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Objective To explore the application value of PCR－reverse dot blot hybridization (PCR－RDB) gene membrane chip technique in genetic diagnosis of hereditary non－syndrome deafness in children.Methods The blood samples(2 mL)were collected from 38 children with congenital deafness,excluding high－risk factors for deaf-ness at Dongguan Rehabilitation School,and then genomic DNA extracted.By using self－designed multiplex－PCR combined with PCR－RDB gene chip technology,20 hot－spot mutations of 4 pathogenic genes of common deafness in Chinese population were detected.Sanger sequencing was used as the gold standard to corroborate the positive samples. Results Among 38 subjects,deafness gene mutations were detected in 16 cases,with a detection rate of 42.11%,and they were all verified by family study.Among 16 cases,6 cases of GJB2 gene mutation(3 cases of homozygote,3 cases of heterozygous),4 cases of SLC26A4 mutation,2 cases of MTRNR (m.1555A＞G)mutation,4 cases of compound muta-tion,but none of GJB3 gene mutations.And their detection rates were 15.79%,10.53%,5.26%,10.53%,and 0,re-spectively.DNA samples from 16 children with deafness gene mutation were corroborated by Sanger sequencing,and the compliance rate was 100%.Conclusions For 20 hot－spot mutations of 4 common deafness pathogenic genes,the matc-hing PCR－RDB gene membroine chip technology was designed and the susceptible gene of congenital deafness children was detected.This technique has some advantages like high detection rate,fast,accurate and economical.It is an ideal method for gene screening on hereditary non－syndrome deafness children and has good clinical application prospects.
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Objective:To establish a double antibody sandwich ELISA assay for detection of human IL-37 in serum.Methods: Mouse anti-human IL-37 monoclonal antibody was used as capturing antibody,rabbit anti-human IL-37 polyclonal antibodies served as detection antibody,HRP labeled goat anti-rabbit IgG employed as second antibody and recombinant human IL-37 protein used as reference standard for the establishment of a doubleantibody sandwich ELISA.The working conditions were optimized,such as sensitivity,linear range,reproducibility and evaluated the serum IL-37 in patients with Dengue fever.Results: The sensitivity of the established ELISA method was 1.465 μg/L approximately.Likewise,the linearity range of this method was about (1.465-46.875) μg/L.Further,the co efficient of variation (CV) of inter-batch and intra-batch in this study were 6.6％ and 11.7％,respectively.Notably,this method could be used in the detection of IL-37 in serum of the patients with Dengue fever,showing that the level of IL-37 in Dengue fever patients was much higher than that in healthy controls.Conclusion: The double antibody sandwich ELISA assay for the detection of human IL-37 was successfully established,which can be apply to detect of human IL-37 in clinical samples.
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We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells,and purified and evaluated for its serological activities.A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide (EAAAK)2 and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The expressed recombinant protein was purified with 6× His purification media,and identified by SDS-PAGE and Western blot,and its antigenicity was analyzed by using an indirect ELISA assay.The recombinant expression vector pET28a-rE was constructed and expressed in BL21 (DE3) successfully,but the recombinant proteins mainly appeared as inclusion bodies.The target protein was obtained with high purity through the purification of affinity chromatography.SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.The established indirect ELISA has high accuracy.This recombinant peptide antigen expressed in E.coli has good potential for serum testing.
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Objective To evaluate the value of detecting HCMV-DNA in urine in the diseases of newborn babies ,by comparing the result between HCMV-DNA in urine and HCMV-IgM antibody in blood in different diseases of the newborn babies .Methods The urine and blood samples were collected from 1 520 infants who were in hospital of the neonate department from January 2013 to December 2014 .The HCMV-DNA in urine was examined by fluorescence quantitative polymerase chain reaction(FQ-PCR) .And HCMV-IgM antibody in blood was examined by the method of chemiluminescence(ECL) .Results In the 1 520 cases ,153 had de-tectable HCMV-DNA in their urine samples with a positive rate 10 .07% ,while only 4 cases were positive of the HCMV-IgM anti-body in blood with a positive rate 0 .27% .The two groups was statistically significant difference (P<0 .05) .In the infant diseases of 1 520 cases ,the positive rate of HCMV-DNA in urine was 82 .6% of the hepatitis syndrome while the positive rate of HCMV-IgM antibody in blood was 3 .85% .The positive rate of HCMV-DNA in urine was 8 .95% in the jaundice symptoms ,while the posi-tive rate HCMV-DNA in urine was 4 .62% in the pulmonary infection .Each group of disease was statistically significant difference in the positive rate of HCMV-DNA (P<0 .05) .Conclusion The detection of HCMV-DNA in urine is more beneficial to the diag-nosis of HCM V infection of infants than the detection of HCM V-IgM antibody in blood ,especially in hepatitis syndrome ,neonatal jaundice and pulmonary infection .
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Objective To retrospectively analyze the result of HCMV‐DNA of breast milk and urine of neonatal hyperbilirubi‐nemia and evaluate the effect on screening neonatal hyperbilirubinemia .Methods Collected 461 cases of neonatal hyperbilirubinemia from January 2014 to December 2014 in our hospital ,which fed with breast milk as observation group ,at the same period collected 450 cases of healthy newborn in our hospital as control group .Tested the their level of HCMV‐DNA in breast milk and urine with Fluorescent Quantitative PCR ,recorded the testing results .Results 239 cases with positive HCMV‐DNA urine were detected in observation group ,accounting for 51 .8% .There were 89 cases of positive HCMV‐DNA urine in control group ,which accounted for 19 .8% ,the comparative difference was statistically significant(P< 0 .01) .There were 367 cases of positive HCMV‐DNA breast milk in observation group ,accounting for 79 .6% .The positive HCMV‐DNA breast milk were detected in 137 cases in control group ,which accounted for 30 .4% ,the comparative difference was statistically significant (P<0 .01) .Conclusion The breast milk of HCMV infection was an important way of neonatal HCMV infection ,the HCMV‐DNA detection of breast milk and urine was of great significance to the diagnosis of neonatal hyperbilirubinemia .
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Deafness is a seriously disabling disease affecting the quality of human life and genetic fac-tors account for a large proportion in the pathogenesis of newborn deafness.With the development of genomics technology,molecular genetics of hearing loss has become a cutting-edge field under investigation in otology. Molecular diagnostic technique plays an important role in exploring the pathogenesis,assisting clinical diagnosis and the prenatal diagnosis.In this review,we introduce the common pathogenic gene mutations and the diagnosis of non-syndromic inherited hearing impairment.
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Objective To retrospectively analyze the result of hemoglobin(Hb)test by using full-automatic Hb electrophoresis and evaluate the its significance in hemoglobinopathy.Methods The data of patients who underwent Hb electrophoresis test and regular blood tests in the hospital from January 2011 to December 2013 were included in the study.The test results were recorded including mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH)and results of Hb electrophoresis test.Final diag-nosis were made for suspected patients by using genetic testing,then disease detection rates and gene coincidence rates and constitu-ent ratios were calculated.Results 12 898 cases were included in the study,after statistical analysis the MCV was(85.32±13.61) fL,MCH was(29.87±6.44)pg.By using automatic hemoglobin electrophoresis,1 315 cases were found to be positive,in which 568 were male,747 were female,the detection rate was 10.19%.In the 1 315 patients,there were 761 cases suspectedα-thalassemia,ac-counted for 5.90%.There were 495 cases of suspectedβ-thalassemia,accounted for 3.84%,11 patients with HbJ(0.08%),15 pa-tients with HbK(0.12%),9 patients with HbG(0.07%),3 patients with HbD(0.02%),21 patients with HbE(0.16%).The sus-pected case′s final diagnosis were made by using genetic testing,α-thalassemia gene′s coincidence rate was 80.55%,β-thalassemia gene′s coincidence rate was 96.77%.Conclusion Automatic hemoglobin electrophoresis detection is of great significance for the di-agnosis of hemoglobinopathy.
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Objective To explore the genotype of patients with glucose-6-phosphate dehydrogenas(G6PD)deficiency in Dong-guan and provide the basis for the clinical diagnosis and prevention.Methods The clinical data of patients who took G6PD activity screening in the hospital were collected from January 2011 to December 2013,the G6PD/6PGD ratios were recorded.469 patients with positive G6PD/6PGD ratio were randomly enrolled in the study,whose mutations were detected by reverse dot blot(RDB)as-say.Results During this period,we measured G6PD activity of 16 464 cases by G6PD/6PGD ratios,there were 672 positive cases, the positive rate was 4.08%.Randomly selected 469 positive samples,detected their genotye by RDB assay.We detected 173 cases of G1376T,141 cases of G1388A,82 cases of A95G,60 cases of G871A,23 cases of G392T,14 cases of C1024T.In addition to that, we also found some rare mutations,such as 6 cases of C1004T,2 cases of T517C,1 cases of C1360T.65 cases of C1311T gene poly-morphism and 96 cases of dual gene mutations were detected.Conclusion The incidence of G6PD deficiency is high and the gene mutation types in Dongguan are both representative for Chinese population and with local heterogeneity.The study on gene muta-tions of G6PD deficiency is benefit for diagnosis and prevention.
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Glucose-6-phosphatedehydrogenase( G6PD) is the main regulatory enzyme of pentose-phos-phate pathway,which plays an important role in maintaining the balance of cell energy and redox reactions in the cell. G6PD deficiency is the most common hereditary erythrocyte enzyme deficiency disease. There are no effective treatments for the disease. Currently,the key of control and treatment is to make a definitive diagnosis in time and keep away from related risk factors of the disease. At present,the main clinical diagnostic method is the detection of G6PD enzyme activity,but it is limited in accuracy of detecting the heterozygote females. It has already been confirmed at home and abroad that G6PD heterozygote is a risk factor of neonatal hyperbilirubi-nemia. Thus,the detection method of different genotypes of G6PD deficiency at the same time is urgently needed in clinical diagnosis. This paper reviews on recent research progress of the G6PD deficiency disease.
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ObjectiveTo investigate the changes and functions of T lymphocyte subsets,immune globulin and complement in children with mycoplasma pneumoniae pneumonia(MPP) on different disease stages.MethodsThe levels of T Iymphocyte subsets of CD3,CD4,CD8 and immunoglobulin ( IgG,IgA IgM),and complement ( C3,C4 ) in the peripheral blood were detected on acute and recovery stages in 28 children with MPP by flow cytometry and immune nephelometry.Twenty-five healthy children were recruited as control group.ResultsAmong these subjects of MPP children on acute stage,the levels of CD3,CD4,CD8,and CD4/CD8 in the peripheral blood were (58.71 ± 11.63)％,(32.36 ± 8.06)％,(28.19±6.23 ) ％ and 1.15 ± 0.41 respectively,and on recovery stage,the levels of CD3,CD4,CD8,and CD4/CD8 were (61.29 ±10.17)％,(34.14 ±7.22)％,(26.47 ±6.01)％,and 1.29 ±0.37 respectively.Both on acute stage and on recovery stage of MPP children,the levels of CD4,CD4/CD8 were significantly lower than those in control group [ (39.53 ± 6.16 ) ％,1.83 ± 0.49 ],and CD8 was significantly higher compared to thecontrol group( 1.83 ± 0.49 ),P＜0.01.CD3 were lower than that in control group [ (63.03 ± 12.32) ％ ] on acute stage (P＜0.01 ),and no significant difference on recovery stage (P＞0.05).During the acute stage of MPP,IgG [ ( 14.50 ±3.86) g/L] and IgM [ ( 1.67 ±0.56) g/L] were obviously higher than those in control group [ (7.92 ± 2.62 ) g/L,( 1.06 ± 0.32 ) g/L,P＜0.01 ],and C3 [ ( 0.83 ± 0.42 ) g/L ] were obviously lower compared to the control group [ ( 1.37 ± 0.33 ) g/L,P＜0.05].There were no significant differences of IgA and C4 between MPP and control groups ( P＞0.05 ).ConclusionChildren with MPP had celhilar immune and humoral immune disorders.Through the detection of T lymphocyte subsets,immunoglobulin and complement,it will be helpful to judge the effectiveness of clinical treatment,which provides a theoretical basis for the clinical application of immune regulators.