Ambiguity results distribution and its solutions of HLA-A, B and DRB1 sequence-based typing / 重庆医学

Objective To investigate the ambiguity results distribution of HLA-A,B and DRB1 gene sequence-base typing in Guangxi population and to propose the way to resolve.Methods HLA-A,B and DRB1 genes of 1 000 donors in the Guangxi branch bank of China'bone marrow bank were genotyped by PCR-SBT,and then the ambiguity results distribution of the three loci was analyzed.The typing ambiguities resultswere resolved by high-resolution polymerase chain reaction-sequence-specific primers(PCR-SSP) and group specific sequencing primer(GSSP) methods,respectively.Results Among 1 000 samples,at least 1 locus in HLA-A,B and DRB1 genes in 96.7％ samples appeared the ambiguity results,in which the proportions of HLA-A,B and DRB1 loci appearing ambiguity results were 65.7 ％,58.8 ％ and 77.2 ％ respectively.For the samples of detected ambiguity results,single using the GSSP method could resolve the ambiguity typing results of 87.37％ HLA-A,93.54％ HLA-B and 60.49％ HLA-DRB1,using high-resolution PCR-SSP could resolve the ambiguity typing results of 12.63 ％ HLA-A,4.76 ％ HLA-B and 15.29 ％ HLA-DRB1,and the rest 1.70 ％ HLA-B and 24.22 ％ HLA-DRB1 ambiguity results were resolved by both GSSP and high-resolution PCR-SSPs method.Conclusion GSSP and high-resolution PCR-SSPs methods have high abilities to solve HLA ambiguity results both locate inside and outside the sequencing region,respectively.GSSP and high-resolution PCR-SSPs methods are supplement for each other,which can effectively resolve the problem of ambiguity results in high resolution HLA typing.

Sequence Analysis and Identification of A Novel HLA-A* 2 4∶3 2 7 Allele Been Designated by the HLA Nomenclature Committee of WHO / 现代检验医学杂志

Objective To do analysis of sequence and identify a novel HLA-A allele in Chinese hematopoietic stem cell do-nors.Methods A rare HLA-A allele was initially detected by Luminex PCR-SSO typing,then the sample was sequenced by sequence-based typing (SBT)and the group-specific sequencing primer (GSSP)to confirm the mutation allele and locus.Re-sults The sequence of the sample results showed that the allele compared with the highest homologous allele HLA-A*24∶02∶01∶01 was the difference in the exon 3 at position 544 G>A,resulting in an amino acid sequence of HLA-A*24∶02∶01∶01 at position 158 change Ala to Thr.Conclusion This allele is a new HLA-A allele and has been designated as HLA-A*24∶327 by the HLA Nomenclature Committee of World Health Organization (WHO).

Resolution of Ambiguous HLA Genotyping in Korean by Multi-Group-Specific Sequence-Based Typing

PURPOSE: To evaluate a multi-group-specific sequence-based typing (SBT) method for resolving ambiguous results from human leukocyte antigen (HLA) genotyping. MATERIALS AND METHODS: A total of 50 samples that showed ambiguous genotypes for at least two HLA loci from HLA-A, -B, -C and -DRB1 by the conventional SBT assay were evaluated using a new SBT test, the AVITA plus assay. The most likely HLA genotypes for the respective samples considering allele frequencies in Korean were concordant between the AVITA and conventional SBT assays. RESULTS: An average of 3.3 loci among the HLA-A, -B, -C and -DRB1 loci per sample gave results with two or more possible allele combinations with the conventional SBT, and 48 (96.0%) out of 50 showed reduced numbers of possible genotypes for at least one HLA locus with the AVITA. A total of 41, 43, 42, and 38 cases among the 50 samples showed ambiguous results for HLA-A, -B, -C, and -DRB1 typing by the conventional SBT, respectively. The average numbers of possible allele combinations for the respective four HLA loci were 8.2, 6.7, 5.9, and 3.2, and they were reduced to 1.5, 2.2, 4.4, and 1.8, respectively, by the AVITA. Ambiguity was resolved by the AVITA in 33 (80.5%), 31 (72.1%), 17 (40.5%) and 28 (73.7%) samples among the ambiguous cases from the conventional SBT for HLA-A, -B, -C, and -DRB1 typing, respectively. CONCLUSION: The multi-group-specific SBT method considerably reduced the number of ambiguous results, and thus may be useful for accurate HLA typing in clinical laboratories.

The correlation of vitamin D level and vitamin D-binding protein gene polymorphism in chronic obstructive pulmonary disease / 中华内科杂志

Objective To assess the correlation of serum 25-hydroxyvitamin D (25-OHD) levels with vitamin D-binding protein (the group-specific component,GC) gene polymorphism in chronic obstructive pulmonary disease (COPD).Methods In a cross-sectional case-control study,250participants,including 116 COPD patients with smoking history and 134 healthy smokers,were investigated.A questionnaire about smoking history,vitamin D intake and comorbidities was collected.General pulmonary function was done by routine.Serum 25-OHD levels were detected by ELISA.The genetic variants (rs4588and rs7041) were genotyped by real time fluorescence polymerase chain reaction (RT-PCR) with TaqMan probe technology.Results The COPD patients had lower serum vitamin D level than the smoker subjects (36.58 nmol/L vs 43.80 nmol/L,P ＜0.001).In the COPD patients,vitamin D level was 39.43 nmol/L in those with percentage of predicted forced expiratory volume in 1 second (FEV1 ％ pred) greater than or equal to 80％.In other groups with FEV1 ％ pred 50％-80％,30％-50％ and lower than 30％,vitamin D levels were 35.32 nmol/L,32.21 nmol/L,26.25 nmol/L respectively (P ＜ 0.01).Moreover,there was a significant relevance of 25-OHD levels with FEV1 ％ pred in both COPD patients and healthy smokers (r2 =1.911; P ＜0.000 1).The mean 25-OHD concentration had a negative correlation with Global Initiative for Obstructive Lung Disease (GOLD) stages.Homozygous carriers of vitamin D-binding protein gene rs7041 T allele were independently related to 25-OHD levels and susceptibility of COPD (P ＜ 0.01 ; OR =2.140,95％ CI 1.157-3.959,P =0.015 respectively).Conclusions Patients with COPD are at high risk of vitamin D deficiency and the severity of COPD is inversely correlated with vitamin D levels.Furthermore,homozygous carrier of rs7041 T allele influences 25-OHD serum levels and is related to susceptibility of COPD,which may be a potential candidate gene for screening COPD.

Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed.

Design of 16 S rRNA-based Oligonucleotide Array Using Group-specific Non-unique Probes in Large Scale Bacteria Detection / 生物化学与生物物理进展

With thousands of sequenced 16 S rRNA genes available,and advancements in oligonucleotide microarray technology,the detection of microorganisms in microbial communities consisting of hundreds of species may be possible.The existing algorithms developed for sequence-specific probe design are not suitable for applications in large-scale bacteria detection due to the lack of coverage,flexibility and efficiency.Many other strategies developed for group-specific probe design focus on how to find a unique group-specific probe that can specifically detect all target sequences of a group.Unique group-specific probe for each group can not always be found.Hence,it is necessary to design non-unique probes.Each probe can specifically detect target sequences of a different subgroup.Combination of multiple probes can achieve higher coverage.However,it is a time-consuming task to evaluate all possible combinations.A feasible algorithm using relative entropy and genetic algorithm (GA) to design group-specific non-unique probes was presented.

Molecular Characterization of Porcine Endogenous Retrovirus gag Genes from Pigs in Korea

Xenotransplantation, as a potential solution to the shortage of human organs, is associated with a number of concerns including immunologic rejection and xenogenic infection. While the pigs are considered the most suitable organ source for xenotransplantation, there is a potential public health risk due to zoonosis. Among the known porcine zoonotic microbes, Porcine Endogenous Retrovirus (PERV) is the most considerable virus. PERV belongs to the Gammaretrovirus and has been divided into three groups (A, B, and C). To characterize the gag of PERVs, we isolated the genomic DNAs from three pig breeds (Birkshire, Duroc, and Yorkshire) and two types of SPF miniature pigs. About 1.5 kb fragments covering full length of gag were amplified and cloned into T-vector. A total of 38 clones were obtained and sequenced. Nucleotide sequences were analyzed and phylogenetic trees were constructed from the nucleotide and deduced amino acids. PERV-A, -B and -C were present in the proportion of 47, 19 and 34%, respectively. Regardless of origin or subgroups, gag clones showed highly homology in nucleotide and deduced amino acid sequences. Deduced amino acids sequence alignments showed typical conserve sequences, Cys-His box and processing sites. Among analyzed clones, about 28% of isolates had the correct open reading frame. To test the functional expression of Gag protein, gag was subcloned into expression vector and confirmed its expression in HeLa cell. This research provides the fundamental information about molecular characteristics of gag gene and functional Gag protein related xenotropic PERVs.

Detection of the New O3:K6 Strain of Vibrio parahaemolyticus by Group-Specific Polymerase Chain Reaction / 대한임상병리학회지

BACKGROUND: Recently, a new serotype O3:K6 has caused a pandemic of Vibrio parahaemolyti-cus infection. The new O3:K6 serovar differs from the old O3:K6 strains at least in 7 base positions within a 1,346 bp region of the toxRS gene involved in the regulation of the virulence. We per-formed a group-specific polymerase chain reaction (GS-PCR) test for detection of the new O3:K6 strains using species-specific primers. METHODS: A total of 48 V. parahaemolyticus were isolated from clinical specimens of patients with diarrhea in different geographic areas of Seoul (Hanyang University Hospital, 20 cases), Inchon (Gachon Medical Center, 26 cases) and Gwangju (Chonnam University Hospital, 2 cases) from 1998 to 2001 in Korea. All isolates were examined for the presence of tdh/trh genes and ure-ase activity. The serovars of isolates were determined by slide agglutination tests with specific anti-sera (O3:K6/O4:K68). A GS-PCR method, detecting the new O3:K6 clone, was used in this study. RESULTS: All these isolates carried the tdh gene but not the trh gene and did not produce urease. The thirty three of the 48 samples (69%) were positive using the GS-PCR method. Thirty of thirty three cases (91%) were O3:K6 using the slide agglutination test. The three cases (9%) were O4:K68. CONCLUSIONS: We confirmed the epidemicity of the new V. parahaemolyticus O3:K6 using the GS-PCR method in Korea.

PREPARATION AND IDENTIFICATION OF ANTI-GC SERUM / 中国法医学杂志

Anti-GC serum was successfully prepared in two New Zealand rabbits immunized with GC protein which was isolated and purified from GC2-2 serum previously in our laboratory. The results of identification showed that the specificity of the home made anti- GC serum and the commercial anti- GC serum (DAKOPATTS) were identical. The titer of the home made anti-GC serum was 128. Three common phenotypes, GC1-1, GC2-1 and GC2-2could be identified by immunoelectrophoresis with the home made anti-GC serum. The concentration of GC protein as low as 3.1 ?g/ml could be detected by double immunodiffusion. In addition the anti-GC serum does not cross react with other human serum proteins.

SIMULTANEOUS PHENOTYPING OF AHSG,PI AND GC WITH IEF AND PATERNITY TEST / 中国法医学杂志

Simultaneous phenotyping of AHSG, Pi and GC by IEF is reported. The results showed that the cumulative discrimination power and the cumulative exclusion probability of paternity of this method were 0.9701 and 0.58.11 respectively. It was proved to be the most efficient method for individual identification among the simultaneous phenotypings of genetic markers.It has been applied to paternity test and the results were satisfactory.

ISOLATION AND PURIFICATION DF Gc PROTEIN FROM HUMAN PLASMA / 中国法医学杂志

Group-specific component(Gc), one of the polymorphic proteins of human plasma,has been isolated and purified from human plasma of Gc 2-2 phenotype by a procedure including DEAE-Sephadex-A50,Sephadex G100 and DEAE-Sephadex-A50 chromatography. The purified Gc identified by PAGE,SDS-PAGE and immunoelectrophoresis was homogeneous and reacted specifically with the commercial anti-Gc serum (DAKO) kit. The molecular weight of the purified Gc was about 58 kd.

Gc SUBTYPING IN THE HAN POPULATION IN CHENGDU AND PHENOTYPING OF Gc IN HUMAN BLOODSTAINS / 中国法医学杂志

0.05).The exclusion probability of Gc is 34.8% and discrimination probability of Gc is 79.44%.There are not any significant difference of the distribution of Gc subtypes between the Hun population in Chengdu and those in Hong Kong and Japanese.The difference of the distribution of Gc subtypes between the Han population in Chengdu and those in Malaysia,Indonesia,India as well as the American caucasians,Belgians,Icelander and West German are sig- nificant. The phenotyping of Gc in 11 bloodstain samples kept in room temperature for twenty weeks were carried out successfully also using PAGIF followed by immunofixation method.