RESUMEN
Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the ap-peared bands in the agarose gel electrophoresis(AGE).With the 3 SNP sites EGFR,c.2573T>G(L858R),EGFR,c.2582T> A (L861Q)and EGFR,c.2155 G>T(G719C)in epidermal growth factor receptor(EGFR)gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6 cases and 2 cases of heterozygotes mutation in the SNP site of L858R among 62 samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condi-tion.
RESUMEN
Hemoglobinopathy is a kind of hereditary hematonosis caused by the disproportion among normal hemoglobins or formation of abnormal hemoglobins.Hemoglobin analysis plays an important role in screening and diagnosing hemoglobinopathy.Emerging methods have been applied continuously to hemoglobinometry since the early 20th century.Nowadays,agarose gel electrophoresis,high performance liquid chromatography and capillary electrophoresis are widely used with mass spectrometry as the latest applied method.All these methods have their own features.Appropriate combination of these methods will lead to more scientific and reliable results.
RESUMEN
ObjectiveTo establish a novel assay for HIV-1 p24 ultrasensitive detection based on Gold Nanoparticle Probe (GNP) and PCR.MethodsSandwich ELISA method was established by a pair of anti-p24 monoclonal antibodies (mAbs),1G12 and 1D4,and was used to detect recombinant HIV-1 p24 antigen.The bio-barcode DNA was 47 bp,selected from genome of Arabidopsis,and formed double-stranded DNA by hybridization with the capture DNA (complementary with bio-barcode DNA) modified with sulfhydryl.Then double-stranded DNA were conjugated on the surface of 1D4-modified gold nanoparticles by sulfhydryl,and the Gold Nanoparticle Probe was produced.1G12 was precoated in the micropaltes,and in the presence of target recombinant HIV-1 p24 protein,a sandwich immuno-complex would form by adding GNP.Then the bio-barcode DNA in the immuno-complex were released by heating as detection signal,and consequently characterized by the polymerase chain reaction (PCR) with synthesized special primers and analyzed by 4％ agar gel electrophoresis,so HIV-1 p24 antigen could be evaluated.The sensitivity comparison between the new assay and ELISA can be done.ResultsSandwich ELISA was used to quantify HIV-1 p24 antigen by monoclonal antibodies 1G12 and 1D4,and the limit of detection (LOD) was 1000 pg/ml.The new GNP assay was established by the same pair of antibodies,combined with PCR and agar gel electrophoresis,and was used to indirectly detect HIV-1 p24 antigen.The band intensity of PCR products paralleled with the quantity of HIV-1 p24 antigen,and the limit of detection (LOD) could reach down to 1 pg/ml.ConclusionThe new assay based on GNP and PCR was efficient in the detection of HIV-1 p24,which is at least 3 orders of magnitude more sensitive than traditional ELISA.
RESUMEN
Objective To detect the Yersinia pestis plasmid and molecular weight along the Qinghai-Tibet Railway.Methods Yersinia pestis plasmids molecular weight detected and analyzed using alkaline lysis,phenol-chloroform extraction of Yersinia pestis plasmid by agarose gel electrophoresis.Results The 18 Yersinia pestis strains of Qinghai-Tibet Railway contained 6×106,45×106,52×106,65×106,92×106plasmid,varing in the range of the 52×106-92×106.Conclusions The Yersinia pestis of Qinghai-Tibet Railway has a standardplasmid graphics,with the biggest Yersinia pestis plasmid changing in a certain regular degree,which providessignificance in the study of plague natural foci of the spatial structure and the genetic.characteristics of Yersiniapestis.
RESUMEN
0.05). Conclusion The changes of serum protein electrophoresis results in the parturient women were mainly presented with the decreased in albumin and ? globin levels, increased percent of ? 1,? 2 and ? globins, increased percent of ? 2 and ? globins markedly related with increased ? and ? lipoprotein. Agarose gel electrophoresis as a screening method for component of serum proteins is valuable.
RESUMEN
Objectives To analyze the various protein components in urine and to define the types of proteinuria. Methods Sodium dodecyl sulfate argarose gel electrophoresis was used to analyze 86 non concentrated urine samples from patients with various renal injury. Results According to the images of urine protein electrophoresis, proteinuria can be classified into five types: glomerular, tubular, mixed, over flow, and physiological. Results from 16 cases of renal biopsy showed glomerular type, in 12 cases mixed type in 3, physiological type in 1. Conclusion This method is highly sensitive, simple, less time consuming and easy for long term preservation of results.