Precise Prediction of Deoxyribonucleic Acid sizes with Transient Isotachophoreis-Capillary Gel Electrophoresis Analysis on a Microchip / 分析化学

The migration-time in transient isotachophoresis (tITP) separation is affected by sample salinity as the dependence of ITP time on sample-zone conductivity.The sample-to-sample variation of migration-time in microchip tITP-CGE analysis is an undesired factor for precise DNA sizing.In this work, a DNA sizing method that based on relative migration-time proportion (RMP) was developed to eliminate the effect of sample salinity on sizing precision.RMP was defined as the ratio of the migration-time difference between the target fragment and the lower marker to that between the upper marker and the lower marker.The RMP values were tested to be reproducible in microchip tITP-CGE separations irrespective of sample salinity.Size of a target DNA was predicted by fitting its RMP value to the equation derived from RMPs of standard DNA ladder vs.DNA sizes.The precision and reproducibility of the sizing method were validated testing multiple standard PCR amplicons.Experimental results showed that the RMP method is simple and reliable, thus well suited to precise DNA sizing with microchip tITP-CGE analysis.

Development of a compact disk type microfluidic chip based on DNA hybridization for phenylketonuria screening / 中华检验医学杂志

Objective To develop a phenylketonuria (PKU) screening method based on a compact disk (CD) type microfluidic chip capable of generating reciprocating flow within the microchannels that facilitate rapid DNA hybridization. Methods This microfluidic device consists of a two-layer structure: a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a bottom glass layer with immobilized hydrogel conjugated DNA arrays. The DNA arrays included R243Q, V245V and the blank control probes. When the CD device was spun, the PCR products were driven into the hybridization channel by centrifugal force. When the rotation of the CD device was stopped, capillary force pulled the PCR products solution to flow back to the channel. After the on-chip hybridization, the hybridization signals were captured on a fluorescence microscope. The specificity, detection limitation and reproducibility of this device were evaluated. Thirty DNA samples from pregnant women with suspected PKU were detected by this device.Then the results were compared with DNA sequencing results. Results With the compact disk type microfluidic chip, the hybridization time could be reduced to 15 min, sample consume could be as low as 1. 5 μl and the detection limitation was 0. 7 ng/μl. With the chip based method, samples of PKU patients and healthy controls were detected and the results were consistent with DNA sequencing results. Five different batches of chips and five micro-channels of each chip were selected to test one PKU patients with V245V mutation. All the results were positive, indicating good reproducibility. Four cases of V245V mutation and 1 case of R243Q mutation were found in 30 suspected PKU carried pregnant women. Conclusion The compact disk microfluidic device has advantages of simple, rapid and highly sensitive, thus is well suited to PKU screening.

Sperm motility sorting using microfluidic chip / 中华检验医学杂志

in vitro fertilization in future.

Diagnostic value of platelet size indices in thrombocytopenia / 中国综合临床

Objective To investigate the role of platelet indices in the differentiation of thrombocytopenia caused by bone marrow diseases and thrombocytopenia caused by non-marrow diseases.Methods Mean platelet volume(MPV),platelet crit(PCT)and platelet size deviation width(PDW)of 76 othromboeytopenia(TP)patients,66 immune thrombocytopenia purpura(ITP)patients,8 secondary thrombocytocritpenia purpura(STP)patients,8 acquired pure megakaryocytic thrombocytopenia purpura(AATP)patients,140 malignant hematological diseases(MHD)(including acute leukemia,aplastic anemia and myelodysplastic syndromes)patients and 100 healthy people were respectively determined by blood cell analyzer Cell-Dyn 3700.Result Compared with control group,the MPV of the patients with MHD significantly decreased,and then the PDW of them increased.For patients with STP,the MVP and PDW markedly increased.The MVP of patients with AATP was normal,and PDW significantly increased.The MPV,PCT and PDW of ITP and TP showed marked difference(P＜0.01).In contrast with the control group,the MPV,PCT and the PDW of patients with hemopathy and patients with ITP were remarkably different(P＜0.01).Compared with the marrow TP and the non-marrow TP or control group,MPV and PDW showed significant difference(P＜0.01).During the effective treatment of patients with ITP,PLT increased and then MPV and PDW decreased.Conclusion The level changes of MPV and PDW,in particular MPV can primarily judge the cause of thrombocytopenia (MPV＜reference value as caused by bone marrow diseases but MPV＞reference value as caused by bone marrow diseases).In addition,during the effective treatment of patients with ITP,with changes of MPV and PDW present 5 days earlier than that of PLT,may used to observe the therapeutic effect.Myelodysplastic syndromedynamic observation of the platelet parameter helps for the differential diagnosis,judgment of the states of the diseases and observation of the effectiveness of therapy of ITP.

Screening of active saccharides interacted with granulocyte colony-stimulating factor using capillary electrophoresis and their effects on leukemia cells / 中华检验医学杂志

Objective To screen out the bioactive sulfate saccharides interacted with granulocyte colony-stimulating factor (G-CSF) and provide some scientific data on their pharmacology. Methods The interactions between G-CSF and 15 sulfate saccharides with various chain lengths and extent of sulfation were studied by capillary electrophoresis. Furthermore, the effects of these G-CSF binding saccharides on the proliferation and differentiation of leukemia cell line and NFS-60 were also investigated. Results Except for the hepatosaccharide, degradable carrageenan, Dextran sulfate (DexS) with a low molecular mass the nonsulfated sancharide and alkaline chitosan oligosaccharide, the rest of saccharides had interact with G-CSF.DexS500, λ carrageenan, ι carrageenan, κ carrageenan and low molecular weight heparin-1(LMWH-1)with anticoagulant activity could inhibit the NFS-60 growth. Compared with the absorbency of the control group (100.00±3.00) after these saccharides interacted with the NPS-60 cells, their relative absorbencies were 31.33±2.08,37.67±2.08,45.00±1.20,43.00±2.65 and 74.67±1.52, respectively. Their differences had a statistical significance(t=27.94,26.71,26.42,26.61,10.54,P<0.01). The morphological analytic results showed that the LMWH and lambda carrageenan as well as DexS with a molecular mass of 500 000 induce effectively promyclocyte to transit metamyelocyte. Flow cytometry results showed enhanced expressions of CD11b, CD18 and CD45 after treatment with the LMWH, lambda carrageenan and DexS500,respectively. Cell cycle analysis indicated that the S phase population decreased after treatment with these saccharides. Conclusions Capillary electrophoresis is an effective medicine-screening technique that is used to screen some of these saccharides which could bind to G-CSF. And it is found that the interactions are correlated with the chain length, the extent and the position of sulfation of these saccharides.

Detection of 16S rRNA methylase genes in Klebsiella pneumoniae / 临床检验杂志

Objective To investigate the prevalence of 16S rRNA methylase genes in Klebsiella pneumoniae isolates from Guangzhou.Methods K-B test was used to determine the resistant rates of these stains.Five 16S rRNA methylase genes,armA,rmtA,rmtB,rmtC,and rmtD,were detected by PCR.Results All 55 K.pneumoniae isolates showed resistant to arbekacin,gentamicin,tobramycin,and neomycin.Susceptibility rates were 5.5%,20.0%,72.7%,and 100% to ceftazidime,ciprofloxacin,piperacillin/tazobactam,and imipenem respectively.ESBLs were positive in 52 of 55 (94.5%) isolates.Among 55 K.pneumoniae isolates,34 were positive for armA and 1 for rmtB.Conclusions In K.pneumoniae resistant to arbekacin,the positive rate of 16S rRNA methylase genes was high,predominantly with armA positive.These strains were highly resistant to some antibiotics.

Dissociation of EDTA-dependent pseudo platelet aggregation by amikacin and its mechanism / 临床检验杂志

Objective To study the dissociation of EDTA-dependent pseudo platelet aggregation by amikacin and to clarify the potential mechanism.Methods At different time points,two concentrations of amikacin (6.5 mg/ml and 10.0 mg/ml) were respectively added to EDTA-K2 anti-coagulated whole blood samples collected from two subjects with EDTA-dependent pseudothrombocytopenia.The aggregated-platelet dissociation was investigated by platelet count and microscopical examination.CD41,CD61,CD62p,PAC-1,and IgG on the platelets of the two patients and of a normal control were determined by the FCS under various conditions.Results When amikacin (6.5 mg/ml) was added within 1 hour after blood collection,it dissociated the EDTA induced platelet aggregation and the platelet counts returned to the normal level. The expression of CD62p,PAC-1,and PA-IgG on the platelets was decreased,yet the expression of CD41 and CD61 had no change.Conclusion Amikacin supplementation (6.5 mg/ml) within 1 hour after blood sampling may dissociate the aggregated platelets caused by EDTA. The mechanism may be associated that amikacin might inhibit the expression of CD62p,PAC-1,and PA-IgG on platelets. The amikacin supplementation would be an inexpensive,effective,and practical method to solve the platelet aggregation induced by EDTA.

Integrons Containing IMP-4 Metallo-?-lactamases(MBLs) Gene among Multiple-drug-resistant Klebsiella oxytoca Strains / 中华医院感染学杂志

OBJECTIVE To analyze the sequence of IMP-4 metallo-?-lactamases(MBLs) encoding gene from clinical isolates of multiple-drug-resistant Klebsiella oxytoca strains and attempt to know the integrons composing the drug resistance gene box.METHODS The antibiotic sensitivity test of multi-resistant K.oxytoca strains was done according to Kirby-Bauer method of CLSI 2005,and the double disk synergy test and Etest were for detecting their MBLs.The Class 1 integrons were detected by PCR.The purified amplicons of Class l integrons were sequenced.The type and order of gene cassettes in integrons were analyzed by searching GenBank.RESULTS The K.oxytoca was resistant to carbapenems,the third-generation cephalosporins,cefoxitin,quinolones,cefoperazone/sulbactam,sulfamethoxazole/trimethoprim,amoxicillin/clavulanate,ticacillin/clavulanate,piperacillin,cefepime,rifampicin and piperacillin/tazobatam,only susceptible to amikacin and polymyxin B.The IMP-4 metallo-?-lactamases,aadA1,AmpC,CTX-M-14,qacE△1-sull and intI1 were positive.CONCLUSIONS Integrons are important molecular mechanism in the development of multidrug resistance.There are resistance gene boxes in them.

Constructing a Competitor of BCR-ABL cDNA by PCR Site-Directed Mutagenesis / 中国实验血液学杂志

A competitive mimic of the cDNA of the BCR-ABL fusion gene was constructed, and its feasibility was testified by capillary electropheresis (CE). The 4 bp-shorter mimic was obtained by PCR amplification using a newly synthesized downstream primer analogous to the former one. Mimics of both types of BCR-ABL cDNA were achieved and the validity was verified with restriction endonuclease. And the products of the coamplification PCR could be easily separated by capillary electrophorisis. The mimic can be used to quantitative detection of BCR-ABL gene through competitive RT-PCR in chronic myeloid leukemia.

Pharmacokinetics of ligustilide in the volatile oil from Angelica Sinensis(Oliv.) Diels in rabbit / 中成药

AIM: To study the pharmacokinetics of ligustilide in the volatile oil from Angelica Sinensis(Oliv.) Diels in the rabbit. METHODS: HPLC method for ligustilide determination in the blood was developed.The HPLC system consisted of C_(18) column using MeOH-H_2O(65∶35,v/v) as mobile phase at a flow rate of 1.0 mL/min and UV detection at 236 nm. RESULTS: Linear calibration curves were obtained over the concentration range of 0.40 ?g?mL~(-1)～10.00 ?g?mL~(-1) for ligustilide.The minimum limit detection was 0.40 ?g?mL~(-1).The recovery of ligusitilide in blood was 90.90% with RSD 2.74%. CONCLUSION: After oral administration of volatile oil,intracorporal process of ligustilide in rabbit accords with 2-compartment model with 1 st order absorption,(2.6638) h and 108.88 h are obtained as t_(1/2?) and t_(1/2?) respectively.