Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【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.

Molecular basis of Del phenotype in RhD negative population of Zhongshan / 中国输血杂志

【Objective】 To conduct serological and molecular study of Del type in RhD-negative donor population in Zhongshan area, so as to improve the diagnosis of Del type. 【Methods】 A total of 102 initially RhD-negative samples, collected from December 2017 to February 2019, were classified by RHCE and PCR-SSP genotyping. And 95 cases of truly negative RhD were confirmed by IAT, 28 cases of Del type were identified by absorption and elution test. The phenotype and genotyping characteristics of Del type in Zhongshan area were summarized based on domestic data of relative literature. 【Results】 Among 102 initially RhD-negative samples by serological test, 95 were truely RhD-negative, 28 were DELRHD 1227A without any other Del allele. Among them, RHCE antigen type were Ccee in 20(71.4%) cases, CCee in 8(28.6%), with no difference in comparison with other regions in China. The frequency of Del in RhD-negative blood donors was 29.5% (28/95), with difference between Shanghai, Taiwan, and Fuzhou, but no difference between Nanchang, Zhejiang, and Wuhan. 【Conclusion】 The study showed that the Del phenotype was closely related to Ce haplotype, and has no difference with other regions in China. The frequency of Del type in RhD negative donors was 29.5%, with regional differences. RHD1227A was the main allele of Del.

Genetic background of RhD negative blood donors / 中国输血杂志

【Objective】 To analyze the genetic background of RhD-negative blood donors by detecting RHD and RHCE genes of those donors. 【Methods】 From March 2021 to May 2022, the blood samples of RhD-negative blood donors, who had been screened out by RhD primary screening and confirmatory experiments in the Yaan Blood Center, were firstly identified whether the RHD allele was completely deleted, then whether there were deletions in 10 exons of non-RHD allele complete deletion samples, finally, the remaining samples without RHD alleles and exon deletions were further analyzed by DNA sequencing. RHCE gene was detected by SSP-PCR method. 【Results】 Among the RHD gene test results of 104 RhD-negative samples, 65 cases were completely deleted (d/d), 33 were RHD partially deleted (one allele deletion), and 6 were without RHD gene deletion. The RHD alleles of 33 samples with partial deletion were detected by 10 exons, 13 had partial exon deletion, with genotype as RHD*D-CE(3-9)-D/d and phenotype as RhD negativity, and the remaining 20 samples had no exon deletion. The exon sequencing results of the non-deletion samples showed RHD*1227A/RHD*1227A in 6 samples, RHD*1227A/d in 19, RHD*3A/d in 1; both of the last two were considered Del by ISBT. The RHCE gene test results showed that all cc genotype blood donors were RhD true negative, while Del blood donors had no cc genotype. 【Conclusion】 Through the genetic background study of RhD negative blood donors, it is found that there is a high proportion of Del with weak expression of RhD antigen, whether this blood type affects clinical blood safety needs further researches.

Association of HLA-C 06 with Psoriasis in a Tertiary Care Hospital.

Prevalence of psoriasis is 1-3% in India. HLA-C*06 has been shown to be strongly associated with psoriasis in different ethnic populations. This study was carried out to determine the association of HLA-C in psoriasis patients in a south Indian ethnic population.METHODSA total of 200 samples were included in the study. In all, 100 psoriasis patients and 100 healthy controls were studied. HLA-C typing was done by PCR-SSP method. Results were analysed statistically using open epi software (2 X 2 table). The Odds ratio (OR), p (probability) value, and 95% confidence interval were the statistical tests applied and analysed.RESULTSA total of 14 different HLA-C alleles were identified in both 100 cases and 100 controls. Among the 14 different HLA-C alleles, the alleles which were found to be strongly associated with psoriasis which were statistically significant were both HLA-C*06 and HLA-C*07. HLA-C*06 was found to be present in 52% of the patients and HLA-C*07 was found to be present in 33% of the patients. HLA-C*06 was found to be strongly associated with the disease in 52% of the patients.CONCLUSIONSThis study confirms HLA-C*06 association with psoriasis which is in concordance with other previous studies.

Identification for rare B(A) blood group / 临床检验杂志

Objective To investigate the serological and molecular identification of 2 rare B( A) blood groups. Methods The ABO blood groups of 2 samples from blood donors were detected by routine serological method. The genotype features was identified by PCR-sequence specific primer (PCR-SSP) and direct sequence analysis. Results The serological results for the 2 blood donors showed the characteristics of B(A) phenotype. The sample 1 was genotyped as BO2 subtype by PCR-SSP and direct sequencing showed B alleles in exon 7, presented nt640 A＞G mutation which was confirmed to be B(A)04/O02 genotype.The sample 2 was genotyped as BO1 sub-type by PCR-SSP and direct sequencing showed B alleles presented nt700 C＞G mutation in exon 7 which was confirmed to be B(A)02/O01 genotype. Conclusion The phenotype of the two samples should be B ( A ) and the genotypes should be rare B(A)04/O02 and B(A)02/O01.

Analysis of serological and genetic test results of rare Ax14 subtypes in a young child / 中国输血杂志

Objectives To identify ABO blood type from a child having discrepant results in forward and reverse ABO blood grouping by serological identification and genetic testing.Methods After routine serological detection with ABO blood group,the ABO gene and ABO blood group-A subgroup genotype were tested by PCR-SSP method.Results The Serological results showed that the specimen was positive type A (anti-A:1 +w),the reverse type is O type (Ac:2 +,Bc:4 +);PCRSSP A-subtype typing showed that the genotype of the child was Ax14/O2.Conclusion Difficult blood type identification sometimes need to combine serology and molecular biology methods to confirm.In this case,the phenotype of the child was Ax,and the genotype was Ax14/O2.

Study on correlation between ankylosing spondylitis and HLA -B27 subgenotype / 国际检验医学杂志

Objective To explore the correlation between different HLA‐B27 subgenotype and ankylosing spondylitis (AS ) . Methods The whole venous blood was collected from the outpatients and inpatients of the orthopedics ,acupuncture and rheuma‐tism departments and HLA‐B27 was qualitatively detected by using the gene analysis method .Among them ,380 cases of AS were HLA‐B27 positive ,and 50 cases of HLA‐B27 positive were selected as the healthy control group .Then the HLA‐B27 subgenotypes were detected by using the sequence specific primers PCR (PCR‐SSP) technology .Results Among 380 cases of AS ,217 cases (57 .1% ) of HLA‐AS B2704 subgenotype ,143 cases (37 .6% ) of HLA‐B2705 subgenotype ,11 cases (2 .9% ) of HLA‐B2707 sub‐genotype and 9 cases (2 .4% ) of HLA‐B2711 subgenotype were detected out ;among 50 cases of HLA‐B27 positive in the healthy control group ,23 cases (46 .0% ) of HLA‐B2706 subgenotype ,21 cases (42 .0% ) of HLA‐B2709 subgenotype ,4 cases (8 .0% ) of HLA B2704 subgenotype and 2 cases (4 .0% ) of HLA‐B2705 subgenotype were detected out ,the differences between the two groups were statistically significant (P=0 .002) .Conclusion The subgenotypes of HLA‐B27 among the AS patients in Baoji area are dominated by the genotype B2704 and B2705 ,which is strongly correlated with the occurrence of AS among Han population in Baoji area ;B2706 and B2709 are the protective subgenotypes in this area .

The characteristic of gene polymorphism of Kell blood group in Zhuang population from Guangxi region in China / 重庆医学

Objective To investigate gene polymorphism of Kell blood group in different Zhuang population from Guangxi region .Methods The genotypes of Kell blood group of 1025 non‐related individuals in different areas of Guangxi Zhuang popula‐tion were analyzed by PCR‐SSP .Results The Kell antigen in all individuals was homozygous ,the gene frequency of K and Jsa was 0 ,while that of k and Jsb was 1 .000 .Conclusion The distribution characteristic of Kell blood group in Guangxi Zhuang population was monomorphism ,which was similar to other Chinese population reported by literatures .

MÉTODOS PARA LA IDENTIFICACIÓN DEL ANTÍGENO DE HISTOCOMPATIBILIDAD HLA-B27: COMPARACIÓN DE CUATRO PROTOCOLOS TÉCNICOS / METHODS FOR IDENTIFICATION OF ANTIGEN HLA-B27 HISTOCOMPATIBILITY: FOUR TECHNICAL COMPARISONS OF PROTOCOLS / MÉTODOS PARA A IDENTIFICAÇÃO DO ANTIGÉNIO DE HISTOCOMPATIBILIDADE HLA-B27: CAMPARAÇAO DE QUATRO PROTOCOLOS TÉCNICOS

Introducción: La asociación del HLA-B27 y las Espondiloartritis, ha hecho evidente que la tipificación del HLA-B27 sea considerada como un apoyo en el diagnóstico de estas enfermedades. Los métodos más empleados para la determinación del antígeno HLA-B27 en los laboratorios clínicos y en investigación son: la microlinfocitotoxicidad (MCTX), la citometría de flujo digital (CMFd), la citometría de flujo análoga (CMFa) y la reacción en cadena de la polimerasa con primers de secuencia específicos (PCR-SSP). Objetivo: Comparar MCTX con la CMFd, la CMFa con la CMFd, y la técnica de CMFd frente a PCR-SSP. Métodos: Se analizaron 4109 solicitudes de HLA-B27 en población con manifestaciones sugestivas de EAS remitidas entre 2009 y 2012 al Hospital Militar Central y al Instituto de Referencia Andino. Se evaluaron las frecuencias obtenidas por Chi cuadrado (X2); para estimar la concordancia metodológica se utilizó el Coeficiente de Correlación Intraclase (CCI). Los análisis se realizaron con el paquete estadístico SPSS V18. Resultados: Al evaluar 467 datos por la técnica de CMFa frente a PCR-SSP, la CMFa mostró 239 resultados entre positivos y en rango indeterminado, de los cuales, luego de ser confirmados PCRSSP, solo 213 demostraron la expresión de HLA-B27 (p<0.05). Se obtuvieron 208 resultados realizados por CMFd y PCR-SSP simultáneamente, observándose una alta correspondencia entre estas técnicas (p<0.05). Para evaluar la concordancia entre la MCTX y CMFd se analizaron 34 datos, revelando un 100% de correspondencia entre esta dos metodologías (CCI=1,p<0.05). Conclusión: La citometría de flujo digital es un método rápido que presenta un desempeño altamente confiable para la identificación de HLA-B27, resultados que se recomiendan confirmar por PCR SSP.

Introduction: The association between HLA-B27 and spondyloarthritis has made clear the fact that identification of HLA-B27 antigen is considered as a support in the diagnosis of these diseases. The most commonly used methods for determination of the HLA-B27 antigen in clinical laboratories as well as in their research, are microlymphocytotoxicity (MCTX), digital flow cytometry (CMFd), analogous flow cytometry (CMFa) and the Single Specific Primer-Polymerase Chain Reaction (PCRSSP). Objective: compare the CMFd against MCTX, CMFa against CMFd and CMFd against PCR-SSP. Methods: 4109 requests for HLA-B27 were analyzed with manifestations suggestive of SpA submitted between 2009 and 2012 at Hospital Militar Central and Instituto de Referencia Andino. To analyze the frequencies Chi square (X2) was evaluated; to estimate the methodological concordance the intraclass correlation coefficient (ICC) was used. All proposed analyzes were performed with SPSS V18. Results: 467 data obtained by CMFa versus PCR-SSP evaluated the CMFA showed 239 results between positive and indeterminate range, which, after being confirmed by molecular biology (PCRSSP), only 213 showed the expression of HLA-B27 (p <0.05). PCR-SSP and CMFd performed 208 results simultaneously, showing a high correlation between these techniques (p <0.05). To evaluate the correlation between CMFd and MCTX, 34 data were analyzed, revealing a 100% match on the positive results from these two methodologies (ICC = 1, p <0.05). Conclusion: The digital flow cytometry is a rapid method that presents a highly reliable for the initial identification of HLA-B27; results confirmed by PCR SSP recommend performance.

Introdução: a associação do HLA-B27 e as Espondilartrite, evidenciou que a tipificação do HLAB27 seja considerada como um suporte no diagnóstico dessas doenças. Os métodos mais usados para a determinação do antígeno HLA-B27 nos laboratórios clínicos e no investigação são: a microlinphocitotoxicity (MCTX), a citometria de fluxo digital (CMFd), a citometria de fluxo análoga (CMFa) e a reação em cadeia de a polimerasa com primers de sequência específicos (PCR-SSP). Objetivo: Comparar MCTX com a CMFd, a CMFa com a CMFd, e a técnica de CMFd com PCRSSP. Métodos: 4109 solicitudes de HLA-B27 em população com manifestações sugestivas de EAS remitidas entre 2009 e 2012 ao Hospital Militar e ao Instituto de Referencia Andino, foram analisadas. Avaliaram-se as frequências obtidas por Chi quadrado (X2); para estimar a concordância metodológica foi utilizado o Coeficiente de Correlação Intraclasse (CCI). Os análises estão feitos com o paquete estadístico SPSS V18. Resultados: A CMFa mostrou 239 resultados entre positivos e em rango indeterminado quando avaliou-se 467 dados com a técnica de CMFa com PCR-SSP. Só 213 deles demostraram a expressão de HLA-27 (p<0.05), depois de ser confirmados PCR-SSP. Foram obtidos 208 resultados por CMFd y PCR-SSP em simultâneo, com uma alta correspondência entre estas técnicas (p<0.05). Para avaliar a concordância entre MCTX y CMFd analisaram-se 34 dados, revelando um 100% de correspondência entre as duas metodologias (CCI=, p<0.05). Conclusão: A citometria de fluxo é um método rápido que tem um desempeno muito confiável para a identificação de HLA-B27, resultados recomendados para confirmar por PCR SSP.

Duffy Blood Group Genotyping in Thai Blood Donors

BACKGROUND: Duffy (FY) blood group genotyping is important in transfusion medicine because Duffy alloantibodies are associated with delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. In this study, FY allele frequencies in Thai blood donors were determined by in-house PCR with sequence-specific primers (PCR-SSP), and the probability of obtaining compatible blood for alloimmunized patients was assessed. METHODS: Five hundred blood samples from Thai blood donors of the National Blood Centre, Thai Red Cross Society, were included. Only 200 samples were tested with anti-Fy(a) and anti-Fy(b) using the gel technique. All 500 samples and four samples from a Guinea family with the Fy(a-b-) phenotype were genotyped by using PCR-SSP. Additionally, the probability of obtaining antigen-negative red blood cells (RBCs) for alloimmunized patients was calculated according to the estimated FY allele frequencies. RESULTS: The FY phenotyping and genotyping results were in 100% concordance. The allele frequencies of FY*A and FY*B in 500 central Thais were 0.962 (962/1,000) and 0.038 (38/1,000), respectively. Although the Fy(a-b-) phenotype was not observed in this study, FY*B(ES)/FY*B(ES) was identified by PCR-SSP in the Guinea family and was confirmed by DNA sequencing. CONCLUSIONS: Our results confirm the high frequency of the FY*A allele in the Thai population, similar to that of Asian populations. At least 500 Thai blood donors are needed to obtain two units of antigen-negative RBCs for the Fy(a-b+) phenotype.

Study on MICA/B gene polymorphism in northern Hunan Han population / 中华微生物学和免疫学杂志

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.

Frequencies of IL10 SNP genotypes by multiplex PCR-SSP and their association with viral load and CD4 counts in HIV-1-infected Thais.

Background: Interleukin (IL)-10 is an immuno-regulatory cytokine, levels of which can be influenced by single nucleotide polymorphisms (SNPs) in the promoter. Some, but not all previous studies have shown associations of IL10 SNPs with HIV-1 disease progression, using markers such as viral load or CD4 count. There are few data on IL10 SNP frequencies and HIV-1 disease in regions where non-B HIV-1 subtypes predominate. Objective: To determine genotypes, haplotypes, allele frequencies and associations with markers of HIV-1 disease progression of IL10 SNPs. Methods: A new multiplexed PCR-SSP assay to detect IL10 SNPs at positions -1082, -819 and -592 was developed and used to determine genotypes and haplotypes in 244 HIV-1 CRF01_AE-infected northern Thais having a median time since HIV-1 infection of 2.7 years. Results: At position -1082 of IL10, AA genotype and A allele were the most common (87.3% and 93.2%, respectively). The -819 CT and -592 CA genotypes were the most prevalent (44.3%), and -819T and -592A were the most prevalent alleles (64.8%). The ATA/ATA was the most common genotype (42.6%) with the most prevalent haplotype of ATA (64.7%). No associations of any of the three IL10 SNPs with CD4+ or CD8+ T cell counts or with viral load were found. Conclusions: This first report of IL10-1082A, -819T and the IL10-592A allele frequencies in HIV-1-infected Thais shows the highest frequencies in HIV-1-infected persons worldwide. The lack of association of IL10 SNPs with CD4+ T cell count and viral load suggest that other genes may influence these markers in HIV-1-infected Thais.

Estudo de HLA classes I e II em trinta pacientes equatorianos com artrite reumatoide em comparação com alelos de indivíduos sadios e afetados com outras doenças reumáticas / Study of class I and II HLA alleles in 30 ecuadorian patients with rheumatoid arthritis compared with alleles from healthy and affected subjects with other rheumatic diseases

INTRODUÇÃO: A artrite reumatoide (AR) é uma doença inflamatória crônica sistêmica autoimune que provém de uma desordem incapacitante. Até hoje, a etiologia da AR é desconhecida. No entanto, já se cogitou a existência de indivíduos geneticamente passíveis de tê-la. Muitos estudos já foram realizados em todo o mundo, como, por exemplo, na Polônia, Argentina, Chile, México, Brasil, Colômbia, entre outros países, com relação à influência entre os alelos HLA-DR e a doença, mas não no Equador. OBJETIVO: O principal objetivo deste estudo foi determinar a participação dos alelos de HLA classes I e II em pacientes com AR. PACIENTES E MÉTODOS: Esta pesquisa foi desenvolvida em 30 pacientes adultos com AR, previamente diagnosticados de acordo com os critérios de classificação do Colégio Norte-Americano de Reumatologia (ACR, 1987) e 28 controles. Para a tipificação de HLA classes I e II, adotou-se a técnica PCR-SSP, e as significâncias estatísticas foram avaliadas pelo teste de Qui-Quadrado. RESULTADOS: O HLA-DR4 está presente em 76,7 por cento dos pacientes, com uma frequência alélica de 45 por cento, enquanto apenas 21 por cento dos sujeitos controle o apresentaram. O teste de Qui-Quadrado confirma que as variáveis HLA-DR4 e RA estão altamente vinculadas (X² = 11,38, P = 0,00074). CONCLUSÃO: Há frequência maior de HLA-DR4 e HLA-DR14. Os resultados encontrados são similares aos encontrados em outros estudos. Porém, seria desejável aumentar o tamanho da amostra para encontrar um maior número de perfis genéticos e de alelos envolvidos.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease that originates from a disabling disorder. To date, the etiology of RA is unknown. However, the existence of genetically susceptible individuals was considered. Many studies have been performed worldwide, for example, in Poland, Argentina, Chile, Mexico, Brazil, and Colombia, among others, regarding the influence between HLA-DR alleles and disease, but not in Ecuador. OBJECTIVE: The aim of this study was to determine the involvement of Class I and II HLA alleles in patients with RA. PATIENTS AND METHODS: This study was conducted in 30 adult patients with RA previously diagnosed, according to the classification criteria of the American College of Rheumatology (ACR, 1987) and 28 controls. For Class I and II HLA typing, we adopted the PCR-SSP, and statistical significances were evaluated by Chi-Square. RESULTS: HLA-DR4 is present in 76.7 percent of patients, with an allele frequency of 45 percent, while only 21 percent of control subjects presented it. The chi-square confirms that HLA-DR4 and RA variables are highly bound (X2 = 11.38, P = 0.00074). CONCLUSION: There is increased frequency of HLA-DR4 and HLA-DR14. The results are similar to those found in other studies. But it would be desirable to increase the sample size in order to find a greater number of genetic profiles and alleles involved.

Otimização de metodologia para o estudo de genes KIR / Methodology optimization for KIR genotyping

Receptores killer cell immunoglobulin-like (KIRs) são moléculas localizadas na superfície de células natural killer (NK) e em subpopulações de linfócitos T codificadas por genes do cromossomo 19q13.4. A interação entre receptores KIR e moléculas antígeno leucocitário humano (HLA) de classe I determina se células NK exercerão ou não sua função citotóxica e/ou secretora de citocinas ou se esta será inibida. Este trabalho teve por finalidade otimizar a metodologia para a genotipagem KIR, baseando-se nas condições descritas por Martin (2004). A técnica utilizada foi a reação em cadeia da polimerase com primers de sequência específica (PCR-SSP) com iniciadores sintetizados pela Invitrogen® e visualização do produto amplificado em gel de agarose a 2 por cento com brometo de etídio. Adaptações foram realizadas e a concentração de alguns reagentes foi alterada, como a do controle interno de 100 nM para 150 nM, iniciadores específicos senso e antissenso de KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 e KIR36.5/36.3 de 500 nM para 750 nM e da solução de MgCl2 de 1,5 mM para 2 mM. As concentrações dos demais reagentes e temperaturas de amplificação foram mantidas. Nessas condições, o uso da Taq DNA polimerase recombinante (Invitrogen®) foi satisfatório. Os resultados das genotipagens de 70 indivíduos foram confirmados por rSSO-Luminex® (One Lambda, Canoga Park, CA, EUA). A tipagem de genes KIR por essa técnica apresentou sensibilidade, especificidade, reprodutibilidade e baixo custo.

The killer cell immunoglobulin-like receptors (KIRs) are molecules expressed on natural killer (NK) cells surface and in T-cell subsets encoded by genes located in chromosome 19q13.4. The interaction between KIR receptors and HLA class I molecules determines if the NK cells will fulfill their cytotoxic function and/or cytokine secretion or if this function will be inhibited. The objective of this work was to optimize KIR genotyping method described by Martin (2004). It was used PCR-SSP (polymerase chain reaction-sequence-specific primers) with primers synthesized by Invitrogen® and visualization of the amplified products on 2 percent agarose gel electrophoresis, containing ethidium bromide. Some adaptations were made and the reagents had their concentrations increased: the internal control from 100 nM to 150 nM, forward and reverse specific primers KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 and KIR36.5/36.3 from 500 nM to 750 nM, and MgCl2 solution from 1.5 mM to 2 mM. Other reagent concentrations and amplification temperatures were maintained. Satisfactory results were obtained with Taq DNA Polymerase Recombinant (Invitrogen®). The results of seventy samples were confirmed by rSSO-Luminex® (One Lambda, Canoga Park, CA, USA). This KIR typing method proved to be accurate, specific, reproducible and cost effective.

Caracterización de la respuesta inmunitaria de bovinos infectados con Mycobacterium bovis en condiciones de campo en el municipio Colón estado Zulia Venezuela / Characterization of the immune response of bovines with Mycobacterim bovis field infection in an area with high incidence of bovine tuberculosis in Venezuela

La tuberculosis bovina es una enfermedad crónica, zoonótica, infecciosa y contagiosa teniendo como agente causal al Mycobacterium bovis inductor de una respuesta inmunitaria diversa. Esta abarca, desde una respuesta celular capaz de controlar la infección, pasando por una potente, más no eficiente respuesta humoral, hasta un estado de no respuesta o anergia, coadyuvante de la diseminación de la micobacteria. En este estudio se evaluaron animales seleccionados por el Instituto Nacional de Salud Agrícola Integral (INSAI), como reactores a la prueba simple de la Tuberculina en una finca con antecedentes de tuberculosis por más de 20 años. A estos animales se le aplicaron las siguientes pruebas: Comparativa del PPD (PPD-B y PPD-A), prueba de Interferón Gamma (INFy) y un ensayo inmunoenzimático para TBC (ELISA-TBC), seguidamente se realizó la inspección post morten en el frigorífico donde se evaluaron y clasificaron las lesiones macroscópicas compatibles con tuberculosis. Los tejidos seleccionados fueron utilizados para estudios bacteriológicos, extracción de ADN y posterior amplificación secuencia específica con cebadores y oligonucleótidos IS6110 específicos para el complejo M. tuberculosis, aplicando la prueba de Reacción en Cadena de la Polimerasa (PCR-SSP) para la identificación definitiva del patógeno. El resultado de estas pruebas permitió observar diferentes patrones de respuesta inmunitaria: celular (PPD+/IFN-y-, PPD+/IFNy+, PPD-/IFNy+), mixto (PPD+ o IFN-y+/ELISA-TBC+) y humoral (ELISA-TBC+). Igualmente se detectaron animales anérgicos, negativos a todas las pruebas inmunológicas, positivos en bacteriología y PCR-SSP. Se pudo establecer la progresión de la enfermedad a partir de la severidad de las lesiones y la edad de los animales. Estos patrones pueden aparecer al inicio de la infección o ser el resultado de la progresión crónica de la enfermedad. Estas diferentes respuestas inmunitarias pueden explicar la permanencia de la infección...

The bovine tuberculosis is a chronic, zoonótic infectious disease and contagious having as causal agent to the Mycobacterium bovis inductive of a diverse immune response. This sandal, from an answer cellular, able to control the infection, happening through powerful, but a nonefficient one, humoral response to a state of not response or anergia, facilitating the dissemination of mycobacteria. In this study animals selected by the Venezuelan National Institute of Integral Agricultural Health (INSAI), like reactors to the simple test of the Tuberculina in a property with antecedents of tuberculosis by but of 20 years. To these animals the following tests were applied: Comparative of the tuberculina (PPD-B and PPD-A), test for Gamma Interferon (INF-y) and a Immun-enzimatic Test for TBC (ELISA-TBC), next was made the inspection post morten in the refrigerator where the compatible macrocospic injuries with tuberculosis were evaluated and classified. The selected weaves were used for bacteriological studies, DNA extraction and later amplification specific sequence with specific boots IS6110 for tuberculoso complex M., applying the test of Chain Reaction of Polimerasa (PCR-SSP) for the definitive identification of the pathogen. The result of these tests allowed to observe different patterns from immune response: cellular (PPD+, PPD+ - INF-y + or INF-y +), mixed (PPD+ and/or INF-y, ELISA-TBC+) and humoral (ELISA-TBC). Also anérgics animals detected themselves, negatives to all the immunological tests, positive in bacteriology and PCR-SSP. It was possible to be established the progression of the disease from the severity of the injuries and the age of the animals. These different immune responses may explain the persistence of infection in farm notwithstanding the implementation of the resolution of official control and eradication of Bovine Tuberculosis in the region.

Evaluation of Two Commercial HLA-B27 Real-Time PCR Kits / 대한진단검사의학회지

BACKGROUND: The standard PCR with sequence-specific primers (SSP) is a widely used method of HLA-B27 typing in clinical practice. The aim of our study was to evaluate 2 Korean HLA-B27 kits with different real-time PCR chemistries. METHODS: To validate the accuracy of real-time PCR kits, we selected 28 HLA-B27-positive samples and 33 HLA-B27-negative samples with a wide range of different HLA-B specificities typed by standard PCR-SSP. The 2 real-time PCR kits used were the AccuPower(R) HLA-B27 real-time PCR kit (Bioneer, Korea) with TaqMan probes and the Real-Q(TM) HLA-B*27 detection kit (BioSewoom, Korea) with SYBR Green I dye for melting curve analysis. RESULTS: All 61 samples typed by PCR-SSP demonstrated a perfect concordance with the 2 real-time PCR assays. It was possible to clearly discriminate between HLA-B27-positive and -negative samples in both real-time assays. CONCLUSIONS: In summary, both real-time PCR assays for HLA-B27 were fast, reliable, well-adapted for routine laboratory testing, and attractive alternatives to the conventional PCR-SSP method.

The polymorphism of HLA-DRB1 and -DQB1 genes in Bai ethnic group in Yunnan province / 中国免疫学杂志

Objective:To investigate polymorphism of human leukocyte antigen (HLA)-DRB1 and -DQB1 genes in Bai ethnic group in Dali,Yunnan province.Methods:Polymerase chain reaction-sequence specific primers (PCR-SSP) were used to determine HLA-DRB1 and -DQB1 alleles in 124 unrelated healthy Bai ethnic individuals living in Eryuan County of the Dali Bai autonomous prefecture,Yunnan province.Results:Among all the 21 DRB1 alleles and 15 DQB1 alleles were identified,the predominant alleles were DRB1*1202(26.61%),DRB1*0901(13.89%) and DRB1*0803(9.92%) on DRB1 locus and DQB1*0301(31.45%),DQB1*0601(10.08%),DQB1*0401(8.06%)and DQB1*0502(8.06%)on DQB1 locus.The most common haplotypes were DRB1*1202-DQB1*0301(20.08%)and DRB1*0803-DQB1*0601(7.19%).Conclusion:The phylogenetic tree constructed according to the HLA-DRB1,-DQB1 allele frequencies of Bais with those of other 10 populations suggests that the Bai ethnic group belongs to the southern group of China,but it keeps genetic distance from others and the HLA genes exhibits a unique profile.This study would provide HLA polymorphism information of Bai for the future investigation on the disease related to the genetic polymorphism.

HLA-B27 Subtypes in Korean Patients with Ankylosing Spondylitis / 대한진단검사의학회지

BACKGROUND: HLA-B27 is strongly associated with ankylosing spondylitis (AS), and its subtypes differ in their ethnic distribution. Studies worldwide have shown that B*2701, B*2702, B*2704, B*2705, B*2707, B*2708, B*2714, B*2715, and B*2719 are AS-predisposing subtypes, whereas B*2706 and B*2709 are reported to be negatively associated with AS. The aim of this study was to investigate HLA-B27 polymorphism and clinical features according to subtypes in Korean patients with AS. METHODS: Two hundred thirty samples from patients with impression of AS were analyzed by polymerase chain reaction using a sequence-specific primers (PCR-SSP) method. Pel-Freez SSP Unitray HLA-B*27 kit (Dynal Biotech, USA) including 16 primers was used to define HLA-B27 subtypes from B*2701 to B*2735. RESULTS: Among 230 samples from patients with impression of AS, 171 were HLA-B27 positive, and among 160 patients diagnosed as AS, 154 (96.3%) were HLA-B27 positive, while 17 patients not diagnosed as AS were HLA-B27 positive. Among 154 HLA-B27 positive patients with AS, 142 (92.2%) were typed as B*2705 and 9 (5.8%) were typed as B*2704. Three cases (1.9%) could be interpreted only variously because of their HLA-B27 homogeneous alleles. Between B*2705 and B*2704, no specific HLA-B27 subtype appeared to contribute to AS susceptibility (P=0.60). Difference in clinical features between B*2705 and B*2704 could not be found in this study (P>0.05). CONCLUSIONS: This study verified that HLA-B27 (96.3%) is strongly associated with AS and identified that the major subtypes of HLA-B27 positive patients with AS in Korea are B*2705 (92.2%) and B*2704 (5.8%).

Relationship between interleukin-18 gene promoter polymorphism and atopic dermatitis in children / 中华皮肤科杂志

Objective To investigate the polymorphism at positions-137 and -607 in the upstream promoter 1 region of exon 2 of interleukin(IL)-18 gene in Han children with atopic dermatitis(AD)in Chongqing,China,as well as its correlation with the development of AD.Methods Blood samples were collected from 82 patients with atopic dermatitis and 100 healthy controls.DNA was extracted from the samples and subjected to test with PCR.The polymorphism of IL-18 gene at positions-137 and -607 in the upstream promoter 1 region was analyzed by polymerase chain reaction(PCR)-sequence specific primers(SSP)and gene sequencing.Genotype frequency was compared between the patients and controls.Resuits A G/C polymorphism(GG,GC and CC genotypes)was identified at position-137 in exon 2 of IL-18 gene,and a C/A polymorphism(CC.CA and AA genotypes)at position-607 of this gene.The frequency of genotype GC at position-137 was significantly higher in the patients than that in the controls(47% vs 27%,P＜0.05;odds ratio=2.33.95% confidence interval 1.26-4.33).Increased frequency of C allele was also noted at the position-137 in the patients compared with the controls(0.24 vs 0.15,P＜0.05;odds ratio=1.76,95% confidence interval 1.04.2.97).Patients with severe AD(SCORAD score＞50)were more likely to carry C allele at position-137 of IL-18 gene than those with mild AD(SCORAD＜20).There was no statistical difference in allele frequency at -607(C/A)among patients with mild,moderate,severe AD and the controls(P＞0.05).Conclusions There is a polymorphism at positions -137 and -607 in the upstream promoter 1 region of IL-18 exon 2.The GC genotype of IL-18 at position -137 may confer the susceptibility to AD in Han children in Chongqing.

HLA-DRB1 genotyping in children with nephrotic syndrome of non-IgA mesangial proliferative giomerulonephritis / 中华微生物学和免疫学杂志

Objective To investigate the association of HLA-DRB1 alleles in Han population of Shanxi childrcn with nephrotic syndrome of non-IgA mesangial proliferative glomerulonephritis (MsPGN). Methods HLA-DRB1 was performed by polymerase chain reaction-sequence specific primers technique, and twenty patients with nephrotic syndrome of non-IgA MsPGN were detected. Results Analysis of the fre- quencies of specific at the HLA-DRB1 loci revealed significantly higher frequencies of HLA-DRB1 * 11 al- leles among the nephrotic syndrome patients of non-IgA MsPGN comparing with controls (22. 50% vs 8.33%, x2= 9. 544, P = 0.002, CI = 1. 674-9.995, RR = 4.09). Nine patients with HLA-DRB1 * 11 all accompanied hematuria, hypertension or short renal insufficiency. Conclusion The results suggested that HLA-DRB1 * 11 alleles contribute to genetic susceptibility to nephritic syndrome of non-IgA MsPGN. The pa- tients with HLA-DRB1 *11 easy accompanied hematuria, hypertension or short renal insufficiency.