The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

Mycobacterium species identification--a new approach via dnaJ gene sequencing

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.     

Quantitative detection of Sphingomonaschlorophenolica in soil via competitive polymerase chain reaction

The 16S ribosomal RNA gene sequence of the pentachlorophenol degrader Sphingomonas chlorophenolica strain RA2 was used to generate specific polymerase chain reaction (PCR) primers for the detection of this strain in soil, whereas a region internal to the two primers was used to provide an S. chlorophenolica strain RA2-specific oligonucleotide probe. The PCR detection system resulted in a 727 bp product detectable via gel electrophoresis and hybridization. It was specific for strain RA2 and its close relative, S. chlorophenolica ATCC 39723, as evidenced by PCR amplifications of a range of bacterial genomic DNAs. Tests of total microbial community DNA obtained from five uninoculated and two RA2-inoculated soils confirmed this specificity for introduced S. chlorophenolica RA2. Strain RA2 could be detected in soil down to a level of 10³ cfu g⁻¹ soil. Two strategies were followed to generate internal standard DNA for competitive PCR. First, a 479 bp MIMICS fragment was obtained based on a previously constructed gene cassette; however, this standard did not reliably quantify RA2 targets. Low stringency PCR performed with a range of bacterial genomic DNAs resulted in the generation of an amplicon with a Paenibacillus azotofixans strain that was slightly smaller than the RA2-derived product. Both products were easily separable via conventional gel electrophoresis. The use of this competitor in a threefold dilution scheme applied to the target DNA allowed for the quantitative detection of RA2-specific target DNA molecules from pure culture and from soil. The fate of strain RA2 in pentachlorophenol-contaminated soil was described using this competitive PCR approach, and the organism was shown to persist at two inoculum levels over prolonged periods of time.     

Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Identification of microorganisms by PCR amplification and sequencing of a universal amplified ribosomal region present in both prokaryotes and eukaryotes

The small ribosomal subunit contains 16S rRNA in prokaryotes and 18S rRNA in eukaryotes. Even though it has been known that some small ribosomal sequences are conserved in 16S rRNA and 18S rRNA molecules, they have been used separately for taxonomic and phylogenetic studies. Here, we report the existence of two highly conserved ribosomal sequences in all organisms that allow the amplification of a zone containing approximately 495 bp in prokaryotes and 508 bp in eukaryotes which we have named the "Universal Amplified Ribosomal Region" (UARR). Amplification and sequencing of this zone is possible using the same two universal primers (U1F and U1R) designed on the basis of two highly conserved ribosomal sequences. The UARR encompasses the V6, V7 and V8 domains from SSU rRNA in both prokaryotes and eukaryotes. The internal sequence of this zone in prokaryotes and eukaryotes is variable and the differences become less marked on descent from phyla to species. Nevertheless, UARR sequence allows species from the same genus to be differentiated. Thus, by UARR sequence analysis the construction of universal phylogenetic trees is possible, including species of prokaryotic and eukaryotic microorganisms together. Single isolates of prokaryotic and eukaryotic microorganisms from different sources can be identified by amplification and sequencing of UARR using the same pair of primers and the same PCR and sequencing conditions.     

Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.     

Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence.

A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana. One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units. The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library. New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved among the different repeated units. Alignment of the consensus sequence for each minisatellite locus allowed the definition of an Arabidopsis thaliana core sequence that shows strong sequence similarities with the human core sequence and with the generalized recombination signal Chi of Escherichia coli. The minisatellites were tested for their ability to detect polymorphism, and their chromosomal position was established.     

Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates

The genome of the Bacillus subtilis 168-type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI-H-G and rrnJ-W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168-type strain and B. subtilis 168-like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168-type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula , Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.     

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.