A single emm gene-specific oligonucleotide probe does not recognise all members of the Streptococcus pyogenes M type 1

Abstract Serological typing of the streptococcal M protein has recently been challenged by a number of unique molecular methodologies based on oligonucleotide recognition of allelic variations within the M protein ( emm ) gene. In these methods, stringent hybridization of an oligonucleotide probe to a polymerase chain reaction amplified emm gene is used as confirmation of specific M type identity. A sample of 17 isolates from 7 previously defined distinct genotypes were tested using a single M1 oligonucleotide probe. Isolates from only three of the genotypes hybridized with the probe. The results demonstrate that a single emm -specific oligonucleotide probe can not identify all members of M type 1, as defined by conventional serotyping using polyclonal antisera.     

Oligonucleotide primers,probes and molecular methods for the environmental monitoring of methanogenic archaea

For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers.     

Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10² CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Kappa-casein alleles in Zebu and cross-bred (1/2 Friesian, 1/4 Jersey, 1/4 Hariana) cattle from India using polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP)

Primers and probes were designed to identify two common alleles CASK-A and CASK-B. A local breed of cross-bred cattle produced to improve the quality and quantity of milk, were studied for the alleles of kappa-casein (κ-casein) gene using polymerase chain reaction and hybridization with sequence specific oligonucleotide probes (PCR-SSPO).     

Isolation of a specific DNA fragment and development of a PCR-based method for the detection of Mycobacterium genavense

The rise of Mycobacterium genavense infections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734-bp fragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses species-specific primers for M. genavense. Followed by a non-radioactive hybridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7-kb sequence was specific for the M. genavense chromosome. The method developed here for M. genavense identification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated.     

多等位基因PCR-SSO反相杂交法及其在HLA-DR_4亚型结构分析中的应用

本文介绍一种适合于多等位基因定型和结构分析的反相顺序特异性寡核苷酸探针杂交方法。将化学合成的多种探针分别经脱氧核苷末端转移酶(TdT)催化,于3′端加上100个以上碱基的Poly(dT)尾,然后将各探针分别以斑点固定于同一张尼龙膜上。用PCR法对该基因位点进行扩增,扩增的同时加入α-~(32)P-dCTP以直接参入放射标记,然后将PCR产物与膜固定探针杂交,以四甲基氯化铵根据探针长度统一洗涤温度。该方法克服了以往对同一份样品进行多个等位基因检测时需要进行多次探针标记和杂交的缺点。我们用该方法对中国人群MHC-Ⅱ类DR4多等位基因进行了研究。     

Inheritance of Pathogenicity of Progeny from an F1 Culture of Melampsora lini

Abstract Races 1 and 400 of Melampsosau bni the causal agent of flax [Lmum usitatissimum]), were crossed and the resulting F² progeny evaluated on near-isogcnic fiax lines. The infection type of the parental cultures, the F¹² and the segregating f² progeny were used to evaluate the inheritance of pathogenicity. Races 1 and 400 as well as the F² and all the F² progeny were avirulent on lines N and P⁴ genes. This indicated both races to be homozygous avirulent. The F² and all the F² progeny were virulent on lines containing L9, M1, M4, and P. This indicated the F to be homuzygous for virulence at these loci. Segregation ratios of the F² cultures fit monogenic ratios on lines with host genes K. L2, L5, L6, L7, L11, M, M2, M3, N1, P1, P2, and P3. Race 1 was avirulent, race 400 virulent and tht F, avirulent on these lines. Several linkage groups were indicated including close linkage at A^p1, A^p2 and A^p3. Segregation on lines L, L3, M5, M6 and cultivars Flor, Dufferin, Linott, and Wishek fit a digenic model. The infection tvpes of the parents, the F¹ and the F², segregations could usually be explained by classical gene-for-gene interactions. Possible exceptions were segregations on L4, L8 and N2 which did not fit a 3: 1 ratio but did fit a 9: 7 ratio. Segregation on L 10 fit a 7: 9 ratio but line L10 may be temperature sensitive. Segregation on L1 indicated a single dominant gene for pathogenicity.     

Empirical establishment of oligonucleotide probe design criteria

Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50-mer probes or -40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.     

Empirical Establishment of Oligonucleotide Probe Design Criteria

Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of ≤85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (≤15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than −30 kcal/mol for 50-mer probes or −40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.     

反向斑点杂交法检测HBV基本核心启动子区A1762T／G1764A突变的实验研究

目的建立一种基于尼龙膜的反向斑点杂交法,用于检测乙型肝炎病毒（HBV）基本核心启动子区（BCP）A1762T／G1764A突变。方法根据我国HBV主要流行的基因型为B和C,从GenBank上查出4种HBVBCP序列。利用在线工具ClustalW进行比对,针对该突变位点设计引物和检测探针。探针经合成和修饰后点在带正电的尼龙膜上。将反向斑点杂交法结合地高辛检测试剂盒用于检测A1762T／G1764A突变,以测序法确定该区域序列的标本为检测对象。结果反向斑点杂交法分别检测5例A1762／G1764病毒株、2例T1762／G1764病毒株、5例A1762／A1764病毒株和4例T1762／A1764病毒株,结果与测序完全相同。结论应用本方法可以快速、准确地HBV相关的热点突变。     

Microarray Analysis of Microbial Virulence Factors

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.