多重PCR检测CSF1PO,TPOX和TH01基因座在中国汉族中的多态性

短串联重复序列（ＳＴＲ）是由几个碱基对作为核心单位串联重复形成的一类ＤＮＡ序列,应用３个ＳＴＲ基因座在同一反应体系中进行互不干扰的多重ＰＣＲ,采用高分辨力的聚丙烯酰胺凝胶电泳分离、银染法显影技术,对我国汉族的ＣＳＦ１ＰＯ,ＴＰＯＸ和ＴＨ０１等３个基因座等位基因的基因频率调查,ＣＳＦ１ＲＯ基因座,观察到９个等位基因,２２个基因型;ＴＰＯＸ基因座,观察到６个等位基因,１４个基因型：ＴＨ０１基因座,观察到６个等位基因,１９个基因型。获得了满意的结果,显示了广阔的应用前景。     

Frequency of HLA-DQA1 alleles in the Japanese population.

One of the HLA class II genes, HLA-DQA1, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQA1 gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQA1 genotype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. The HLA-DQA1 genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.     

A population genetic survey of the haptoglobin polymorphism in Melanesians by DNA analysis.

We have determined the haptoglobin (Hp) genotypes of 831 Melanesians from Vanuatu, Papua New Guinea, and New Caledonia by Southern blot analysis of DNA extracted from umbilical cord and peripheral blood samples. There was complete agreement between these genotypes and the protein phenotype in cases where both were determined, and genotyping was possible in cases where no serum haptoglobins were measurable. Subtyping of Hp1 alleles using restriction enzymes showed that Melanesians, like Mongoloids and Australian Aboriginals, have only the Hp1S allele. Three cases of Hp Johnson were found in Vanuatu, and further restriction mapping supported a partial gene triplication model for the structure of this variant. We also report a new common BclI restriction enzyme polymorphism upstream of the Hp1 gene. The advantages of using DNA for haptoglobin typing are discussed.     

全自动血型仪的应用评价

目的了解全自动血型鉴定系统用于检测ABO血型和Rh(D)血型的效果。方法采用深圳爱康AK03A型数字血型仪对北海市中心血站2010年5月—2011年12月无偿献血者20702份样本进行ABO血型和Rh(D)血型鉴定,并与手工试管法进行对比验证。结果发现正反定型不一致的有75例,ABO初筛血型错误的36例,Rh(D)阴性的53例。结论全自动血型鉴定系统检测血型快捷、方便、高效,能降低检测人员的工作强度,但判读结果时需要与手工试管法结合进行验证,才能得出正确的结果。     

辽宁新品系绒山羊微卫星标记与经济性状相关关系的研究

利用微卫星技术对我国特有遗传资源150头辽宁新品系绒山羊的11个微卫星位点进行了研究,对其进行群体遗传学特性分析,并利用SAS程序下的GLM分析了体重、产绒量和绒细度3个经济性状与标记基因型的关系,结果表明：红LSCV13位点,AA和BC型为体重性状的优势基因型,AB型为绒产量性状的优势基因型。在CSSM11付点,AD和BE犁为绒产量性状的优势基凶型,AA型为绒细度性状的优势基因型。在IDVGA64位点DE为体重性状的优势基因型,BD和CC型为绒产量性状的优势基凼犁,BC和DE型为绒细度性状的优势基因型。在BMS2782何点,BB型为体重性状的优势基凼型,BC和DE型为绒产量性状的优势基凶型,CD型为绒细度性状的优势基因型。     

Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material

Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.     

Immunogenetic studies of rhesus monkeys. 8. A new reagent of the G blood group system and an example of the founder principle.

A new reagent called G-3' has been discovered which detects the product of the G3 allele, regardless of the other G allele in the genotype. With this new reagent we can delineate nine of 10 genotypes of the G blood group system. The explicit detection of G3 revealed an example of the founder principle in that the frequency of this allele between the feral and laboratory-reared monkeys was significantly different.     

Multiple-locus variable-number tandem repeat analysis of Dutch Bordetella pertussis strains reveals rapid genetic changes with clonal expansion during the late 1990s

Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected.     

用全基因组测序评价VNTR分型在泛耐药结核分枝杆菌传播中的应用

近年来,随着技术的进步、测序成本的降低,全基因组测序(whole-genome sequencing,WGS)技术开始应用于结核分枝杆菌传播的研究。本研究采用基于单核苷酸多态性(single nucleotide polymorphism,SNP)的分型方法评价多位点数目可变串联重复序列(variable-number tandem-repeat,VNTR)分型判断泛耐药结核分枝杆菌(extensively drug-resistant Mycobacterium tuberculosis,XDR-TB)传播及成簇特征的准确性。对2003-2009年重庆市肺科医院诊断的55例XDR-TB菌株分别进行9＋3个位点的VNTR分型和WGS分析,分别构建系统进化树,并比较两种方法判断成簇的一致性与差异。VNTR分型方法鉴定出45个基因型,其中39株为单一基因型,16株(29.1%)分别归入6个基因簇。规定菌株间差异不超过12个SNP即为成簇,WGS将20株(36.4%)分为5个簇。两种方法判断成簇的一致性为 63.6%。与WGS相比,VNTR分型的灵敏度为 40.0%,特异度为 77.1%。相比于WGS,VNTR分型特异度较高,但仅凭其结果可能会错误估计XDR-TB的传播性。因此,规定菌株间相差不超过12个SNP即有近期传播关系是否适用于XDR-TB,有待进一步研究。     

Evaluation of DRB1 high resolution typing by a new SSO-based Luminex method

HLA testing is an essential part of the process to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow, peripheral blood or cord blood and the DNA typing in high resolution is now recommended as the Scientific Societies also describe in their standards. Recently the new PCR-Luminex HLA typing method, based on the reverse sequence specific oligonucleotide probes coupled with a microsphere beads in an array platform, has been well established. We report the data from 146 samples previously typed to a four digits level and used to evaluate the accuracy, sensitivity and performance of the new high definition DRB1 by PCR-Luminex kit. One hundred and forty-six samples from unrelated healthy donors, haematological patients or external proficiency tests were used in this study. The Luminex high definition DRB1 typing represents a versatile method and may be easily introduced in the routine, particularly when the technical team has already acquired experience on the technique. Only few HLA allelic combinations need an additional typing by PCR–SSP or SBT to solve the ambiguous results thus reducing the time necessary to produce a final report.     

Assessment of DNA fingerprinting for rapid identification of outbreaks of systemic candidiasis.

DNA fingerprinting was assessed as an improved typing system for Candida albicans aimed at speeding the implementation of cross infection control measures in outbreaks of systemic candidiasis. The study was carried out with 45 previously characterised isolates from five different outbreaks and with 96 unrelated isolates from a mixed control population. Sixteen different genotypes were produced. Results were obtainable within days, reproducibility was high, and there was good discrimination among different outbreaks. Compared with existing typing systems DNA fingerprinting provides a robust system that may be used rapidly to identify outbreaks of nosocomial candidiasis in laboratories with no specialist skill in typing C albicans.     

Immunogenetic studies of rhesus monkeys. 8. A new reagent of the G blood group system and an example of the founder principle

A new reagent called G-3' has been discovered which detects the product of the G ³allele, regardless of the other G allele in the genotype. With this new reagent we can delineate nine of 10 genotypes of the G blood group system. The explicit detection of G ³revealed an example of the founder principle in that the frequency of this allele between the feral and laboratory-reared monkeys was significantly different.     

The multi-locus VNTR-analysis in studies of the population structure of Yersinia pestis in natural foci

A collection of Yersinia pestis strains was investigated by the multi-locus VNTR analysis. All 9 used locuses were diverse, although they differed between themselves by the quantity of genotypes displaying 4 to 13 variations in the sample. The diversity index (DI) ranged from 0.18 (ms21) to 0.86 (ms46); 8 locuses had DI > 0.5. The statistical processing showed 55 individual genotypes in a group of 81 examined strains, which denoted a high discriminative potentiality of the typing system (DP = 0.98). On the basis of the cluster analysis, the genotypes were shared between 11 main groups. The strains belonging to one genotype group were found to originate, as a rule, from one natural focus. The suggested scheme of typing and of creating the databases of genotypes of plaque agent can be used to establish, with a high probability degree, the source of strains.     

Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay.

The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.     

Three new blood groups of rhesus monkeys

Three new blood group systems, called “T,” “U,” and “V,” have been identified in the rhesus monkey (Macaca mulatta). Each system consists of a single antigenic factor (blood group) detected by a monospecific alloimmune reagent that agglutinates erythrocytes. The antisera that detect these blood groups were obtained following a series of alloimmunizations and absorption fractionizations of the resulting antisera to produce operationally monospecific typing reagents. Analyses of family data indicated that each blood group was controlled by an autosomal dominant gene and that each system was independent of previously defined systems. With the addition of these new blood groups, we can identify 16 different blood group systems and well over one hundred million possible phenotypes in this species.     

Mapping of the bovine blood group systems J,N', R', and Z show evidence for oligo-genetic inheritance

Genes determining the bovine erythrocyte antigens were mapped by linkage analysis. In total 9591 genotypes of 20 grandsire families with 1074 sires from a grand-daughter design were elucidated for the genes determining the erythrocyte antigens EAA, EAB, EAC, EAF, EAJ, EAL, EAM, EAN', EAR', EAS, EAT', and EAZ according to standard paternity testing procedures in the blood typing laboratories. Linkage analyses were performed with 248 microsatellite markers, eight SSCP markers and four polymorphic proteins and enzymes covering the 29 autosomes and the pseudoautosomal region of the sex chromosomes. The number of informative meioses for the blood group systems ranged from 76 to 947. Blood group systems EAM and EAT' were non-informative. Most of the erythrocyte antigen loci showed significant linkage to a single chromosome and were mapped unequivocally. The genes determining erythrocyte antigen EAA, EAB, EAC, EAL, and EAS were mapped to chromosomes 15, 12, 18, 3, and 21, respectively. Lod-score values ranged from 11.43 to 107.83. Moreover, the EAF system could be mapped to chromosome 17. However, the EAN' system previously known as part of the EAF system could be mapped to chromosome 5. In addition, the blood group systems EAJ, the new EAN', EAR', and EAZ, showed significant linkage to microsatellite markers on various chromosomes and also to other blood groups. The appearance of a single blood group system might be therefore either dependent on the existence of other blood group systems or because of an interaction between different loci on various chromosomes as is known in humans and in pigs.     

Associations between marker genotypes, halothane reaction, creatine kinase activity and meat quality characters in a sample of German Landrace pigs

Routine blood typing of German Landrace pedigree populations and an earlier study revealed very low frequencies of the favourable alleles at the marker loci Phi, Pgd and H. The hypothesis was that in this population the whole linkage group of favourable alleles at the halothane and neighbouring marker loci may have been lost as a consequence of intense selection for leanness and type. The present study of 1050 German Landrace pigs at the Relliehausen experimental station, where some effort has been made to maintain a higher frequency of the favourable alleles PhiA (0.48), H- (0.43) and PgdA (0.70) gave quite different results. The frequency of halothane-positive pigs found by using a severe test was only 30%. Only 5.4%, 8.8%, 13.4% and 13.9% of animals with PhiA/A, H-/-, PgdA/A and PhiA/B genotypes respecitively were halothane-positive. Forty to sixty per cent of pigs with these marker genotypes could therefore be expected to be homozygous halothane-negative (N/N) animals. Creatine kinase activity and three selected meat quality characters showed highly significant differences between the A/A and the B/B genotypes for the marker loci Phi and Pgd, with the heterozygotes being intermediate. These differences are greater than those observed between halothane-negative and halothane-positive phenotypes. The only other consistently superior marker genotype in this population was the H blood group genotype H-/-. In contrast to findings from Sweden and Switzerland, the postalbumin locus Po2 and the suppressor locus S for the A-O blood groups did not exhibit useful marker qualities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of an allele-specific polymerase chain reaction to the direct determination of ABO blood group genotypes.

The allele-specific polymerase chain reaction (ASPCR) procedure has proven a powerful tool for the detection and analysis of known genetic polymorphisms. Here, we present a novel application of the ASPCR technique to determine the ABO genotypes of individuals without the need of family analysis. The method introduces a new strategy for primer design that permits the identification of the different ABO genotypes according to the molecular size of allele-specific amplification products. Four primer sets, each specific for a different set of ABO alleles, are mixed in one reaction and the amplification products are resolved on a polyacrylamide gel. Forty-one individuals belonging to various families, whose ABO phenotypes were previously determined serologically, were typed with this new variation of the ASPCR technique. A 100% correlation between the serology and the ASPCR data was found. The Mendelian segregation of ABO alleles was also demonstrated in families. The method is rapid, simple, reproducible, and specific. Potential applications include gene mapping, genetic disease diagnosis, HLA typing, paternity testing, and forensic science.     

Detection of Genetic Diversity in Campylobacter jejuni Isolated from a Commercial Turkey Flock Using flaA Typing,MLST Analysis and Microarray Assay

Campylobacter is genetically highly diverse and undergoes frequent intraspecific recombination. Turkeys have been identified as an important reservoir for Campylobacter jejuni which is of public health significance. The assessment of the genetic diversity among Campylobacter population is critical for our understanding of the epidemiology of this bacterium. The genetic profiles were different according to the molecular typing methods used. The performance of established flaA genotyping, multilocus sequencing typing (MLST) and DNA microarray assay based on the ArrayTube™ technology was evaluated using 14 Campylobacter jejuni isolated from a commercial turkey flock. The flaA typing was performed using PCR-RFLP with restriction enzymes Sau3AI, AluI, a ‘composite’ flaA analysis of AluI and Sau3AI and DdeI. The 14 isolates were differentiated into 3, 5, 7 and 9 genotypes, respectively. Entire flaA gene and short variable region (SVR) sequences were analysed. Sequencing of the entire flaA provided 11 different genotypes. flaA-SVR sequence analysis detected 8 flaA alleles and 4 flaA peptides. One new flaA allele type (528) was identified. MLST analysis represented 10 different sequence types (STs) and 5 clonal complexes (CCs). The microarray assay recognised 14 different genotypes. The discriminatory indices were 0.560, 0.802, 0.857, and 0.912 for flaA-RFLP depending on the used enzymes, 0.890 for flaA-SVR, 0.967 for entire flaA sequencing, 0.945 for MLST and 1.00 for the DNA microarray assay. The flaA gene was genetically stable over 20 passages on blood agar. In conclusion, the different typing tools demonstrated a high level of genetic heterogeneity of Campylobacter jejuni in a turkey flock, indicating that a single flock can be infected by multiple genotypes within one rearing cycle. DNA microarray-based assays had the highest discriminatory power when compared with other genotyping tools.     

Identification of five major and two minor genotypes of varicella-zoster virus strains: a practical two-amplicon approach used to genotype clinical isolates in Australia and New Zealand

Whole genome phylogenetic analysis in this study resolved a total of five major genotypes among the 22 varicella-zoster virus (VZV) strains or isolates for which complete genomic sequences are available. Consistent with earlier publications we have designated these genotypes European 1 (E1), European 2 (E2), Japanese (J), mosaic 1 (M1), and mosaic 2 (M2). Single nucleotide polymorphism (SNP) analysis performed in a whole-genome alignment revealed that VZV isolates of all five genotypes can be accurately genotyped using SNPs from two amplicons: open reading frame 22 (ORF22) and either ORF21 or ORF50. This modified approach identifies all of the genotypes observed using any of the published genotyping protocols. Of 165 clinical varicella and zoster isolates from Australia and New Zealand typed using this approach, 67 of 127 eastern Australian isolates were E1, 30 were E2, 16 were J, 10 were M1, and 4 were M2; 25 of 38 New Zealand isolates were E1, 8 were E2, and 5 were M1. VZV strain diversity in eastern Australia is thus broader than has been described for any other region, including Europe, Africa, and North America. J strains were far more prevalent than previously observed in countries other than Japan. Two-amplicon typing was in complete accord with genotypes derived using SNP in multiple ORFs (ORFs 1, 21, 22, 38, 50, 54, and 62). Two additional minor genotypes, M3 and M4, could also be resolved using two-amplicon typing.