PCR Detection of DNAs of Animal Origin in Feed by Primers Based on Sequences of Short and Long Interspersed Repetitive Elements

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.     

Chromosomal Assignment of Short cDNA Sequences by PCR Using Overlapping and Tailed Short Primers

Overlapping primers and tailed short primers are effective agentsfor mapping very short cDNA sequences. By using such primers,human cDNAs as short as 32 nucleotides in length can producePCR bands. Using these and other primers of ordinary size, 44cDNAs were assigned to chromosomes, of which 24 were assignedto single chromosomes, and 2 were assigned to two chromosomesand two were assigned to three chromosomes, respectively. Amongthe 24 cDNAs, all of which matched GenBank entries, 6 cDNAswere observed to map to the same chromosomes as reported previously.     

A Gene with Specific and Global Effects on Recombination of Sequences from Tandemly Repeated Genes in Saccharomyces Cerevisiae

The preservation of sequence homogeneity and copy number of tandemly repeated genes may require specific mechanisms or regulation of recombination. We have identified mutations that specifically affect recombination among natural repetitions in the yeast Saccharomyces cerevisiae. The rrm3 mutation stimulates mitotic recombination in the naturally occurring tandem repeats of the rDNA and copper chelatin (CUP1) genes. This mutation does not affect recombination of several other types of repeated genes tested including Ty elements, mating type information and duplications created by transformation. In addition to stimulating exchange among the multiple CUP1 repeats at their natural chromosomal location, rrm3 also increases recombination of a duplication of CUP1 units present at his4. This suggests that the RRM3 gene may encode a sequence-specific factor that contributes to a global suppression of mitotic exchange in sequences that can be maintained as tandem arrays.     

PCR Detection of Salmonella enterica Serotype Montevideo in and on Raw Tomatoes Using Primers Derived from hilA

Salmonellae have been some of the most frequently reported etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of primers derived from hilA and sirA, positive regulators of Salmonella invasive genes, were developed to identify Salmonella enterica serotype Montevideo on and in tomatoes. Based on examination of 83 Salmonella strains and 22 non-Salmonella strains, we concluded that a pair of hilA primers detects Salmonella specifically. The detection limits of the PCR assay were 10¹ and 10⁰ CFU/ml after enrichment at 37°C for 6 and 9 h, respectively. When the assay was validated by detecting S. enterica serotype Montevideo in and on artificially inoculated tomatoes, 10² and 10¹ CFU/g were detected, respectively, after enrichment for 6 h at 37°C. Our results suggest that the hilA-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonellae in or on fresh produce.     

Molecular Characterization of High Plant Species Using PCR with Primers Designed from Consensus Branch Point Signal Sequences

A novel method is introduced for producing molecular markers in plants using single 15- to 18-mer PCR primers designed from the short conserved consensus branch point signal sequences and standard agarose gel electrophoresis. This method was tested on cultivated peanut and verified to give good fingerprinting results in other plant species (mango, banana, and longan). These single primers, designed from relatively conserved branch point signal sequences within gene introns, should be universal across other plant species. The method is rapid, simple, and efficient, and it requires no sequence information of the plant genome of interest. It could be used in conjunction with, or as a substitute for, conventional RAPD or ISSR techniques for applications including genetic diversity analysis, phylogenetic tree construction, and quantitative trait locus mapping. This technique provides a new way to develop molecular markers for assessing genetic diversity of germplasm in diverse species based on conserved branch point signal sequences.     

Tandemly Repeated Sequences in the Mitochondrial DNA Control Region and Phylogeography of the Pike-PerchesStizostedion

DNA sequences from the mitochondrial DNA control region are used to test the phylogeographic relationships among the pike-perches,Stizostedion(Teleostei: Percidae) and to examine patterns of variation. Sequences reveal two types of variability: single nucleotide polymorphisms and 6 to 14 copies of 10- to 11-base-pair tandemly repeated sequences. Numbers of copies of the tandem repeats are found to evolve too rapidly to detect phylogenetic signal at any taxonomic level, even among populations. Sequence similarities of the tandem repeats amongStizostedionand other percids suggest concerted evolutionary processes. Predicted folding of the tandem repeats and their proximity to termination-associated sequences indicate that secondary structure mediates slipped-strand mispairing among the d-loop, heavy, and light strands. Neighbor-joining and maximum parsimony analyses of sequences indicate that the genus is divided into clades on the continents of North America and Eurasia. Calibrating genetic distances with divergence times supports the hypothesis thatStizostediondispersed from Eurasia to North America across a North Pacific Beringial land bridge approximately 4 million years before present, near the beginning of the Pliocene Epoch. The North AmericanS. vitreumandS. canadenseappear separated by about 2.75 million years, and the EurasianS. luciopercaandS. volgensisare diverged by about 1.8 million years, suggesting that speciation occurred during the late Pliocene Epoch.     

Stability of Tandemly Repetitive Subelement PCR Patterns in Trichophyton rubrum over Serial Passaging and with Respect to Drug Pressure

Trichophyton rubrum is the most significant agent of dermatomycoses worldwide, primarily causing tinea pedis and tinea unguium. PCR analysis of tandemly repetitive subelements (TRS) within the rDNA nontranscribed spacer region is a major tool for molecular typing of T. rubrum. The aim of this study was to investigate the stability of TRS PCR patterns by analyzing isogenic strains of T. rubrum. Twenty-seven groups of isogenic T. rubrum strains were examined, each composed of an original clinical isolate and its 3 subcultures, maintained on a drug-free medium, a medium containing fluconazole and itraconazole. TRS typing was performed for the original strains and their subcultures grown after 12 passages, at 4-week intervals, on respective media. To add more objectivity to the results, TRS typing for each of the isogenic strain was performed three times, using DNA isolated from three different colonies. Among 27 groups of isogenic strains, all but one were exclusively composed of strains with identical TRS-1 and TRS-2 PCR patterns. In one group, 3 isolates from the last, twelfth passage had identical TRS-1 PCR profiles (type 1), yet different TRS-2 PCR profiles, as compared with the original strain (type I vs. type II). The mechanism underlying the genotype switch was a deletion of a single repeat unit in the TRS-2 locus, as evidenced by sequence analysis. In the interpretation of TRS typing results, microevolutionary events need to be taken into account, urging drawing epidemiological conclusions with caution and in conjunction with other genotyping data and traditional contact tracing information.     

Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The fusion of two fragments by this method takes less than 0.5 day. The method is expected to facilitate various kinds of complex genetic engineering projects with enhanced efficiency.     

Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences

Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer’s specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and ecological niche of Thaumarchaeota.     

Use of Repetitive DNA Sequences and the PCR To Differentiate Escherichia coli Isolates from Human and Animal Sources

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.     

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V₃, V₃-V₅, and V₆-V₈ hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V₃ and V₃-V₅ primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.     

A Universal Method for the Identification of Bacteria Based on General PCR Primers

The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.     

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Characterization of Nocardia asteroides Isolates from Different Ecological Habitats on the Basis of Repetitive Extragenic Palindromic-PCR Fingerprinting

Thirteen isolates of Nocardia asteroides from both soils and aquatic samples (lake and moat sediments, as well as scum from activated sludge), together with a type strain and two known clinical isolates of this species, were characterized by repetitive extragenic palindromic-PCR fingerprinting with the BOX-A1R primer. The resulting DNA fingerprint patterns proved to be strain specific, and cluster analysis distinguished the soil isolates, the aquatic isolates, and the known strains as being in separate groups.     

Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of the Cryptosporidium genome were evaluated for the detection of C. parvum, the agent of human cryptosporidiosis, and C. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridis couldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.     

Rapid Diversification of Marine Picophytoplankton with Dissimilar Light-Harvesting Structures Inferred from Sequences of Prochlorococcus and Synechococcus (Cyanobacteria)

Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions. Received: 18 January 1997 / Accepted: 18 May 1997     

Identification of Burkholderia andropogonis with a Repetitive Sequence BOX Element and PCR

Burkholderia andropogonis is the causal agent of bacterial leaf stripe in sorghum. Strict import quarantine regulations of numerous countries mandate a need for development of a rapid, reliable, and cost-effective diagnostic technique for the identification of B. andropogonis. Using a primer corresponding to the bacterial repetitive BOX element and PCR, we developed a DNA fingerprint which differentiated B. andropogonis from other phytobacterial pathogens.     

Molecular Typing of Staphylococcus epidermidis and Other CNS with Repetitive Element Sequence-Based PCR

The chromosomal distribution of the repetitive DNA sequence found in Mycoplasma pneumoniae (REP-MP2) provides an ideal target for detecting DNA fragment patterns specific to individual Staphylococcus epidermidis and S. haemolyticus strains. A REP-MP2 sequence-based PCR (rep-PCR) was developed and applied to CNS isolates. We identified a 450 bp genomic DNA fragment which was common and specific to S. epidermidis isolates and not found in other CNS. In addition, S. epidermidis isolates showed several bands that could be grouped into 14 different fragment patterns. Similarly, S. haemolyticus isolates were classified into 10 groups. Significant correlations between the typing patterns of S. epidermidis and resistance to oxacillin (P<0.05), gentamicin (P<0.01), erythromycin (P<0.02), and sulfamethoxazole-trimethoprim (P<0.001) were found. The rep-PCR method is a rapid and reproducible discriminatory means for molecular typing of S. epidermidis and other CNS.     

Comparative and Evolutionary Analysis of the Cytochrome b Sequences in Cyprinids with Different Ploidy Levels Derived from Crosses

The mitochondrial cyt b genes in the allotetraploid and triploid crucian carp as well as triploid common carp were isolated and completely sequenced. Their DNA sequences were compared with those derived from the cyt b genes of the red crucian carp, Japanese crucian carp, and common carp with MEGA 1.0 software. Phylogenetic analysis revealed the sister relationships between allotetraploid and diploid red crucian carp, between the triploid crucian carp and diploid Japanese crucian carp, and between triploid common carp and diploid common carp. Our results indicated the cyt b genes in the allotetraploid, triploid crucian carp, and triploid common carp were maternally inherited. Through maternal inheritance, the cyt b gene in the F11 tetraploid displayed extremely high similarity to that in the female parent red crucian carp after 11 generations (from F1 to F11 hybrids). Since the establishment of the new tetraploid stocks has great significance in analyzing evolutionary theory of vertebrate and in improving aquaculture industry, analysis of the cyt b gene and the elucidation of the variation of the cyt b gene DNA in different cyprinids prove that cyt b is a useful genetic marker to monitor the variations in the progeny of the crosses.     

Sensitive and Specific Detection of Pseudomonas avellanae using Primers based on 16S rRNA Gene Sequences

A rapid polymerase chain reaction (PCR)-based procedure was developed for the detection of Pseudomonas avellanae , the causal agent of hazelnut ( Corylus avellana ) decline in northern Greece and central Italy. The partial sequence of the 16S rRNA gene of P. avellanae strain PD 2390, isolated in central Italy, was compared with the sequence coding for the same gene of P. syringae pv. syringae type-strain LMG 1247_t1. Primers PAV 1 and PAV 22 were chosen, and after the PCR, an amplification product of 762 base pairs was specifically produced only by 40 strains of P. avellanae isolated from northern Greece and central Italy. No other bacterial species among those tested showed an amplification product under optimized PCR conditions. The adding of 4% BLOTTO (10% skim milk powder and 0.2% NaN₃) in the PCR mixture proved essential in order to avoid interference of hazelnut extracts during the amplification. The procedure proved more effective than repetitive PCR with ERIC primer sets in diagnosing apparently healthy hazelnut trees as infected. This technique could be of great help for screening the hazelnut propagative material as well as for monitoring the wild C. avellana trees growing in the woods near the infected hazelnut orchards.