M protein gene (emm type) analysis of group A beta-hemolytic streptococci from Ethiopia reveals unique patterns

The genetic diversity of group A streptococcal (GAS) isolates obtained in 1990 from Ethiopian children with various streptococcal diseases was studied by using emm gene sequence analysis. A total of 217 GAS isolates were included: 155 and 62 isolates from throat and skin, respectively. A total of 78 different emm/st types were detected among the 217 isolates. Of these, 166 (76.5%) belonged to 52 validated reference emm types, 26 (11.9%) belonged to 16 already recognized sequence types (st types) and 25 (11.5%) belonged to 10 undocumented new sequence types. Resistance to tetracycline (148 of 217) was not correlated to emm type. Isolation rate of the classical rheumatogenic and nephritogenic strains was low from cases of acute rheumatic fever (ARF) and acute glomerulonephritis (AGN), respectively. Instead, the recently discovered st types were overrepresented among isolates from patients with ARF (3 of 7) and AGN (9 of 16) (P < 0.01) compared to isolates from subjects with tonsillitis and from healthy carriers (10 of 57 and 16 of 90, respectively). In contrast to rheumatogenic strains from the temperate regions, more than half of the isolates from ARF (four of seven) carried the genetic marker for skin preference, emm pattern D, although most of them (six of seven) were isolated from throat. Of 57 tonsillitis-associated isolates, 16 (28%) belonged to emm pattern D compared to <1% in temperate regions. As in other reports emm patterns A to C were strongly associated with throat, whereas emm pattern D did not correlate to skin. This first large-scale emm typing report from Africa has demonstrated a heterogeneous GAS population and contrasting nature of GAS epidemiology in the region.     

Application of polymerase chain reaction (PCR) to HLA-C locus typing

Polymerase chain reaction/oligonucleotide typing was used to identify HLA-Cw*0601 (Cw6) in patients with psoriasis and psoriatic arthritis. The assignment of HLA-Cw*0601 was established by the concordant presence of codons for alanine (position 73), lysine (position 80) and tryptophan (position 97). The frequencies of all three codons were increased in the patient groups.     

半定量聚合酶链反应在PCR-RFLP酶切法测定基因多态性中的应用

目的探索将半定量聚合酶链反应方法，应用于PCR-RFLP酶切法以提高基因多态性测定精确性和可重复性。方法PCR扩增81例人维生素D受体基因BsmI位点多态性片段，分别在酶量不同的两种酶切体系中测定基因型；PCR产物经半定量后，再次用上述两种体系酶切。考察4次测定的一致性。结果经产物半定量后用PCR-RFLP法测定VDR基因BsmI多态性，不同酶切系统测得基因多态性的一致性明显优于未经半定量的结果。结论应用半定量PCR方法可明显提高PCR-RFLP酶切法的精确性和可重复性。     

Application of the polymerase chain reaction (PCR) in the diagnosis of Hb S-beta(+)-thalassemia

Isoelectric focusing of the hemolysate prepared from a two-year-old American black boy with microcytic hypochromia showed the presence of a high percentage (63.3%) of such Hb variant as Hb S, while the levels of Hb A, Hb F and Hb A2 were 20.0%, 12.7%, and 4.0%, respectively. The ratio of the non-alpha-chain to the alpha-chain of the biosynthesized globin chains was 0.49. The variant was identified as Hb S by amino acid analysis of the abnormal peptide (beta T-1) and digestion of DNA amplified by the polymerase chain reaction with enzyme Eco 81 I. This was further confirmed by DNA sequencing. DNA sequencing of a beta-gene without the beta s-mutation revealed a nucleotide change of T to C in the polyadenylation signal sequence AATAAA 3' to the beta-gene, resulting in beta(+)-thalassemia. These results are consistent with the existence of a beta s-gene and a beta(+)-thalassemia gene in trans.     

Positive immunoglobulin gene rearrangement study by the polymerase chain reaction in a colonic adenocarcinoma.

The detection of clonal rearrangements of the immunoglobulin heavy chain gene by the polymerase chain reaction provides a rapid method to differentiate monoclonal from polyclonal B-lymphocyte proliferations. It has been shown to be highly specific and so far, no false-positive results have been described. A case of a poorly differentiated colonic adenocarcinoma that showed a "false positive" clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction technique is reported. DNA contamination was unlikely because of the strict adherence to the laboratory polymerase chain reaction protocol and also the repeated demonstration of the same amplified band in a separate experiment using DNA extracted from another piece of tumor tissue. The apparent monoclonal immunoglobulin heavy chain gene rearrangement in the first polymerase chain reaction may be related to a combination of the paucity of lymphoid cells in the tissue sample and the presence within this small number of lymphocytes of a clonal reactive cell population. It is, therefore, important to correlate the routine microscopic and immunohistochemical findings in the interpretation of polymerase chain reaction results, especially when working with nonlymphoid tumors and lymphocyte-poor lesions.     

Application of polymerase chain reaction to detect activated oncogenes in hematological malignancies

Polymerase chain reaction (PCR) was applied to detect the structural change accompanying the activation of oncogenes in hematological malignancies and preleukemic states. Point mutation of N-ras oncogene was examined by oligonucleotide differential hybridization coupled with PCR. Five out of 17 AML patients were shown to have mutated N-ras gene. These mutations could be used as a genetic marker to diagnose the residual malignant cells. Philadelphia chromosome in CML was examined by cDNA synthesis and PCR with successful results. PCR was shown to be a highly versatile and sensitive method which would be invaluable in clinical diagnosis.     

Detection of giardine gene in local isolates of Giardia duodenalis by polymerase chain reaction (PCR)

Giardia duodenalis is an intestinal parasite that causes diarrhoea and malabsorption in children. The parasite also infects AIDS patients with a weak immune system. A study was carried out on six local isolates of Giardia duodenalis (110, 7304, 6304, M007, 2002 and 6307) from faeces of Orang Asli patients admitted to the Gombak Hospital. WB, a reference pathogenic strain from human and G. muris from a wild mouse, were commercially obtained from the American Type Culture Collection (ATCC). All the isolates were cultured axenically in TYI-S-33 medium. Two sets of primers were used for the techniques: primers LP1 and RP1 and primers LP2 and RP2. The sets of primers amplified giardine gene of 171 bp and 218 bp in sizes respectively. The study showed that the two sets of primers could detect G. duodenalis to the genus and species level specifically.     

Rapid identification of beta-hemolytic streptococci by fluorescence in situ hybridization (FISH)

Rapid identification of pathogenic, beta-hemolytic streptococci is important for treatment decisions. We evaluated fluorescence in situ hybridization (FISH) for this purpose using 23 reference strains, 157 clinical isolates, and 80 blood cultures showing streptococci in the Gram stain. With a sensitivity and specificity in excess of 99%, FISH proved to be suitable for rapid identification of beta-hemolytic streptococci in a diagnostic laboratory.     

Prevalence of the amylase-binding protein A gene (abpA) in oral streptococci

Salivary amylase binds specifically to a number of oral streptococcal species. This interaction may play an important role in dental plaque formation. Recently, a 585-bp gene was cloned and sequenced from Streptococcus gordonii Challis encoding a 20.5-kDa amylase-binding protein (AbpA). The goal of this study was to determine if related genes are present in other species of oral streptococci. Biotinylated abpA was used in Southern blot analysis to screen genomic DNA from several strains representing eight species of oral streptococci. This probe hybridized with a 4.0-kb HindIII restriction fragment from all 13 strains of S. gordonii tested. The probe did not appear to bind to any restriction fragments from other species of amylase-binding oral streptococci including Streptococcus mitis (with the exception of 1 of 14 strains), Streptococcus crista (3 strains), Streptococcus anginosus (1 strain), and Streptococcus parasanguinis (1 strain), or to non-amylase-binding oral streptococci including Streptococcus sanguinis (3 strains), Streptococcus oralis (4 strains), and Streptococcus mutans (1 strain). Primers homologous to sequences within the 3' and 5' ends of abpA yielded products of 400 bp following PCR of genomic DNA from the Southern blot-positive strains. Several of these PCR products were cloned and sequenced. The levels of similarity of these cloned products to the abpA of S. gordonii Challis ranged from 91 to 96%. These studies reveal that the abpA gene appears to be specific to S. gordonii and differs from genes encoding amylase-binding proteins from other species of amylase-binding streptococci.     

Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers

This study describes a new approach to the determination of all known mannan-binding lectin (MBL) mutations. The distribution of known variants of the MBL gene in a population of healthy unrelated Danes was determined and the genotype was correlated with the plasma MBL concentrations. The following genetic polymorphisms were studied: three point mutations in the promoter region at position -550 (H/L variants), -221 (X/Y variants), -70 (nt C or T), one point mutation in the 5' untranslated (UT) region at position +4 (P/Q variants) and three point mutations located at codons 52, 54 and 57 in exon 1 of the MBL gene, at nucleotide positions 223, 230 and 239, respectively. To perform genotyping, we designed sequence specific primers for a polymerase chain reaction (PCR-SSP). PCR-SSP is a powerful technique for the discrimination of alleles resulting from single base substitutions and is a widely used technique. Another major advantage of the PCR-SSP method is its ability to determine whether sequence motifs are in cis or trans. The frequencies of variants in exon 1 obtained by PCR-SSP were completely comparable to results obtained by previously described PCR methods, restriction fragment length polymorphism (RFLP) and site-directed mutagenesis (SDM). This PCR-SSP method is performed with standard laboratory equipment and has the capacity to detect all genetic variants in 100 samples in 2 days at an estimated total cost of GBP 11 per sample. Analysing the correlation between MBL haplotype and plasma MBL levels, we confirmed that three different structural variants, B, C and D and the promoter haplotypes HY, LY and LX have a dominant effect on the concentration of MBL. The HY haplotype is associated with the highest plasma concentration, the LY haplotype with intermediate levels and the LX haplotype with the lowest levels. The LX haplotype was found to be associated with very low levels of MBL similar to those found in association with the structural B genotype. The gene frequencies of variants in the MBL gene in the Danish population studied correspond to previous reports on Caucasian populations.     

多重聚合酶链式反应（MPCR）用于霍乱弧菌检测的研究

本文首先从实用的角度探讨用三对特异的引物对不同血清群霍乱弧菌进行ＭＰＣＲ检测的可能性。这三对引物具有很大的应用价值，从一次扩增反应中可以得出有关菌株的毒力因子和血清群的信息。检测的敏感性可达到１０ＣＦＵ。实验还提示将引物数量改为五条寡核苷酸链来进行ＭＰＣＲ能得出同样的结果。对Ｐ３、Ｐ４引物扩增形成的６１７ｂｐ的ＤＮＡ片段进行酶切分析表明，埃尔托型霍乱弧菌Ｏ１３９群霍乱弧菌的ｔｃｐＡ基因有很大的相似性，至少在ＰｓｔⅠ和ＢｇｌⅡ酶切位点上表现相同，但两者均与古典型霍乱弧菌不同。     

In situ polymerase chain reaction and its applications to the study of endocrine diseases

Conclusion In situ PCR is a new and exciting technology that is already providing a mechanism to gain insights into disease pathogenesis. As with other emerging technologies, its true strengths and weaknesses are becoming clearer with time.In situ PCR encompasses several different techniques, not all of which are equally applicable to different starting materials. It appears to be most effective for low-copy DNA or RNA detection in single-cell preparations after controlled fixation and pretreatment. However, the exact quantification of results still remains somewhat problematic. Directin situ PCR yields significantly less specific results than indirectin situ PCR and is— with currently used protocols—not applicable in tissue sections. Protocols need to be improved significantly to render them applicable to routinely processed specimens. Clinical application of this technology in routine laboratories must await resolution of its current limitations. Its impact in endocrine pathology will be most marked where conventional ISH fails owing to low sensitivity. It is also reasonable to believe that other methods, such as the self-sustained sequence replication (3SR) [14] or refined ISH procedures, may soon become suitable for studies on archival materials.     

Evaluation of clonal immunoglobulin heavy chain rearrangements in Hodgkin's disease using the polymerase chain reaction (PCR)

We have correlated histologic type of Hodgkin's disease, degree of Hodgkin and Reed-Sternberg cell infiltration, percentage of Hodgkin and Reed-Sternberg cell positivity for latent membrane protein, immunophenotype of Hodgkin and Reed-Sternberg cells, and immunoglobulin heavy chain (IgH) gene rearrangements detected by polymerase chain reaction (PCR) in 56 unselected Hodgkin's disease cases. Two protocols were used for amplification of IgH gene using Fr2 or Fr3 V-region primers, in conjunction with nested primers directed to the JH region. PCR products were run on polyacrylamide gels. Immunohistochemical studies were performed on paraffin sections using monoclonal antibodies for CD20 and latent membrane protein, and polyclonal antibody to CD3. Using both primer combinations we detected a definitive clonal band in 23.2% of the Hodgkin's disease cases. Clonal IgH rearrangements were detected in 23.6% of nodular sclerosis type and in 28.5% of mixed cellularity type. Using a highly sensitive method such as PCR, more than 20% of unselected cases of Hodgkin's disease were found to contain B-cell clonal proliferations, but there was no correlation between histological and immunological parameters and molecular analysis results.     

Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction.

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to detect the gene for aerolysin in strains of Aeromonas hydrophila and to screen for identical genes in A. caviae, A. sobria, and A. veronii isolated from patients with diarrheal disease. Primers targeted a 209-bp fragment of the aer gene coding for the beta-hemolysin and detected template DNA only in the PCR using nucleic acid (NA) from hemolytic strains of A. hydrophila which were also cytotoxic to Vero and CHO cells and enterotoxic in suckling-mouse assays. PCR amplification of NA from hemolytic A. sobria or nonhemolytic A. hydrophila and A. caviae strains was consistently negative. Primer specificity was determined in the PCR by using NA extracted from 56 strains of bacteria, including hemolytic Escherichia coli and Listeria monocytogenes as well as several recognized enteric pathogens defined in terms of their toxigenicity. The detection limit for the aerolysin gene by PCR amplification was 1 ng of total NA. The PCR clearly identified aerolysin-producing strains of A. hydrophila and may have application as a species-specific virulence test because other hemolytic Aeromonas species tested were negative.     

Detection of murine cytomegalovirus (MCMV) DNA in skin using the polymerase chain reaction (PCR)

We used the polymerase chain reaction (PCR) and primers for the immediate early gene of murine cytomegalovirus (MCMV) to detect MCMV DNA in skin harvested from mice during acute infection. MCMV DNA was also detected in DNA extracted from spleen and salivary gland of MCMV-infected mice, but not in the skin, salivary gland, or spleen of uninfected, seronegative mice. Detection of MCMV DNA in skin provides direct evidence that skin can serve as a vehicle for transmission of MCMV. This observation is relevant to humans, such as burn patients, who receive skin allografts that may be infected with cytomegalovirus.