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OBJECTIVES@#To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification.@*METHODS@#The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios.@*RESULTS@#When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method.@*CONCLUSIONS@#The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.
Subject(s)
Humans , Forensic Medicine , DNA/genetics , Real-Time Polymerase Chain Reaction , Magnetic Phenomena , DNA Fingerprinting/methods , Microsatellite RepeatsABSTRACT
Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1-6)for 2 h,under 20％ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.
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@#Objective To investigate the feasibility of using magnetic beads to locate small pulmonary nodules. Methods Twelve rabbits were randomly divided into two groups, 6 in each group. One group underwent thoracotomy after anesthesia and the other group underwent percutaneous puncture under the guidance of X-ray. One and two cylindrical tracer magnets (magnetic beads) with a diameter of 1 mm and a height of 3 mm were injected adjacent to the imaginary pulmonary nodules in left lung in each group. The magnetic beads beside the imaginary nodules were attracted by a pursuit magnet with a diameter of 9 mm and a height of 19 mm. The effectiveness of localization by magnetic beads were determined by attraction between tracer and pursuit magnets. Results All processes were uneven in 12 rabbits. There was micro hemorrhage and no hematoma in the lung tissue at the injection site of the magnetic beads. When tracked with the pursuit magnets, there was one bead divorce in cases that one bead was injected, but no migration or divorce of the magnetic beads in cases that two magnetic beads were simultaneously injected to localize the small pulmonary nodules. Conclusion The feasibility of using magnetic beads to locate small pulmonary nodules has been preliminarily verified.
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Plant virus diseases are one of the major diseases restricting erop production.Timely identification of their pathogen and development rules is the prerequisite for effective control of their large- scale spread.However, long cycle, tedious steps and strict detection environment were the disadvantages existing in the detection technology of plant virus disease.In this study, Tobacco Mosaic virus (TMV) was used as a model to he extract UNA based on CMBs-ACPtmv , which was design based on the principle of complementary base pairing.Meanwhile, the experimental conditions were optimized and analyzed, including the preparation conditions of functionalized magnetic beads, the reaction conditions during extraction, and the sensitivity, stability and other properties of the method.The results showed the ability to capture RNA of CMBs-ACPtmv were best when prepared with 4 fxmol capture probe (ACPTMV ) and 0.08 mg carboxyl magnetic beads (CMBs) ; After 3 min of extraction, CMBs-ACPtmv has the best RNA extraction effect, but when the extraction temperature of CMBs-ACPtmv was changed, its extraction capacity showed no significant change; In the comprehensive performance evaluation, the sensitivity of CMBs-ACPjjjv can reach 2.5 ng/fxL, and the detection stability is good.Compared with conventional RNA extraction technology, CMBs-ACPimv has outstanding advantages in detection time and sample consumption.The functional magnetic beads extraction method established in this study is fast, safe and simple.It can achieve rapid extraction of plant virus RNA with simple equipment, which has a broad application prospect.
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Objective To ascertain the short-term effects of traditional Chinese medicine(TCM) auricular magnetic beads application for treatment of poor vision in primary school students. Methods In 2017, a randomized sampling method was adopted in this study.A total of 458 students with a naked eye visual acuity ≤4.9 were screened from a primary school in the jurisdiction.Of them, 230 students were informed by the parent′s informed consent to receive the TCM ear acupoint application, and the remaining 228 students served as the control group. Results The average eyes visual acuity of intervention group was 4.842, increasing to 4.848 or 4.859 after 6 or 12 weeks′ treatment by auricular magnetic bead application.After statistically analysis, there is a significant difference in short-term effect of auricular magnetic bead application on eyesight improvement for primary school students. Conclusion The TCM auricular magnetic bead application proves to have a short-term effect on the poor eyesight of the students, and the operation is simple and safe.It is conducive to improving students′ participation in poor eyesight control and achieving family and school participation in TCM prevention and treatment of myopia.
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Natural products have been a major source of leading compounds in drug discovery. How to effectively screen active compounds from complex matrix remains an interesting topic. In this review, we comprehensively summarized advanced liquid chromatography based approaches in natural products screening, including pre-column, on-column and post-column screening methods. Their advantages, disadvantages and prospect are also discussed.
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OBJECTIVE@#To observe the effect of acupoint stimulation on the quality of recovery in patients with radical thyroidectomy under the concept of enhanced recovery after surgery (ERAS).@*METHODS@#A total of 62 patients with radical thyroidectomy were randomized into an observation group and a control group, 31 cases in each one. In both of the two groups, general anesthesia with tracheal intubation was applied, the same anesthesia induction and maintenance medication were given. In the observation group, auricular point pressing with magnetic beads was adopted at bilateral shenmen (TF) and transcutaneous electrical acupoint stimulation (dilatational wave, 2 Hz/100 Hz in frequency, 6 to 12 mA) was performed at bilateral Hegu (LI 4) and Neiguan (PC 6) from 30 min before anesthesia induction to the end of the anesthesia. In the control group, medical adhesive plaster was pasted at bilateral shenmen (TF) and the electrodes were plastered at bilateral Hegu (LI 4) and Neiguan (PC 6) with no corresponding stimulation. In both of the two groups, visual analogue scale for anxiety (VAS-A) score was observed to evaluate the anxiety severity before anesthesia induction; the total intraoperative dosages of sufentanil, remifentanil and propofol were recorded; the numerical rating scale (NRS) score was used to assess the pain severity of instant time (T0) and 30 min (T1) of entering post-anesthesia recovery room (PACU), motor and static mode at 2 h (T2), 6 h (T3), 12 h (T4), 24 h (T5) after surgery; time of first anal exhaust, time of getting out of bed after surgery, total hospitalization time and the incidences of postoperative nausea and vomiting were observed; the quality of recovery was assessed by the 40-item quality of recovery score (QoR-40).@*RESULTS@#The VAS-A score and the total intraoperative dosage of remifentanil in the observation group were reduced compared with the control group (0.05). The time of first anal exhaust and getting out of bed after surgery in the observation group were advanced than those in the control group (0.05). Compared with the control group, the QoR-40 score was increased in the observation group (<0.05).@*CONCLUSION@#Acupoint stimulation can improve the preoperative anxiety in patients with radical thyroidectomy, reduce the intraoperative anesthetic dosage and postoperative pain, advance the time of anal exhaust and getting out of bed, improve the quality of postoperative recovery and enhance the recovery process.
Subject(s)
Humans , Acupuncture Points , Enhanced Recovery After Surgery , Postoperative Nausea and Vomiting , Thyroidectomy , Transcutaneous Electric Nerve StimulationABSTRACT
@# Abstract：Objective To establish a sensitive，rapid and convenient method for the detection of dengue antigen and assist clinical diagnosis of dengue. Methods In this paper，we developed a rapid detection method for dengue antigen based on microfluidic immune magnetic beads. Solidwork software was used to design microfluidic chip，which was prepared by mechanical processing and chemical sealing. Immunomagnetic beads of dengue antibody were prepared by chemical coupling reaction. Using HRP ⁃TMB ⁃H2O2 as color system，dengue NS1 antigen was detected on microfluidic chip carrier by double antibody sandwich method. Finally，57 clinical samples were tested by the novel method and traditional ELISA kit，and the accuracy of the method was analyzed，and the advantages and disadvantages of the two methods were compared. Results 20 minutes was needed to detect dengue NS1 antigen by using the novel ELISA method，and the reaction system only needed 10 μg beads and 10 μL samples. In the verification experiment，the method could distinguish the negative from the positive obviously. The positive sample had color rendering，while the negative and blank samples had no color rendering. In terms of detection performance，the coincidence rate between the new ELISA method and the traditional ELISA method reached 100%. Conclusion The novel ELISA detection platform had the advantages of simple，rapid，reagent and sample saving，high sensitivity，good stability and high accuracy，and could be used for the detection of dengue antigen.
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ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.
Subject(s)
Humans , Plague/microbiology , DNA, Bacterial/genetics , Nucleic Acid Amplification Techniques/methods , Magnetics/methods , Yersinia pestis/isolation & purification , Yersinia pestis/classification , Yersinia pestis/genetics , DNA, Bacterial/isolation & purification , DNA, Bacterial/chemistry , Polymerase Chain Reaction , Sensitivity and Specificity , Immunomagnetic Separation , DNA Primers/genetics , Nucleic Acid Amplification Techniques/instrumentation , Magnetics/instrumentationABSTRACT
Objective To compare the effects of TRIzol and magnetic beadsmethods on quantitative detection of hepatitis C virus (HCV) RNA. Methods Serum samples and genotype information of 117 patients with positive HCV infection were collected. HCV RNA was extracted from serum samples by TRIzol method and magnetic bead method, respectively. And then the viral load of HCV RNA was detected by quantitative PCR to compare the difference between the two methods. Results qPCRresults showed that a good linear correlation existed between TRIzol and magnetic beads methods: y=0. 978x+0. 063 (R2=0. 973). Bland-Altman statistical analysis showed that the average logarithmic value of viral load ofHCV RNA extracted by TRIzol method was slightly lower than that of magnetic beads method, without significant difference (P>0. 05). There were no significant difference among the genotypes 1a, 1b, 2a, 3a or 6a between the two methods (P>0. 05). Conclusion TRIzol method is comparable to magnetic beads method in HCV RNA quantitative detection, with less samplevolume and lower cost, indicating that t might be widely used for developing ktt and HCV RNA clinical detection in China.
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Objective To evaluate the forensic application of TE-MAGS technology based on magnetic beads kit on TECAN pipetting platform and establish the automated DNA extraction system of case work samples. Methods Sensitivity test: 10 different DNA samples from 0.1ng to 1ng were prepared with a commercial standard DNA 9947A diluted into 200μL TES. DNA samples were purified by the TE-MAGS technology automatically on the TECAN pipetting platform and then typed using the IdentifilerTM Kit and get the profile of STR with the software GeneMapper ID-X; the power of purification was tested with a trail that purified 1ng DNA mixed with humus acid and hemachrome. Comparative test: 304 casework samples were divided into two purified by TE-MAGS technology and silicon beads respectively to compare the power of purification and the possibility of forensic utility. Results Sensitivity test: 0.3ng and more imported DNA can obtain a good quality of DNA profile compared to the lower imported DNA with dropout of STR peaks (0.1ng and 0.2ng). The power of purification of the TE-MAGS technology was not affected by humus acid and hemachrome. The comparison result between automatic TE-MAGS technology and manual silicon beads extraction methods from 304 casework samples showed that the former's success rate(50%) was higher than the latter(40.8%). Conclusion The established DNA purification method of TE-MAGS technology automatic DNA extraction system in this study was obviously advantaging at the aspect of success rate, stability, and uniformity and suited to application in the forensic utility future.
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Objective The magnetic beads direct adsorption method was used to extract the cell-free DNA (cfDNA) from three kinds of human humoral biological samples, including urine, saliva and blood, as to provide a reference for forensic cfDNA research and forensic inspection. Methods The cfDNA was isolated from humoral samples by centrifuging, and the cfDNA was extracted with the method of magnetic beads direct adsorption. Then the samples were sequentially amplified with Identifiler-Plus amplication kit, and the STR genotyping was detected by ABI 3500 Analyzer. Results The cfDNA was detected from all the three kinds of samples. The detection rate of cfDNA from the blood samples was 100%, the saliva was 90%, and the urine was 70%. Conclusion The results suggest that human humoral biological samples contain cfDNA. What's more, the magnetic beads direct adsorption method can be used to extract cfDNA efficiently and conveniently.
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Objective This study aimed to assess the efficiency and purification of the Trace DNA extraction with a quantified method for the magnetic bead-based DNA extraction as performed on the Tecan Automated systems with TE-MAGS magnetic separator. Methods Serial dilutions of standard commercial DNA 9947A were used with the total DNA contents, 0.1ng, 0.2ng,0.3ng, 0.4ng,0.5ng, 0.6ng, 0.7ng, 0.8ng, 0.9ng,1ng,diluted progressively and a 1ng DNA (standard commercial DNA 9947A) admixed with 6 common DNA-PCR inhibitors were extracted on the Automated systems and then performed via Fluorogenic probe quantitative PCR and STR genotype for the quantification analysis of recovery and purification. Results The recovery rate of standard 9947A DNA diluted with 0.1~1ng was 38.92~60.01%, and 0.3ng and more DNA could observed the full STR profiles. For the different PCR inhibitors, above 94.5% of bile acid, collagen and urea were efficient removal during the purification process, and the hemoglobin, melanin and humic acid removal efficiency were about 97.5%, 97.85%, 82.14%, respectively.Conclusion Our results suggested that The TE-MAGS magnetic bead-based DNA extraction was suitable for the extraction of trace DNA with high recovery efficiency and purification ability.
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Objective To compare magnetic beads kit,agrose gel recovery kit and heparinase I three methods to purify the micro DNA from crude heparin, then use q-PCR to identify the species origins and select the best method.Methods Using magnetic beads kit,agrose gel recovery kit and heparinase I to purify micro DNA from crude heparin and combined the porcine,bovine and ovine identification kits to identify the species origins and conformed the minimum detection limit of different percentage of ovine crude heparin in porcine crude heparin.Results Three pretreatment methods all can solve the pretreatment difficulties and we found that the haparinase was the best method; the minimum detection limit was 0.01%of ovine crude heparin in porcine crude heparin.Conclusion The heparinase method is the best pretreatment method and can successfully solve the pretreatment difficulties.Heparinase combine the porcine, bovine and ovine identification kits can identify the species origins from crude heparin.
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Aims: The present study aimed to develop a new approach for detecting Salmonella species at picogram levels using magnetic bead (MB) aggregation through loop-mediated isothermal amplification (LAMP). Methodology and results: For the first time to our knowledge, Salmonella LAMP amplicons were analyzed using MB aggregation. LAMPs were conducted with a simple heat block, and the results were compared with those obtained with conventional LAMP-MB techniques. Furthermore, the volume and concentration of MB solutions were optimized. Our method detected Salmonella genomic DNA at a low picogram level (1 pg/µL). The specificity of this method was also examined using other bacterial species. Owing to specific Salmonella primers, the use of LAMPs approach was time effective; because these amplicons could be utilized after 20 min instead of the 1 h needed for conventional methods. Furthermore, LAMP-positive amplicons were rapidly detected within 5 min. Conclusion, significance and impact study: The determination of DNA in biological samples is a recent keystone in genomic analysis techniques. Salmonella is a foodborne pathogen that causes many diseases and, in extreme cases, death. Accordingly, detecting Salmonella has become a vital issue for food safety and security. Combining DNA and MBs on paper helped us to develop a new method for label-free, non-immobilized, naked eye detection of Salmonella. The process is very specific owing to the use of exact primers and does not require heavy or expensive instrumentation. In the future, this method could be applied to biosensors as well as in biomedical and molecular diagnostic fields.
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SalmonellaABSTRACT
BACKGROUND: The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice. METHODS: Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs. CONCLUSION: 57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.
Subject(s)
Adult , Animals , Humans , Mice , Cell Transplantation , Human Embryonic Stem Cells , Teratoma , Testis , Transplants , TretinoinABSTRACT
Objective To construct a rapid genetic DNA extraction method, with nano magnetic beads, self-designed reagents system and extracting process. Method Part I: DNA extraction from old blood cotton swab sample with self-designed DNA extraction kit, then quantiifed by UV spectrophotometer. The method was further optimized on the preliminary results. Part II: All kinds of difficult DNA sample were tested with optimized kit, to detect the applicability of the kit. Result By improving the experimental condition, the extraction effects of different DNA sample is good, meanwhile, the extraction cost is relatively low.
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Objective:To investigate the feasibility of real-time fluorescence quantitative PCR in the identification of Fritillariae Cirrhosae Bulbus.Methods:The DNA of samples was extracted by magnetic beads ,the primers were amplified by real-time fluores-cence quantitative PCR , and the Cq value and amplification curve were used to determine the samples .Results:The Cq values for five batches of Fritillariae Cirrhosae Bulbus were lower than 30, and the curve had obvious growth period .No Cq value was shown for Fritil-lariae Ussuriensis Bulbus , Fritillariae Thunbergii Bulbus and Bolbostemmatis Rhizoma with straight line curves .Conclusion:The meth-od is simple,feasible and effective in the identification of Bulbus Fritillariae Cirrhosae with high accuracy and good reproducibility .
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Objective To establish a simple method to extract the methylated ctDNA in urine using magnetic beads as solid phase adsorption carri?er with a specific design reagent system and extraction process,and evaluate its application feasibility for methylated gene detection in urine sample . Methods Fourty cases of methylated ctDNA were extracted in urine using magnetic beads. After methylated modification,the concentration and pu?rity of DNA was determined by ultraviolet spectrophotometer. Results The extraction of 50 mL urine can gain 61?200 ng/mL methylated ctDNA, and the OD260/280 was 1.8 ± 0.05. Conclusion There are methylated ctDNA exist in the urine which can be extracted by magnetic beads. The estab?lished extraction method is simple,effective,and with high purity.
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Objective To study the application of proteomics in detecting differentially expressed proteins in renal clear cell carcinoma (RCCC) patient's urine in order to improve the diagnosis rate of RCCC.Methods From Mar.2010 to May.2010,the urine samples of 11 RCCC cases were collected,including 10 males and 1 female with average age of 63 (46-78) years.All patients were finally diagnosed as RCCC by post-operative or biopsy pathology.The normal control urine samples were collected from 10 males with average age of 29 (25-32) years.WCX beads combined with matrix assisted laser desorption ionization time of flighl mass spectrometry (MALDI-TOF MS) technique was applied in detecting differentially expressed proteins in RCCC patient's urine to find out differentially expressed proteins.And genetic algorithm was utilized to establish a diagnosis model.Results 160 differentially expressed proteins in RCCC patient 's urine were detected,and 1 was in significant difference,P=0.0304.ClinProTools 2.2 software was utilized with genetic algorithm to find out 13 differentially expressed proteins to establish a diagnosis model,and the sensitivity and specificity rate was 100％ after cross validation.Conclusions The diagnosis model established by genetic algorithms has high sensitivity and specificity rate,and can improve the diagnosis of RCCC.