 0×10~6CFU/mL and had no cross reaction with 9 common respiratory pathogens with a good stability.Conclusion The prepared colloidal gold immunochromatographic test strip can detect Pa rapidly within 15 min,with high specificity and good stability.
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The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Subject(s)
Animals , Antibodies, Viral , Chickens , HN Protein/metabolism , Newcastle Disease/prevention & control , Newcastle disease virus/metabolism , Oryza/geneticsABSTRACT
Vibrio splendidus is an opportunistic pathogen in aquaculture. It can infect a variety of aquaculture animals and has caused huge losses to the aquaculture industry. In this study, a novel and efficient method for detecting V. splendidus was developed by combining the exonuclease Ⅲ amplification strategy with a nucleic acid test strip developed based on gold nanoparticles-labeled DNA probe. The results could be directly visualized by naked eyes, and this system overcame the difficulty in preparation of the monoclonal antibody used in conventional immunostrip. Upon optimization of experimental conditions, the detection limit of the strip was 5 ng/mL for the synthetic oligonucleotide DNA fragment and 10 ng/mL for the actual genomic DNA sample of V. splendidus. This test strip was more sensitive compared with the PCR method and was specific for the detection of V. splendidus. The rapid preparation of nucleic acid strip and the efficient detection of V. splendidus open a new way for the prevention and control of aquatic diseases.
Subject(s)
Animals , DNA Probes , Gold , Metal Nanoparticles , Vibrio/geneticsABSTRACT
New technological advances have paved the way for significant progress in automated urinalysis. Quantitative reading of urinary test strips using reflectometry has become possible, while complementary metal oxide semiconductor (CMOS) technology has enhanced analytical sensitivity and shown promise in microalbuminuria testing. Microscopy-based urine particle analysis has greatly progressed over the past decades, enabling high throughput in clinical laboratories. Urinary flow cytometry is an alternative for automated microscopy, and more thorough analysis of flow cytometric data has enabled rapid differentiation of urinary microorganisms. Integration of dilution parameters (e.g., creatinine, specific gravity, and conductivity) in urine test strip readers and urine particle flow cytometers enables correction for urinary dilution, which improves result interpretation. Automated urinalysis can be used for urinary tract screening and for diagnosing and monitoring a broad variety of nephrological and urological conditions; newer applications show promising results for early detection of urothelial cancer. Concomitantly, the introduction of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has enabled fast identification of urinary pathogens. Automation and workflow simplification have led to mechanical integration of test strip readers and particle analysis in urinalysis. As the information obtained by urinalysis is complex, the introduction of expert systems may further reduce analytical errors and improve the quality of sediment and test strip analysis. With the introduction of laboratory-on-a-chip approaches and the use of microfluidics, new affordable applications for quantitative urinalysis and readout on cell phones may become available. In this review, we present the main recent developments in automated urinalysis and future perspectives.
Subject(s)
Automation , Cell Phone , Creatinine , Expert Systems , Flow Cytometry , Mass Screening , Mass Spectrometry , Microfluidics , Microscopy , Semiconductors , Specific Gravity , Urinalysis , Urinary Tract , Urinary Tract InfectionsABSTRACT
Objective To research and develop the immunochromatographic detection technology of upcon-verting nanoparticles(UCNP)-based rapid fluorescence quantitative procalcitonin (PCT ) detection .Methods UCNP (NaYF4 :Yb3+ ,Er3+ ) was prepared by solvothermal method ,which was the compound of rare earth yt-terbium and erbium with four fluorine sodium yttrium .UNCP was conducted the modification and silica (SiO2 ) packing by using the reverse microemulsion method .Then the PCT determination method was estab-lished by using the dry-type immunochromatographic technology .Results The water-soluble UCNP with good dispersibility was successfully prepared by using the reverse microemulsion method .The lowest detection limit for detecting PCT by UCNP-based dry-type immunochromatographic technology was 0 .02 ng/mL ,the linear range was 0 .05 -44 .00 ng/mL ,the intra-batch and inter-batch coefficients variable(CV) were <10％and 13％ respectively .Conclusion UCNP-based immunochromatography technology is a rapidly and sensitively effective method for quantitatively detecting serum PCT .
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Procalcitonin (PCT) is the precursor of calcitonin related to the severity of human bacterial infection. We made a test strip by coupling anti-PCT to quantum dot, in order to develop a highly sensitive and convenient PCT testing product. The anti-PCT titer had reached 10⁷ because of the stability by coupling anti-PCT with quantum dot. The detecting linear range of the experiment was 0.15 to 120 μg/L, the sensitivity was 0.007 μg/L, the recovery range was 91% to 113%, and the intra- and inter-assay coefficient of variation was less than 8%. Comparing the homemade fluorescence-detected test strip with PCT ELISA kit on sale, we got accurate results which could mostly accomplish the test of clinical samples.
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A fluorescent immunochromatographic test strip based on the quantum dots submicrobeads (QBs) was developed for quantitative detection of chloramphenicol (CAP). In this method, monoclonal antibody of CAP and OBs complex fluorescent probe was first prepared using 1-ethyl-3-( 3-dimethylaminopropyl ) carbodiimide / N-hydroxysuccinimide coupling approach, then complete antigen CAP-HS-BSA was synthesized and sprayed on nitrocellulose membrane as test line (T line). Similarly, goat anti-mouse antibody was sprayed as control line (C line). The time required for the analysis was 15 min, and the limit of detection (LOD) for CAP was 0. 1 μg / L, with a working range of 0. 1 - 100 μg / L. In spiked milk samples, the test strip demonstrated high recoveries in the range from 93. 3% to 97. 9% with relative standard deviations of less than 7% .
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The quality evaluation of traditional Chinese medicine can be divided into two aspects, effectiveness and safety. The existing methods for evaluating the quality of traditional Chinese medicine(TCM) are mostly based on the testing instruments, which can not meet the practical needs of simple, rapid and on-site in production and life. Immunoassay, characterized by simple, rapid, sensitive, specific, low-cost and high-throughput, is widely used in the fields of clinical diagnosis, environmental pollution monitoring, food safety testing, and other fields. In recent years, immunoassay technology has been gradually applied in the field of quality control of TCM, involving quantitative detection of effective components of TCM, detection of harmful substances in TCM, and detection of exogenous pollutants in TCM. This paper summarizes the principle of the wide application of ELISA and colloidal gold immunostrip technology and its application in quality evaluation of TCM, this technique has a good application prospect in the field of rapid detection of the quality of TCM. In this paper, the principles of the widely used ELISA and colloidal gold immune test strip technology as well as their application in quality evaluation of TCM were reviewed, and the results showed that this technique had a good application prospect in the field of rapid detection of the quality of TCM.
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The residue of the pesticides affects seriously the quality and safety of traditional Chinese medicine. Pesticide residue has caused ever-growing attention of people at home and abroad. Rapid detection techniques used for rapid screening of pesticide residues have expanded in a fast progress. As one of the fast development methods of rapid detection, visualization test strip based on nanoparticle has received much concern in recent years. This article focused on the classification of detection test strips and key factors on the fabrication of nanoparticle-based visualization test strips used in small molecule pesticides. Moreover, a wide application of nanoparticles-tagged test strips on pesticide residue was reviewed including single residue detection, multi residue detection, as well as quantitative analysis. Finally, the future application of visual test strip for detecting of pesticide residues in traditional Chinese medicine was forecasted, intending to provide the reference for rapid detection techniques on pesticide residues screening in herbal medicine industry.
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Objective To explore the effect of a novel colloidal gold immunochromatographic test strip for detection of group B streptococci (GBS).Methods A total of 202 cases of swab of vagina or neck of uterus were collected,and they were detec-ted by novel strip and control strip to evaluate their clinical applications.Results Sensitivity of novel strip was about 105 CFU/ml and the detection time was about 5 to 8 minutes,and it showed better sensitivity and shorter detection time com-pared with control strip.In the 202 cases of clinical samples,the detection results of 197 cases were in consistent with the control strip,however,the detection results of 5 cases were not in consistent.The positive coincidence rate and negative coin-cidence rate were 97.5% and 97.54% respectively,and the total coincidence rate and Kappa value were 97.52% and 0.948 respectively.The consistency test showed no significant difference between this strip and control strip.Conclusion This method was a effective technology for diagnosing of infection caused by GBS,and had high value in clinical application.
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Objective To evaluate the capability of four tests for identification of the in vitro suscepti-bility of tigecycline against Acinetobacter and Enterobacteriaceae isolates.Methods Disk diffusion test was per-formed to detect the sensitivity of 158 Acinetobacter and 339 Enterobacteriaceae isolates to tigecycline.The mini-mum inhibitory concentrations ( MICs) of tigecycline for non-sensitive isolates were detected by using broth dilu-tion method ( BDM) , MIC Test Strip ( MTS) and agar dilution method.The differences with antimicrobial sus-ceptibility among the four different methods were evaluated.Results Tigecycline showed good antibacterial ac-tivity against both non-sensitive Acinetobacter and Enterobacteriaceae isolates with most of the MIC50 values in the sensitivity range of (0.5-2) mg/L and all of the MIC90 values of 4 mg/L.The MIC50 and MIC90 values measured by BDM were respectively 1 mg/L and 4 mg/L.The sensitivity rates presented by the results of BDM were re-spectively 87.1%and 70.2%based on the standards made by Food and Drug Administration (FDA) and Euro-pean Committee on Antimicrobial Susceptibility Testing ( EUCAST) .Agar dilution method indicated that most of the MICs of tigecycline to Acinetobacter and Enterobacteriaceae isolates were two dilutions higher than those de-tected by BDM with essential agreement (EA) rate of 56.5%.Both the very major error (VME) and the major error (ME) values were 0 and the categorical agreement (CA) rate was 46.8%according to the FDA standard.The VME and CA values were 0.8% and 24.2% based on EUCAST standard.Compared with agar dilution method, MTS showed better results in determining the susceptibility of Acinetobacter and Enterobacteriaceae iso-lates to tigecycline with MIC50 and MIC90 values of 1.5 mg/L and 4 mg/L, which was similar to the capability of BDM.Referring to the FDA and EUCAST standards, the sensitivity rates were 83.1% and 21.0%, the CA rates was 81.5%and 29.8%, and the EA rate was 71.8%.Most of the results tested by MTS were one dilution higher than those by BDM.FDA standard showed better correlation than EUCAST standard.Disk diffusion method showed the ME, mE, VME and CA values were respectively 19.4%, 71.8%, 0 and 8.9%according to FDA standard.Conclusion Disk diffusion method, MTS and agar dilution method all showed differences with BDM in susceptibility testing.The capability of MTS was similar to that of BDM.The results evaluated by FDA standard were better than those by EUCAST standard.The in vitro susceptibility of bacteria to tigecycline could be tested by disk diffusion method using FDA standard for evaluation, and confirmed with MTS if isolates were resistance or intermediate strains.The BDM could be performed for further confirmation if necessary.
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Objective To investigate the feasibility of screening for esophageal cancer with survivin colloidal gold immunochromatographic test strip. Methods The serum samples from 158 esophageal cancer patients and 146 healthy individuals were tested with survivin colloidal gold immunoohromatographic test strips. Results 20 nm-diameter colloidal gold solution was prepared and survivin polyclonal antibodies with different concentrations were labeled to the gold particles. It showed that the optimal concentration for labeling was 12 μg/ml. The prepared survivin colloidal gold immunochromatographic strip teat showed positive rates of survivin in esophageal cancer group and control group were 51.9 % (82/158) and 15.1% (22/146) respectively (χ~2 = 45.7, P < 0.05). No statistical differences were observed in age groups (between 42-54 years: 47.1%; between 56-68 years: 40.9%; between 69-81years: 63.9%, χ~2= 1.11, P > 0.05), gender groups (male: 50.4%, female: 58.1%, χ~2= 0.59, P > 0.05) , primary site groups (upper segment: 56.3%, middle segment: 46. 1%, lower segment: 32. 1%, χ~2 = 2.64, P > 0.05), differentiation groups (well-differentiation: 56.3%, moderate differentiation: 43.2%, poor-differentiation: 32.7%,χ2= 1.63, P >0.05)and lymph node metastasis groups (with metastasis:43.3%, without metastasis:43.4%, χ~2 = 0.00, P > 0.05). The sensitivity, specificity and accuracy of survivin colloidal gold immunochromatographic test strip was 51.9% (82/158), 84.9% (124/146) and 67.8% (206/304) respectively. Conclusions Survivin colloidal gold immunochromatographic test strip is fast, simple, easy-to-read. It could be used as a valuable tool for screening of high-risk patients with esophageal cancer.
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Objective To evaluate the consistency and diagnosis importance among three assays of Micral-Test strip,DCA 2000 analyzer and radioimmunoassay.Methods A total of 133 random urine samples of patients with diabetes were determined positive or questionable positive by Micral-Test strip,from whom urine samples within 8 hours during the nighttime were also collected.Both the two urine samples were determined by three assays for detection of microalbuminuria.Results(1)The value of Alb and AER determined by radioimmunoassy was significantly lower than that by DCA2000 analyzer,of which the sensitivity and specificity is 100%and 86.2%,?=0.812.(2)According to the reading scales of Micral-Test strip,the area under ROC curve(AUC)of random and 8 h urine samples were 0.900 and 0.934 respectively.(3)The AUC of random ACR and Alb was 0.952 and 0.923.The AUC of 8h urine ACR and Alb were 0.965 and 0.958.Conclusion The diagnosis importance among three assays is all dependable.Micral-Test strip is convenient for screening.ACR cannot substitute for AER as a diagnosis standard.And the value determined by DCA 2000 analyzer is more precise than that of radioimmunoassay.
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PURPOSE: This study was performed to test the clinical usefulness of the glucose test strip method for early detection of pulmonary aspiration in tube fed patients. METHOD: The subjects for the study were 36 patients who were receiving enteral feedings and 39 patients who were not given enteral feedings. For the analysis, the tube fed patients were divided into two groups (clinically significant aspiration and no aspiration) according to criteria. RESULT: The mean glucose concentration of tracheal secretions from non enteral fed patients was 26.35mg/dl and were lower than those concentrations found in tube fed patients (32.75mg/dl). The mean glucose concentration of the aspiration group was 45.60mg/dl and the glucose concentration of the non aspiration group was 19.93mg/dl. The difference was statistically significant (t=2.163, p=. 038). More subjects in the no aspiration group (73%) than the aspiration group (56%) had glucose concentrations below 20mg/dl. After deleting the cases that had samples containing blood, glucose concentrations of tracheal aspirates were lower in both groups. CONCLUSION: The glucose level of the aspiration group was significantly lower than the no aspiration group and more subjects in the aspiration group had a glucose level higher than 101mg/dl. Therefore, the glucose test of tracheal secretions in tube fed patients could be a desirable test for screening for tracheal aspiration. Especially the patient who is showing repeatedly high glucose levels should not be given feedings until reassessment is completed.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Enteral Nutrition/adverse effects , Glucose/analysis , Intubation, Gastrointestinal/adverse effects , Pneumonia, Aspiration/diagnosis , Reagent Strips , Trachea/metabolismABSTRACT
Blood glucose levels were measured in 89 healthy term neonates during the first 72 hours using the SureStep, a newly developed reagent test strip method by LifeScan. The blood samples were obtained by heel-stick puncture and blood glucose concentrations were monitored at birth(0), 2, 4, 6, 12, 24, 48, and 72 hours after birth. Mean and standard deviation of their measurement were compared according to postnatal hours and type of delivery. Comparison of significance between mean plasma glucose levels were made with the Wilcoxon rank sum test and significance level of 0.05 was used to determine which pair-wise comparisons were significantly different. The mean plasma glucose concentrations of first 6 hours were significantly lower than those of 12, 24, 48, 72 hours. This finding indicates that plasma glucose stabilization in healthy fullterm neonates can be expected with the feedings. The mean plasma glucose concentration at birth in the neonates of cesarean section (64.5+-8.06 mg/dl) was significantly lower than that of vaginal delivery (80.3+-20.7 mg/dl), but there were no significant differences after 2 hours. This may be due to the difference in prenatal care of obstetric department of Horamae hospital (C/S: midnight NPO and Hartmann solution, V/D: NPO with labor pain and 5% dextrose solution intravenously). In summary, the changes in perinatal care, especially prenatal fluid therapy and time of first feeding should be considered in defining neonatal hypoglycemia. Our data suggest that hypoglycemia should he defined as below 40 mg/dl during the first 6 hours and below 55 mg/dl thereafter.
Subject(s)
Female , Humans , Infant, Newborn , Pregnancy , Blood Glucose , Cesarean Section , Fluid Therapy , Glucose , Hypoglycemia , Labor Pain , Parturition , Perinatal Care , Plasma , Prenatal Care , Punctures , Reagent StripsABSTRACT
No abstract available.