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Objective To establish and optimize a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Escherichia coli and its microbial toxin.Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction,and the optimized LAMP system was used for the detection.Results Primers targeting shiga toxin (stx) gene and O157 antigen gene rfbe were designed.The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs,10 mmol/L MgSO4,0.4 mol/L betaine,1 μl 10 × Bst DNA polymerase Buffer,8 U Bst DNA polymerase fragment,2 μl DNA template,and the ratio of inner-primer (FIP and BIP) and outerprimer (F3 and B3) were 8∶ 1.Time and temperature for LAMP was 60 min,60 ℃.The sensitivity was 103 times higher than polymerase chain reaction (PCR),reached 5 × 101 CFU/ml.When LAMP was applied to 19 reference strains,102 EHEC strains,the specification was 100％ while identification rate of rfbe,stx1 and stx2 gene reached 100％,95.2％,92.9％.Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin.
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The patient was a 21 days-old baby girl,admitted to Guangdong Women and Children Hospital because of "poor intake,seldom crying and no activity in 1 day".The major clinical manifestations included hypotonia,aggravation of the conscious disturbance,pancytopenia,intractable acidosis and hyperammonemia,so,inherited metabolic disorders should be considered.Screening of inherited metabolic diseases with blood and urine samples,genetic test and active treatments were carried out.After targeted next-generation sequencing,a novel homozygotic frame shift mutation in PCCB gene:c.838_839insC (L280Pfs * 11) was identified,which was validated by Sanger sequencing.This mutation had not been reported in the mutation database,and bioinformatic analysis of this mutation indicated disease-causing.So,the diagnosis of propionic acidemia was identified.The baby was in a critical condition,and despite active treatment,her conscious disturbance was aggravated,and the spontaneous breathing disappeared.Subsequently,the baby died of pneumonia.Propionic acidemia is a relatively common genetic metabolic disease in newborns.The severity and the clinical phenotypes of propionic acidemia varied,which often made the diagnosis difficult.When the baby is presented with developmental delay,hypotonia,recurrent convulsion and vomiting,etc,which can't be explained by common diseases of children,propionic acidemia may be considered.Next generation sequencing analysis of the complicated cases can easily to pinpoint a disease-causing gene,which lays a solid foundation for accurate diagnosis and treatment of the patients.
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Objective To investigate the application value of fetal umbilical cord blood hemoglobin analysis in the prenatal diag ‐nosis of thalassemia .Methods 113 couples were the carriers of the same gene type of thalassemia ,moreover the females were in the pregnant period of 24 - 30 pregnant weeks and performed the prenatal diagnosis .The fetal umbilical cord blood hemoglobin compo‐nents were analyzed by the full automatic capillary electrophoresis technique ,meanwhile the fetal thalassemia gene was detected .Re‐sults Among 113 fetuses ,the umbilical cord blood HbBart′s level in 11 cases of severe α thalassemia was 85 .0% - 95 .5% ,which in 9 cases of intermediate type α thalassemia was 22 .0% - 39 .5% ;the umbilical cord blood HbA level in 6 cases of severe β thalas‐semia was 0% - 0 .4% ,which in 17 cases of light type β thalassemia was 2 .1% - 12 .5% .Conclusion The fetal umbilical cord blood hemoglobin analysis could be used for rapid prenatal diagnosis of severe α ,β and intermediate type α thalassemia ,which can serve as a supplementary method for the prenatal diagnosis of thalassemia .
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Objective To compare the effect of three β-thalassemia prenatal screening strategies in Guangdong province. Methods A total of 13 284 hospital-delivered couples and 13 369 newborns were recruited from 91 hospitals in 21 counties or districts of Guangdong province from June to December 2012. Mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A2 (Hb A2) were tested for all the couples, and all the couples and newborns were detected by 17 types ofβ-globin gene mutations. The effect of three β-thalassemia prenatal screening strategies were compared as following:(1) MCV/MCH with Hb A2 serial screening(SS):Hb A2 was tested if the woman′s MCV3.5, it meant positive. And if the woman wasβ-thalassemia carrier and her husband′s Hb A2>3.5, it meant couple positive. (2) MCV/MCH with Hb A2 parallel screening(PS):if the woman′s MCV3.5 pg, it meant couple positive. And the husband would be tested forβ-globin gene mutations if the woman was β-thalassemia carrier. (3) MCV/MCH with Hb A2 serial screening for couples(SSC):if one of the couple or both of them had MCV3.5, it meant couple positive. Results (1) For the SS strategy, the sensitivity was 92.69%(583/629);the specificity was 99.87%(12 638/12 655); the positive predictive value was 97.17%(583/600);and the negative predictive value was 99.64%(12 638/12 684). The results ofβ-globin gene mutations tested showed that the rate ofβ-thalassemia carriers was 4.74%(629/13 284) in the 13 284 pregnant women, and it was 4.29%(570/13 284) in their husbands. (2) The SS strategy detected 27 (0.20%,27/13 284) β-thalassemia carrier couples. For the SS strategy detecting β-thalassemia carrier couples, the missed diagnosis rate was 11.11%(3/27);the sensitivity was 88.89%(24/27);the specificity was 100.00%(27/27); the positive predictive value was 100.00%(24/24); and the negative predictive value was 99.98%(13 257/13 260). (3) When using the SS strategy for 13 369 offsprings, there were 582β-thalassemia carriers (4.35%,582/13 369), including 578 (99.31%,578/582) minorβ-thalassemia, 3 (0.52%,3/582) intermediaβ-thalassemia and 1 (0.17%,1/582) major β-thalassemia. The SS strategy detected 25 fetuses who neededβ-thalassemia prenatal diagnosis. (4) For the PS strategy, the sensitivity was 98.09%(617/629); the specificity was 88.73%(11 229/12 655); the positive predictive value was 30.20%(617/2 043); and the negative predictive value was 99.89%(11 229/11 241). (5) When using the PS strategy for theβ-thalassemia carrier couples, the sensitivity was 100.00%(27/27);the specificity was 95.55%(12 667/13 257);the positive predictive value was 4.38%(27/617);and the negative predictive value was 100.0%(12 667/12 667). (6) The PS strategy detected 28 fetuses who needed β-thalassemia prenatal diagnosis in 13 369 offsprings. (7) For the SSC strategy, the sensitivity was 93.80%(590/629); the specificity was 95.75%(12 117/12 655); the positive predictive value was 52.30%(590/1 128); and the negative predictive value was 99.68%(12 117/12 156). When the SSC strategy was used for the husbands, the sensitivity was 92.28%(526/570); the specificity was 95.27%(12 112/12 714);the positive predictive value was 46.63%(526/1 128); and the negative predictive value was 99.64%(12 112/12 156). (8) When the SSC strategy was used inβ-thalassemia carrier couples, the sensitivity was 100.00%(27/27);the specificity was 91.69%(12 156/13 257);the positive predictive value was 2.39%(27/1 128);and the negative predictive value was 100.00%(12 156/12 156). (9) The SSC strategy detected 28 fetuses who neededβ-thalassemia prenatal diagnosis. Conclusions All the three β-thalassemia prenatal screening strategies had good effect in clinical practice and public health. While in the high-prone area of β-thalassemia, MCV/MCH with Hb A2 parallel screening and MCV/MCH with Hb A2 serial screening for couples stratigies were better.
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<p><b>OBJECTIVE</b>Diagnosis and prenatal diagnosis to a family of hemoglobin variant with α-thalassemia.</p><p><b>METHODS</b>Whole blood cell analysis, hemoglobin analysis by capillary zone electrophoresis (CZE), Gap-PCR, polymerase chain reaction-reverse dot blot (PCR-RDB) assay and DNA sequencing.</p><p><b>RESULTS</b>Hb Zurich Albisrieden with α°-thalassemia lead to severe anemia. The genotype of fetus is also Hb Zurich Albisrieden with α°-thalassemia.</p><p><b>CONCLUSION</b>Abnormal hemoglobin with α-thalassemia may lead to severe anemia, Prenatal diagnosis of thalassemia has the vital significance for eugenic birth.</p>
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Adult , Child, Preschool , Female , Humans , Male , Pregnancy , Young Adult , Base Sequence , Fetal Diseases , Blood , Diagnosis , Genetics , Hemoglobins, Abnormal , Genetics , Metabolism , Molecular Sequence Data , Prenatal Diagnosis , alpha-Thalassemia , Blood , Diagnosis , Embryology , GeneticsABSTRACT
Objective To devolope a method for extracting DNA from dried blood spots (DBS)and optimizing the operating procedure,which could be applied to clinical gene diagnosis of thalassemia.And the cross contamination of DBS punching and the storage stability of DBS were studied.Methods A total of 1 50 blood specimens were collected,and DBS were prepared.Circles (3 mm in diameter)were punched in the DBS,and eluted with lysis buffer.The eluting method and operating procedure were opti-mized.Genomic DNA extracted from the elution solution by magnetic beads,and were performed thalassemia gene test.Finally jud-ging whether the results of DBS and whole blood were consistent.Two methods of thalassemia gene test were used in DBS and the compatibility of DBS processing method was verified.Judging whether there was cross contamination of DBS punching by the thalassemia gene test results of blank hole which were punched in the blank filter paper between thalassemia positive DBS.The DBS storage stability in thalassemia gene test was verified by detecting the DBS which were dry stored at room temperature for 6 and 9 months.Results 5 circles (3 mm in diameter)DBS were vibrating eluted at 55 ℃ for 1 hour,the DNA concentration extracted from the elution solution was 10-20 ng/μL,which was dissolved in 50 μL solution,and the DNA quality was good.The thalassemia gene test results of DBS and whole blood were the same,and the DBS results of two thalassemia gene test methods were the same too. The cross contamination of DBS punching was not detected in thalassemia gene test.The DBS which were dry stored at room tem-perature for 6 and 9 months could be stably performed thalassemia gene test.Conclusion DBS could be used to perform thalassemia gene test,which is accurate,convenient and stable.It is an ideal way for specimen referral of thalassemia gene test.
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Objective To compare the effect and cost of three different α-thalassemia prenatal screening strategies used in Guangdong, China, and to provide evidence for α-thalassemia prevention. Methods In total, 13 284 hospital-delivery couples and 13 369 newborns/fetuses (offspring) from 21 counties or districts of Guangdong Province were included in this study, who were treated from June to December 2012. Mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A2 (Hb A2) were detected in the couples, and 6 types ofα-globin gene mutations were found in all couples and newborns. The strategies were MCV/MCH and serum Hb A2 (protocolⅠ) or parallel screening based on pregnant women (protocolⅡ), and serum screening based on couples (protocolⅢ). The validity and reliability of the three strategies were then compared using the Chi-square test. Results The sensitivity and the specificity of pregnant women who wereα-thalassemia carriers in protocolⅠwere 74.82%(1 352/1 807) and 74.11%(8 506/11 477), and were 89.82%(1 623/1 807) and 48.60%(5 578/11 477) in protocol Ⅱ , respectively. And 1.67% (221/13 284) couples were bothα-thalassemia carriers by the gene test. The rate of missed diagnosis in bothα-thalassemia carrier couples in protocolsⅠ,ⅡandⅢwas 50.68%(112/221), 11.76%(26/221) and 11.31%(25/221), respectively. In couples who needed prenatal diagnosis, the rates of missed diagnosis, sensitivity, specificity, positive predictive value, and negative predictive value were 17.46%(11/63), 82.54%(52/63),98.35%(13 003/13 221), 19.26%(52/270) and 99.92%(13 003/13 014) in protocolⅠ;4.76%(3/63), 95.24%(60/63), 88.18%(11 658/13 221), 3.70%(60/1 623) and 99.97%(11 658/11 661) in protocolⅡ;and 3.17%(2/63), 96.83%(61/63), 59.31%(7 842/13 221), 1.12%(61/5 440) and 99.97%(7 842/7 844) in protocol Ⅲ , respectively. The diagnosis of severeα-thalassemia was not missed in all three screening strategies. The mean cost of protocols Ⅰ, Ⅱ and Ⅲ for detecting a couple who needed prenatal diagnosis was 37 049.23, 50 836.00 and 40 321.64 RMB, respectively. Conclusions The three screening protocols have good efficiency in screening forα-thalassemia. However, protocolsⅡandⅢare preferred when financial conditions permit.
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Objective To study the diagnostic utility of hemoglobin electrophoresis in neonatal cord blood screening for thalassemia. Methods Between January 2012 and December 2013, 14032 core blood samples which were from different 21 Women and Children Hospitals in Guangdong were performed for the neonatal screening with hemoglobin electrophoresis. The positive samples of hemoglobin electrophoresis were recalled for genetic testing. Results Out of 1445 (11.07%) positive samples of hemoglobin electrophoresis , 1075 (54.08%) cases were suspected for α-thalassemia, 478 (3.41%) cases were suspected for β-thalassemia, 127 (0.91%) cases were suspected for abnormal hemoglobin. With the genetic testing, 967 cases were diagnosed as α-thalassemia, 404 cases were diagnosed asβ-thalassemia. The coincidence rate ofα-thalassemia andβ-thalassemia were 89.95%and 82.96%, respectively. Besides, 124 cases were diagnosed as abnormal hemoglobin, including 38 cases of Hb E, 28 cases of Hb Q, 21 cases of Hb D, 19 cases of Hb New York, 13 cases of Hb J, and 5 cases of Hb J. Conclusion Hemoglobin electrophoresis was definitely helpful in the neonatal cord blood screening for thalassemia and abnormal hemoglobin.
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Objective To conduct the amplification,cloning,bioinformatics analysis,prokaryotic expression and purification of enterovirus 71 VP1 gene segment and to initially confirm the biological activity of the recombinant expression product.Methods A pair of specific primers was designed according to GenBank EV71 sequence,viral RNA as a template was extracted from the throat swab specimens in the EV71 patients.EV71 VP1 gene was amplified by RT-PCR.After enzyme digestion,the expression vector pET28a was inserted.The prokaryotic expression vector of pET28a-EV71 VP1 was constructed.Then the E.coli DH5a transforma-tion was performed.IPTG was adopted for induction expression.The expression results were analyzed by using SDS-PAGE and Western blot.The bioinformatics analysis of the sequenced results was performed by the software.Expressed protein was purified and the plates were coated,ELISA was used to test the VP1 specific IgG antibody in serum samples of EV71 positive and COX A16-positive patients.Results The BLAST alignment showed that the homology of the objective gene EV71 VP1 was 99% com-pared with other strains(JQ766207.1)in GenBank.EV71 VP1 protein was about 32×103 ,which mainly existed in the form of in-clusion body.The bioinformatics analysis showed that EV71 VP1 protein was a hydrophilic protein,without transmembrane region and N-terminal signal peptide sequence,the tertiary structure existed.The ELISA results showed that the specific IgG OD value in EV71-positive patients was(2.425±0.521),OD value in COX A16 positive patients was(1.205 ±0.314),the normal control OD value was(0.353±0.128).The sensitivity and specificity of EV71 VP1 protein detection were 84% and 88% respectively.Conclu-sion The pET28a-EV71 VP1 expression vector is successfully constructed;the preliminary analysis on the serum of the infected patients by ELISA shows that the obtained objective protein has higher sensitivity and specificity,which is initially confirmed to have biological activity and can be further used for the related study on EV71 diagnosis and vaccine.
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0.05).Ob-Rb was also expressed in the endometrium of mouse throughout the implantation process,but the expressions were significantly different in the different phases of the implantation process (P