The detection of HLA-B27 gene and gene typing of ankylosing spondylitis patients by fluorescence PCR / 国际检验医学杂志

Objective To investigate the clinical application value of fluorescence polymerase chain reaction (PCR) .in the human leucocyte antigen-B27(HLA-B27) gene and gene typing detection of ankylosing spondy-litis (AS) patients .Methods A total of 43 clinical blood samples of AS and 56 samples of healthy controls were collected in Shenzhen Futian hospital for rheumatic diseases from January 2014 to March 2015 .HLA-B27 gene was detected by flow cytometry .HLA-B27 gene and gene typing was also detected by the fluorescence PCR method .Results Among 43 samples ,40 samples were HLA-B27 positive(93 .02%) by flow cytometry while 39 samples were HLA-B27 positive (90 .70%) by fluorescence PCR .The total coincidence rate was 97 .50% .Among 39 positive samples ,32 samples were HLA-B2704 positive (82 .05%) and 7 samples were HLA-B2705 positive (17 .95%) .Conclusion The fluorescence PCR is an accurate method to detect HLA-B27 gene and presents high consistency with flow cytometry .It can also detect the HLA-B27 gene typing .It may have great clinical application value and prospects .

Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction / 北京大学学报(医学版)

Objective:To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome.Methods:Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type Ⅳ collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type Ⅳ collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome.A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study.Their genotypes were unknown.Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification.Genomic DNA was isolated from peripheral blood leukocytes in all the participants.Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction.When a deletion removed exon 1 of COL4A5 gene was identified,the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes,a promoter shared by COL4A5 and COL4A6 genes,and three reference genes in a single reaction.Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions.Results:Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification.Deletions were identified in 6 of the 20 patients,including two large deletions removing the 5'part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6,two large deletions removing more than 30 exons of COL4A5 gene,one large deletion removing at least 1 exon of COL4A5 gene,and one small deletion involving 13 bps.No duplication was found.Conclusion:Our results show that multiplex competitive fluorescence polymerase chain reaction is a good alternative to classical techniques for large deletion genotyping in X-linked Alport syndrome.

Genetic polymorphisms of HPRTB, DXS6803 and DXS6809 STR loci in Tianjin Han female population / 天津医药

Objective To investigate genetic polymorphisms of HPRTB, DXS6803 and DXS6809 STR loci in Tianjin Han female population, and to provide experimental data in the prenatal diagnosis of aneuploidies accurately and rapidly. Methods A total of 150 blood samples were collected in Tianjin Han population. QF-PCR and capillary electrophoresis were used in this study. The relevant data were analyzed by ABI Prism GeneMapper v3.0 software. Two homozygotes were se?lected from each locus for sequencing. The frequencies of the genotypes were checked using Chi-square test to verify Hardy-Weinberg Equilibrium. Data of genetic polymorphisms were calculated by PowerStatsV12 software. Results A total of 150 samples were successfully amplified in 24 hours. The 10, 6 and 10 alleles and 22, 12 and 29 genotypes were found respec?tively in HPRTB, DXS6803 and DXS6809 loci. The most common alleles were 14, 13 and 14. The higher frequencies of gen?otypes were 14-14, 12-13 and 13-14. No significant deviations from the Hardy-Weinberg equilibrium were observed in these three STR loci (χ2=10.554, 5.783 and 15.355, respectively, P>0.05). Values of He were 0.748, 0.649 and 0.806 for these three STR loci respectively. Values of Ho were 0.607, 0.700 and 0.713 respectively. Values of PIC were 0.706, 0.599 and 0.775 respectively. Values of PD were 0.894, 0.814 and 0.931 respectively. And values of PE were 0.299, 0.428 and 0.449 respectively. Conclusion HPRTB, DXS6803 and DXS6809 STR loci were highly polymorphic, which are favorable genetic markers on chromosome X and can be used in rapid prenatal genetic diagnosis.

Comparison of two kinds of HLAˉB27 detection method in diagnosis of ankylosing spondilitis / 国际检验医学杂志

Objective To investigate the clinical application value of fluorescence PCR in vitro diagnosis(IVD)reagent kits in the HLA-B27 detection by comparing 2 kinds of HLA-B27 IVD reagent kit approved by CFDA.Methods A total of 573 clinical blood samples were collected and detected for HLA-B27 by the approved reagent kits based on the fluorescence PCR technique and the flow cytometry.The samples with inconsistent testing results by the two kits were further confirmed by the PCR sequencing.At the same time,about 5% samples of the positive results detected by the fluorescence PCR method were extracted for conducting the re-testing.Results Among 573 samples,191 samples were HLA-B27 positive and 382 cases were HLA-B27 negative by flow cytome-try;the same samples had 194 cases of HLA-B27 positive and 379 cases of HLA-B27 negative by real-time PCR.With flow cytome-try as reference of the final results,the positive coincidence rate of the two kinds of kit was 96.33%(184/191),the negative coinci-dence rate was 94.76%(362/382),27 samples had inconsistent results from the two kinds of assay(accounting for 4.71% of the to-tal number of samples),the total coincidence rate was 95.29% [(184+362)/573],the Kappa value was 0.896(P =0.02);the chi-square test P =0.021,the two kinds of testing method had the high consistency,but the differences existed in the testing results. The re-testing results by PCR sequencing(including 27 samples with inconsistent results by two kinds of kit)were entirely consist-ent with the fluorescence PCR testing results.Conclusion Compared with the authority method flow cytometry for HLA-B27 tes-ting in clinic,the fluorescence PCR kit may present more accurate judging ability for the HLA-B27 testing on the basis of ensuring the higher consistency of the testing results,is easier compared with the sample preparation and operating procedures,and has the stronger clinical application value and prospects s.

An Integrated Nucleic Acid Extraction Microchip for Real-time Polymerase Chain Reaction Micro Total Analysis System / 分析化学

A real-time polymerase chain reaction ( PCR ) micro total analysis system (μ-TAS ) integrated nucleic acid extraction, PCR amplification and real-time-fluorescent PCR detection on a same microfluidic chip was prepared for the fully automated and on-chip analysis of nucleic acid. The proposed method had the advantage such as low sample consumption, fast analysis and simple operation and so on. Micromachining technology was used to fabricate the anodic molds of integrated nucleic acid extraction microfluidic chip. A polydimethylsiloxane (PDMS) substrate with 3D channels was manufactured by a combination of molds and an injection molding method. The glass substrate and the chip were bonded together using a plasma treatment. The μ-TAS included a microfluidic control device whose micro fluidic velocity ( 0-10 mL/min ) could be adjusted, a TEC platform which the precision of temperature control was 0. 1℃ and a CCD detection module. The DNA of human blood was extracted by using a silica gel membrane method on the microfluidic chip. The processes of DNA extraction and detection were preset in the μ-TAS. Human blood lysate ( 20 μL ) were driven to the extraction chamber and was then washed. The fluidic drive speed was 2 mL/min. DNA and PCR reagents were mixed and then were driven into the PCR chamber. The fluidic drive speed was 1 mL/min. The GAPDH gene in extracted genome DNA was amplified by PCR and detected. The amplified product was verified by melting analysis. The results of nucleic acid extraction method on the chip were compared to those obtained using a standard manual centrifuge extraction method. The amplification curves were obvious. Ct values of the chip method were 25 . 3 and 26 . 9 . The denaturation temperature of all the melting was 89 . 9 ℃. The results validated that the chip-based method and device realized the extraction of nucleic acid, amplification and detection automatically.

Fluorescence PCR for detecting ureA gene of Helicobacter pylori in feces / 中华临床感染病杂志

Objective To evaluate the application of fluorescence PCR in detecting ureA gene of Helicobacter pylori(HP)in feces.Methods Fluorescence PCR was used to detect ureA gene of HP in feces from 50 patients,including 23 confirmed by gastric biopsy urease test and histological staining.Bacterium culture and serum antibody detection were also performed, and chi-square test was used to compare the sensitivity,specificity,positive predictive value and negative predictive value among three methods.Results The sensitivity,specificity,positive predictive value and negative predictive value of fluorescence PCR were 1.00,0.96,96.O%and 100.0%,while those for HP culture were 0.78,1.00,100.0%and 84.0%,and thee for serum antibody detection Was 0.96,0.74,76.O%and 95.0%.There were significant differences in sensitivity and negative predictive value between PCR and bacterium culture (X2=5.60 and 4.44,P<0.05),and significant differences in specificity and positive predictive value between PCR and serum antibody detection(X2=5.28 and 4.08,P<0.05).Conclusion ureA gene detection in feces by fluorescence PCR is of value for the diagnosis of HP infection.

ApoE genotyping by real-time fluorescence PCR / 临床检验杂志

Objective To establish a new effective method by using real-time polymerase chain reaction (PCR) to detect single nucleotide polymorphism (SNP) typing for rapid identifying apolipoprotein E alleles.Methods To determine alleles of human apolipoprotein E genetic polymorphism at Cys112Arg locus was detected by PCR melting curve analysis with fluorophore SYBR Green I. In order to increase the speciality of SNP assays, high fidelity Taq polymerase was used. The reliability of SNP typing was validated by comparison with the results of direct DNA sequencing.Results Each sample was determined by double tubes, and two melting curves were analysis. As compared the Tm value of samples with the Tm of standard substance, the apoE genotype of samples was determined. The apoE genotype of 30 samples were E3/3 (27/30) and E3/4 (3/30) respectively, which was accordant with the results of PCR-RFLP and DNA sequencing.Conclusion The presented allelic assay was specific, easy to operate and applicable for discrimination of apolipoprotein E genotyping of human blood.

Five Pathogen Mixed Infection in STDs of Female Genitourinary Tract:Analysis and Status / 中华医院感染学杂志

OBJECTIVE To study the status of five pathogen mixed infection in sexually transmitted diseases(STDs) and analyze the clinical meaning.METHODS We detected five common pathogens by fluorescence polymerase chain reaction,which were Neisseria gonorrhoeae(NG),Chlamydia trachomatis(CT),Ureaplasma urealyticum(Uu),human papilloma virus(HPV),Candida albicans(Ca),and herpes simplex virus.RESULTS We analyzed the status of infection among 4 601 patients,got 279 mixed infection cases,accounted for 6.1% in all cases;and the population with ages from 21 to 40 years was accounted for 89.6%. CONCLUSIONS Fluorescence polymerase chain reaction is a simple,rapid,high sensibility technique for quantitation testing of STDs pathogens,and we should pay great heed to its effective control.

Study on Quantified Lyophilized serum of Hepatitis B Virus DNA in Fluorescence PCR Detection / 中华检验医学杂志

Objective To study suitability of a series of lyophilized serum with definitive HBV DNA value in fluorescence quantitative COBAS Amplicor HBM kit and a sample with 106 copies/ml HBV DNA was prepared, and sent to various manufactures which would be asked to detect the samples using their own kits. Then a calibration curves from CT values of the series to the corresponding concentrations was compared with that obtained from the external standard-calibration curve with the manufactures series. Results The standard-calibration curve with the series of lyophilized serum showed an excellent correlation (