应用PCR—SSP技术对粒细胞抗原NA进行基因分型

根据ＮＡ基因序列,合成ＮＡ特异性引物,通过ＰＣＲ－ＳＳＰ技术,特异性地扩增ＮＡ１和ＮＡ２,建立ＮＡ基因分型方法,同时对１２０例浙江汉族人群进行ＮＡ基因型的检测。结果表明ＰＣＲ－ＳＳＰ技术对ＮＡ的基因分型方法简单、翰林折汉族人群的ＮＡ基因型ＮＡ１为０．５２５,ＮＡ２为０．４７５。     

NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers

BACKGROUND: The granulocyte antigens NA1 and NA2 are often targets of granulocyte antibodies causing immune neutropenia. Currently, NA typing relies on the properties of the typing sera or antibodies and the techniques used. Therefore, the technique of polymerase chain reaction with sequence-specific primers (PCR-SSP) was adapted for DNA-based NA typing and was used for determining the NA gene frequencies in the German population. STUDY DESIGN AND METHODS: The genomic DNA of 160 unrelated healthy individuals was typed for NA1 and NA2 by PCR-SSP. In 60 granulocyte samples, the NA phenotype was additionally determined by the antigen capture assay and the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. RESULTS: Results of the antigen capture assay and PCR-SSP correlated precisely, whereas nine individuals were typed heterozygous only by GIFT. The gene frequencies were 0.35 for NA1 and 0.65 for NA2. CONCLUSION: The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR-SSP. In contrast to GIFT, which showed an error rate for NA typing of 15 percent, PCR-SSP and the antigen-capture assay are more reliable methods of NA typing of granulocytes.     

Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers

D_el (D-elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti-D eluate obtained after an adsorption-elution test using anti-D antibodies. We studied here the molecular genetic status of D_el by using polymerase chain reaction with sequence-specific primers (PCR-SSP).
We screened 306 serologically apparent D-negative Japanese donors comprising 102 D_el types for exons 7, 4 and 10 of the RHD gene. No PCR product was found in all 204 non-D_el samples from the D-seronegative donors. However, PCR products were found in all 102 D_el samples and all 70 D-seropositive samples tested by the three PCR methods for exons 7, 4 and 10 analysis.
D_el was found with CCee, CcEe and Ccee, but not with CCEe, CcEE, ccEE, ccEe or ccee phenotype. The incidences of D_el in the samples with the serological phenotypes CCee, CcEe and Ccee were 80.0% (4/5), 68.2% (45/66) and 61.6% (53/86), respectively.
The results provide evidence that D_el samples have exons 4, 7 and 10 of an RHD gene present in some form. This is consistent with the evidence that D antigen is present on the cells although only detected by antibody adsorption and elution. The PCR-SSP method in the present study is useful to confirm D_el among serologically apparent D-negative samples.     

Determination of neutrophil antigen gene frequencies in five ethnic groups by polymerase chain reaction with sequence-specific primers

BACKGROUND: The granulocyte antigens NA1 and NA2 are the two recognized allelic forms of Fc gamma receptor IIIB. These antigens are clinically relevant, because they are the most frequent targets of neutrophil antibodies in alloimmune neonatal neutropenia, transfusion-related acute lung injury, and chronic benign autoimmune neutropenia of infancy. STUDY DESIGN AND METHODS: A genotyping assay for NA1 and NA2 using polymerase chain reaction with sequence-specific forward and reverse oligonucleotide primers has been developed and validated. Genomic DNA was isolated from the peripheral blood of 478 unrelated individuals of five ethnic groups and used as template for NA genotyping. RESULTS: A validation study of 22 serologically typed samples (2 NA1/NA1, 10 NA1/NA2, and 10 NA2/NA2) was performed. A concordance rate of 100 percent (22/22 samples) was observed between the genotyping assay and serologic typing. In the genotyping study conducted, the NA1 and NA2 gene frequencies observed were 0.31 and 0.69 for African Americans, 0.30 and 0.70 for Asian Indians, 0.37 and 0.63 for whites, 0.53 and 0.47 for Hispanics, and 0.55 and 0.45 for Native Americans, respectively. CONCLUSION: Polymerase chain reaction with sequence-specific primers provides a simple and rapid alternative to neutrophil antigen typing by serologic tests. The NA1 and NA2 gene frequencies observed in Asian Indians and African American populations are similar to those observed in white populations, while those observed in Native American and Hispanic populations are more similar to those previously reported for Asian populations.     

序列特异性引物PCR法检测Rh血型E/e基因型

目的　建立Rh血型E/e基因分型的方法，以检测人群中E/e基因频率。方法　采用快速盐析法抽提样本DNA，采用PCR—SSP方法检测E/e基因。结果　在本研究的RhD阳性人群中E基因频率为0.223，e基因频率为0.777。RhD阴性人群中E基因频率为0.040，e基因频率为0.960。结论　人群中以e基因为主，Rh阳性与阴性人群中E和e基因频率存在明显的差异。     

Rapid determination of platelet alloantigen genotypes by polymerase chain reaction using allele-specific primers

BACKGROUND: The technique of allele-specific polymerase chain reaction (PCR) amplification has been adapted for DNA-based human platelet alloantigen typing. STUDY DESIGN AND METHODS: Sequence-specific primers were used to discriminate between the alleles encoding the six major human platelet alloantigens in a series of patients and normal blood donors. RESULTS: This technique allows the direct determination of platelet antigen genotypes from genomic DNA after PCR amplification and agarose gel electrophoresis. It offers significant advantages over previously described techniques for alloantigen identification, as the additional analytical steps of restriction enzyme digestion or dot blot hybridization are not required. The results obtained with this technique correlated precisely with those derived from serologic typing and allele-specific oligonucleotide hybridization. CONCLUSION: The use of allele-specific PCR for platelet alloantigen typing should facilitate the development of DNA-based typing in other regional blood centers and clinical laboratories.     

Human neutrophil antigen-4a gene frequencies in an Australian population,determined by a new polymerase chain reaction method using sequence-specific primers

Human neutrophil antigen-4a (HNA-4a) is a high-frequency (99% in the USA) neutrophil antigen, which has recently been linked to a case of alloimmune neonatal neutropenia. We have devised a new polymerase chain reaction sequence-specific primer (PCR-SSP) method to assess HNA-4a genotype, and used it to determine the HNA-4a gene frequencies in an Australian population. The gene frequency was found to be 0.906, which is the same as in the American population. The PCR-SSP genotyping method perfectly correlates with serological phenotyping and is efficient for screening large numbers of samples.     

Rapid typing of the human Fc gamma receptor IIA polymorphism by polymerase chain reaction amplification with allele-specific primers

BACKGROUND: The human Fc gamma receptor IIa (Fc gamma RIIa) is expressed in two polymorphic forms, Fc gamma RIIa-H131 and Fc gamma RIIa-R131, that differ by the replacement of histidine by arginine at position 131. This replacement is caused by a single-nucleotide exchange of A–>G. The resulting receptor forms differ in their binding to human IgG2 and mouse IgG1, which may lead to a different immunologic defense to bacterial polysaccharides and encapsulated bacteria. STUDY DESIGN AND METHODS: A rapid and easy polymerase chain reaction(PCR) method of genotyping the Fc gamma RIIa was developed. Allele-specific primers discriminate between the Fc gamma RIIa-H131 and the Fc gamma RIIa-R131 forms of the receptor. The results were compared with those obtained by another DNA-based genotyping method, in which PCR-amplified DNA was hybridized with allele-specific oligonucleotides, and with a functional phagocytosis assay using mouse IgG1-coated red cells as target antigens. RESULTS: The genotypes deduced from the PCR with allele-specific primers were in complete accordance with those obtained by the data from the hybridization of PCR-amplified DNA with allele- specific oligonucleotides. Furthermore, the Fc gamma RIIa genotypes of 28 individuals in all cases corresponded to the functional phenotypes. CONCLUSION: The use of PCR with allele-specific primers provides a rapid and easily performed method for the determination the Fc gamma RIIa polymorphism.     

Inosine-containing primers in human papillomavirus detection by polymerase chain reaction.

A PCR protocol has been developed using inosine-containing primers for human papilloma virus (HPV) detection. The HPV16 L1 region from CaSki-positive cells was efficiently amplified by a single reaction and directly analyzed by agarose gel electrophoresis. The method was found to be sensitive and reproducible, and it easy to use for HPV typing and screening.     

Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism: an evaluation of four quantitative methods

BACKGROUND: Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage, and this feature is relevant in investigating RHD mosaicism and chimerism. STUDY DESIGN AND METHODS: Monoplex and duplex real-time PCRs, uncalibrated and calibrated, were tested. RHD zygosity was determined for 72 samples and compared with serology-based prediction of RHD zygosity. Additionally, a range of constructed RHD dosages were compared with determined RHD dosages. Finally three samples, Days 1, 90, and 120, from a patient with loss of D were included. RESULTS: All setups enabled differentiation between hemi- and homozygous samples. Results from calibrated methods had the smallest variation and enabled differentiation at the mean +/- 3SD. Incongruity between determined and predicted RHD zygosity was found in three samples. PCR-sequence-specific priming specific for the hybrid downstream Rhesus box was performed on those samples confirming the real-time PCR results. Determined RHD dosage equaled constructed RHD dosage in both calibrated real-time PCRs. Patient samples showed RHD with a mean of 1, 5, and 8 percent relative to GAPDH in all samples. CONCLUSION: Duplex calibrated real-time PCR was a robust and exact method for determination of RHD zygosity and applicable in evaluation of RHD mosaicism and chimerism. This was illustrated with patient samples, where this assay revealed for the first time signs of mosaicism in both expression of D antigens and in RHD dosage.     

Genotyping of KEL1 and KEL2 of the human Kell blood group system by the polymerase chain reaction with sequence-specific primers

BACKGROUND: Kell is a major antigenic system in human red cells, with more than 20 identified antigens. KEL1 and KEL2 are two opposing low- and high-frequency alleles. Immunization to KEL1 is clinically significant, because anti-KEL1 can cause severe reactions to transfusion of incompatible blood, as well as hemolytic disease of the newborn. At the nucleotide level, the difference between the KEL2 and KEL1 alleles is a single-base change within exon 6 that results in the substitution of methionine (ATG) for threonine (ACG) at position 193. STUDY DESIGN AND METHODS: An assay using polymerase chain reaction and sequence-specific primers to genotype for the KEL1 and KEL2 alleles has been developed. It uses two allele-specific forward primers for either KEL1 or KEL2 and a single reverse-consensus primer. RESULTS: A validation study of 42 serologically typed samples (5 KEL:1,-2 [K+k-]; 23 KEL:1,2 [K+k+]; and 14 KEL:-1,2 [K-k+]) was performed. A concordance rate of 100 percent (42/42 samples) was observed between polymerase chain reaction with sequence-specific primers and serologic typing. CONCLUSION: This rapid, nonradioactive, Kell system genotyping assay does not require the additional steps of probe hybridization or restriction enzyme digestion. This application of polymerase chain reaction with sequence-specific primers should prove particularly useful in Kell system genotyping of amniotic cells to identify pregnancies at risk for hemolytic disease of the newborn.     

Evaluation of human papillomavirus-consensus primers for HPV detection by the polymerase chain reaction

Cervical cancer is one of the most frequently found cancers in women and appears to have a viral aetiology. Substantial evidence points to the human papillomaviruses (HPV) as the infectious agents and there is considerable interest in identifying and accurately typing the viruses. Since HPVs now comprise more than 100 different HPV types, the polymerase chain reaction (PCR) has been the preferred methodology for virus identification and typing on isolated DNA. In that context, five commonly employed PCR consensus primers have been evaluated for the detection and typing of HPV. The five consensus primer pairs were derived from the consensus sequences of either the L1 and E1 open reading frames. All primers exhibited approximately equal sensitivity, as defined by the ability to detect HPV DNA, on a series of standard HPV DNA-containing preparations. However, the five primer pairs performed differently on 24 HPV-positive and 34 HPV-negative samples obtained from cervical scrapes which had been typed by type-specific PCR for HPV 6/11, 16, 18 and 33. The values for agreement between identification of samples by a HPV type-specific PCR and the consensus primer PCR were 78, 84, 91, 93 and 98%. Three samples, which were positive with only one of the five consensus primer pairs and were negative with the PCR for HPV types 6/11, 16, 18 and 33, contained other HPV sequences or HPV-related sequences as determined by DNA sequence analysis. To our knowledge, this report represents the first extensive comparison of five different consensus primers in a polymerase chain reaction for the detection of HPV. Our results suggest that PCR typing for human papillomaviruses requires more than one consensus primer pair to identify all HPV-infected samples.     

Comparison of different primers for rapid detection of Salmonella using the polymerase chain reaction.

Salmonella is the leading cause of food-borne diarrhoeas in the US. In recent years polymerase chain reaction (PCR) has become the method of choice for rapid and sensitive detection of Salmonellae in contaminated foods. As a result, several different primer sets have been reported for use in PCR-based assay systems. In order to identify an optimal primer set from among the wide range of primers reported in the literature, we synthesized five different pairs and evaluated their relative performance in PCR under uniform assay conditions using a common panel of the target (Salmonella) and non-target (non- Salmonella) bacterial strains. Of the five sets of primers tested, the one designed on the basis of a 199 bp repeat sequence of S. weltevreden[Jitrapakdee et al. (1995) Molecular and Cellular Probes 9, 375-382] gave optimal results with most bacterial strains examined.     

Selection for 3' end triplets for polymerase chain reaction primers

The 3' end of a primer is a key component of PCR primer design. Many recommendations for the composition and sequence of the 3' end have been suggested based on theoretical considerations, but have not been verified experimentally. We analyzed 3' end triplets of PCR primer sequences obtained from refereed journal articles, to test those recommendations and to make empirical recommendations for primer design. The frequencies of the 64 possible 3'end triplets among 2137 PCR primers from the VirOligo database were not uniformly distributed. From the analysis, we found that unfavored and preferred 3' end triplets existed, and that the apparent preferences were not due to base compositions in viral genome sequences. Comparison of the sequences preferred by practitioners to those recommended, suggested that no single recommendation is entirely satisfactory. We suggest that recommendations be replaced with a scoring system incorporating empirical frequencies such as those reported here.     

用序列特异性引物多聚酶链反应的方法进行大样本、多基因的多态性鉴定

目的:建立多个基因同时进行多态性分析的序列特异性引物多聚酶链反应的方法,以满足其在临床检验中进行基因多态性鉴定时的应用。方法:实验于2005-10/2006-05于河南省新乡医学院分子生物研究室完成。进行临床普通血液常规检测的患者血样200份(由新乡医学院第三附属医院提供),采用临床血样,提取DNA后采用多聚酶链反应扩增,并以扩增后的样品进行琼脂糖凝胶电泳,以是否出现相应的扩增条带作为基因分型的标准。应用优化后的序列特异性引物多聚酶链反应的方法分析以下基因的多态性:肿瘤坏死因子α-308A/G和-238G/A变异、白细胞介素6-174G/C变异、细胞色素P4502D6(CYP2D6)*10B外显子第188位的C/T变异。并由此优化序列特异性引物多聚酶链反应的方法,以达到应用同一个多聚酶链反应扩增程序,可同时对多个基因、多个临床样本的基因多态性同时进行分析,且基因型清晰,分型快速、准确。结果:①用20 g/L的琼脂糖凝胶电泳可看到每泳道内可有2条或1条扩增产物,其中阳性对照β珠蛋白基因的扩增片段长268 bp,此条带可以出现、减弱或者不出现。②其中肿瘤坏死因子α-308A/G存在G/G和A/G两种基因型;肿瘤坏死因子α-238G/A位点可检测到的基因型有G/A、G/G和A/A3型;白细胞介素6-174G/C变异位点可检测到的基因型均为G/G纯合子;细胞色素P4502D6*10B外显子第188位的C/T位点可检测到的基因型为C/C、C/T、T/T3型。结论:优化后的序列特异性引物多聚酶链反应适合对大样本,多个基因的单核苷酸位点变异进行多态性分析,快速、准确、成本低。     

Laboratory diagnosis of chancroid using species-specific primers from Haemophilus ducreyi groEL and the polymerase chain reaction

To enhance laboratory identification of Haemophilus ducreyi, the causative agent of the genital ulcer disease chancroid, a polymerase chain reaction (PCR) assay was developed using target DNA sequences from the essential H. ducreyi gene, groEL. Positive reactions were obtained in this PCR assay with 139 isolates of H. ducreyi from patients in worldwide locations from the 1940s to the 1990s. In contrast, 24 other bacterial species were negative. When genital ulcer specimens from 162 African patients with clinically diagnosed chancroid were evaluated, 66 were culture positive. The sensitivity of PCR as compared with culture was 89% (59 of 66), and specificity was 79% (76 of 96). However, representative samples of the 20 culture-negative, PCR-positive specimens were confirmed as positive by a second PCR assay using different H. ducreyi-specific primers. Thus, combined results of culture and PCR detected H. ducreyi in 86 specimens, with resolved sensitivities of 92% (79 of 86) for PCR, and 77% (66 of 86) for culture. These results suggest that PCR assays for H. ducreyi have great potential for augmenting or replacing problematic cultural techniques.     

Detection of erythromycin resistance by the polymerase chain reaction using primers in conserved regions of erm rRNA methylase genes

Genes belonging to different erm DNA hybridization classes were selectively amplified by polymerase chain reaction with a pair of oligonucleotides that corresponded to conserved amino acid motifs in known ERM methylases. Identification of the resistance mechanism was possible despite substantial nucleotide sequence diversity among the erythromycin resistance genes.