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【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.
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【Objective】 To explore the polymorphism of HPA-1-6w, HPA-15 and 32bw-35bw in platelet donors in Deyang, Sichuan, and estimate whether to include the detection of 32bw-35bw in the platelet bank. 【Methods】 Polymerase chain reaction with sequenced based typing (PCR-SBT) was used to sequence the HPA-1-6w, HPA-15 and 32bw-35bw loci of 205 platelet donors in Deyang. Allele frequencies were calculated by the direct counting method. The frequencies of HPA-1-6 and 15 alleles in northern and southern Chinese, Japanese and Australian population were compared, and those HPA loci and HPA-32bw-35bw were searched in the Chinese Millionome Database (CMDB) and genomAD to obtain the polymorphism data. Then the Chi-square test was performed with the data of this study through GraphPad Prism 9 software. 【Results】 The allele frequencies of HPA-1b, 2b, 3b, 5b, 6bw and HPA-15b were 0.005(2/410), 0.037(15/410), 0.471(193/410), 0.020(8/410), 0.010(4/410) and 0.461(189/410), respectively, b allele of HPA-32bw-35bw and HPA-4 was not detected. Statistical significance was observed between the HPA-1b allele frequency of this study and northern Chinese, Australian population and genomAD global population sample (P< 0.05, 0.005 vs 0.014 vs 0.145 vs 0.122). The frequency of HPA-2b alleles in this study, Japanese population and genomAD global population samples was 0.037 vs 0.120 vs 0.100, with statistical difference(P<0.05). Comparison of HPA-5b and HPA-6bw allele frequencies with those of genomAD global population showed a statistical difference (P<0.05, 0.020 vs 0.089 and 0.010 vs 0.000 008, respectively). 【Conclusion】 The polymorphisms of HPA-1-6w and HPA-15 of donors in Deyang has characteristics of the southern Chinese. The frequencies of HPA-32bw-35bw were extremely low, which could be excluded from the platelet bank in Deyang.
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The objective of this study was to evaluate the genetic diversity of ball-DRB3 gene in the Colombian creole Hartón del Valle cattle. A total of 93 Hartón del Valle (HV), 30 Lucerne (LUC), and 30 Holstein (HOL) animals were evaluated for DRB3 gene polymorphism using the PCR-SBT method. The number of alleles found were 27, 17 and 14 for HV, LUC, HOL, respectively. The most frequent alleles were DRB3*1101 (14.5%) in HV; LUC DRB3 * 0701 in LUC; and *1107, *1501 and *2301 in HOL (11.7%). Expected heterozygosity values were higher than observed, which resulted in a low fixing index, positive F IS (significant only for HV), and no significant deviations from Hardy- Weinberg equilibrium for LUC. The molecular analysis of variance reflected little variation between breeds (3%) and a moderate genetic differentiation (F ST = 0.056 p<0.01). Regarding sequence, LUC was the most diverse breed, no neutral selection is occurring, and none of the breeds is in selection equilibrium. Comparison with reports on other breeds showed high genetic diversity in Colombian breeds. We conclude that HV has high genetic diversity in BoLA-DRB3 locus. The cause of this polymorphism may be due in part to the origin of HV. This high variation can be maintained by selection equilibrium.
El objetivo de esta investigación fue evaluar la diversidad genética del gen BoLA-DRB3 en el ganado criollo Colombiano Hartón del Valle. En 93 animales Hartón del Valle (HV), 30 Lucerna (LUC) y 30 Holstein (HOL) se evaluó el polimorfismo del gen DRB3 usando la metodología PCR-SBT. Se encontraron 27 alelos en la raza HV, 17 en LUC y 14 en HOL. El alelo más frecuente en HV fue el DRB3*1101 (14,5%), en la raza LUC los alelos DRB3*0701, *1107, *1501 y *2301 al igual que el alelo DRB3*0701 en la raza HOL tuvieron la frecuencia más alta (11,7%). Los valores de heterocigocidad esperada fueron más altos que la observada, lo que se tradujo en un valor bajo del índice de fijación, valores de F IS positivos solo significativo en el HV y desviaciones del equilibrio de Hardy-Weinberg no significativos en LUC. El análisis de varianza molecular reflejó poca variación entre razas (3%) y una diferenciación genética moderada (F ST= 0,056 p<0,01). A nivel de secuencia se resalta que, la raza más diversa es LUC, que no se está presentando selección neutral y que ninguna de las razas se encuentra en equilibrio de selección balanceadora. La comparación con otras razas reportadas, mostró alta diversidad genética en las razas colombianas. Se concluye que la raza HV tiene alta diversidad genética en el locus BoLA-DRB3, que el origen del polimorfismo puede deberse en parte al origen de la raza y que esta alta variación puede ser mantenida mediante equilibrio de selección.
O objetivo desta pesquisa foi avaliar a diversidade genética do gene BoLA-DRB3 no gado crioulo colombiano Hartón del Valle. O estudo se fez com 93 animais Hartón del Valle (HV), 30 Lucerna (LUC) e 30 Holandês (HOL). Avaliou-se o polimorfismo do gene DRB3 utilizando a metodologia PCR-SBT. Encontraram-se 27 alelos na raça HV, 17 em LUC e 14 em HOL. O alelo com maior frequência em HV foi o DRB3*1101 (14,5%), na raça LUC os alelos DRB3*0701, *1107, *1501 e *2301 ao igual que o alelo DRB3*0701 na raça HOL tiveram a maior frequência (11,7%). Os valores de heterocigocidade esperada foram maiores que a observada, o que se traduz em um valor baixo do índice de fixação, valores de F IS positivos só foram significativos no HV e desvio do equilíbrio de Hardy- Weinberg foram não significativos em LUC. O Analice de variância molecular reflexou pouca variação entre raças (3%) e uma diferenciação genética moderada (F ST= 0,056 p<0,01). No nível de sequência destacasse que, a raça mais diversa é LUC, que não está apresentando seleção neutral e que nenhuma das raças encontra-se em equilíbrio de seleção balanceadora. Se comparar com outras raças reportadas, as raças colombianas exibiram uma alta diversidade genética. Conclui-se que a raça HV tem alta diversidade genética no locus BoLA DRB3, que a origem do polimorfismo pode se dever em parte àorigem da raça e que essa alta variação pode ser mantida mediante o equilíbrio de seleção.
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Objective To compare PCR-SBT to IMS-ELISA in the HLA-B27 detection in the ankylosing spondylitis (AS)pa-tients.Methods Simultaneously,PCR-SBT and IMS-ELISA were used to detect the HLA-B27 expression in peripheral blood samples which were suspected patients with AS from 120 cases.Chisquare test of paired design and the area under curve of receiver operating characteristics of SPSS17.0 software were used to evaluate the value of PCR-SBT and IMS-ELISA in HLA-B27 detection of AS patients.Results Among 120 cases of suspected patients with AS,the positive rates of HLA-B27 detected by PCR-SBT and IMS-ELISA were 45.83%(55/120)and 37.50% (45/120),respectively.There was statistical difference between the two methods in the HLA-B27 detection (χ2 =59.455,P =0.000).The sensibility and spe-cificity of PCR-SBT were 96.36% and 96.92%,respectively.While the sensibility and the specificity of IMS-ELISA were 69.09% and 89.23%,respectively.Area under the curve of two methods were 0.966 and 0.792,respectively.Conclusion In comparison with IMS-ELISA,the sensibility and the specificity of PCR-SBT in HLA-B27 detection were higher in AS diag-nosis,that is to say,PCR-SBT is better in HLA-B27 detection and AS diagnosis.
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Objective To study on the characterization of MICA/B genetic polymorphism in a northern Chinese Hunan Han population.Methods Ninty-five unrelated individuals were involved in this study and MICA/B genotypes were determined by two methods:PCR-sequence-specific primers (PCR-SSP) and PCR-sequence-based typing (PCR-SBT).Results In northern Hunan Han population,eleven MICA alleles were found,among which MICA * 010 (28.95％),MICA * 008 ∶ 01 (20.53％) and MICA * 002 ∶ 01 (15.79％) were the common alleles.Five MICA-STR(short tandem repeat) alleles were found,among which MICA * A5 (37.89％) and MICA * A5.1 (21.05％) predominated.In this population,ten MICB alleles were found.The common alleles were MICB * 005 ∶ 02/* 010 (58.42％),MICB * 002 ∶ 01 (10.00％),and MICB * 008 (7.89％).Two kinds of MICA-MICB haplotypes were MICA * 004-MICB * 004 ∶ 01 and MICA * 010-MICB * 005 ∶ 02/010 in significant linkage disequilibrium.This study also showed MICA/B gene with high polymorphism in different populations.Conclusion MICA/B alleles distribution in northern Hunan Han population with its unique characteristics.
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Objective:To study the HLA-DPB1 allele polymorphism in Ewenki from Inner Mongolian.Methods:HLA-DPB1 allele polymorphism in normal Ewenki were determined by PCR with sequencing-based-typing(SBT).Results:20 HLA-DPB1 alleles were observed and compared with other ethnic groups,the allele frequency of HLA-DPB1*02012(24.4%) and DPB1*0402(22.6%),DPB1*0401(20.2%),DPB1*0501(10.7%) are highest,while others are lower.Conclusion:The distributions of HLA-DPB1 alleles frequencies in normal Ewenki from Inner Mongolia has a unique style.It is most important to further study anthropology and related to illness in Ewenki nationality.