Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction.

Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase enzymes of marine species have shown that these proteins have diverged to the point where they have only short regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae bv. albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods.

The polymerase chain reaction was used to selectively amplify sequences within the cholera toxin operon from Vibrio cholerae O1. Oysters, crabmeat, shrimp, and lettuce were seeded with V. cholerae and then homogenized or washed with alkaline peptone water, followed by short-term (6- to 8-h) enrichment. A detection limit of as few as 1 V. cholerae CFU per 10 g of food was obtained with amplification reactions from crude bacterial lysates. The method is extremely rapid and obviates the need for DNA isolation from a variety of complex food matrices.     

Cholera toxin gene polymerase chain reaction for detection of non-culturable Vibrio cholerae O1

Cholera enterotoxin is a major antigenic determinant for virulence of Vibrio cholerae O1 which can enter into a viable but non-culturable (N-C) state, not detectable by conventional culture methods, yet remain capable of producing enterotoxin and potentially pathogenic. PCR was applied in the current study to detect the chilera toxin (ctx) gene of N-C cells, thus eliminating the necessity of culture. Sets of oligonucleotide primers were designed, based on the ctxAB operon of V. cholerae O1, to detect the presence of the ctx gene. DNA from both culturable and N-C cells of V. cholerae O1 was amplified by PCR using sets of primers flanking 302-, 564- and 777-bp fragments of the ctx gene. The PCR method employed was capable of detecting the ctx gene in N-C V. cholerae in aquatic microcosms and in diarrheal stool samples from three patients who had distinct clinical symptoms of cholera but were culture-negative for V. cholerae O1 and non-O1 and enterotoxigenic Escherichia coli. Forty cycles of a two-step reaction (30 s each at 94 and 60°C) were optimal and more time efficient than a three-step PCR described previously. The procedure, from the point of heating microcosms or broth culture samples to observation on gels, requires < 4 h to complete.J.A.K. Hasan, A. Huq, M.A.R. Chowdhury, and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD, USA. M. Shahabuddin is with the National Institute of Health. Bethesda, MD, USA. L. Loomis is with New Horizons Diagnostics Corporation, Columbia, MD, USA.     

Detection of beta-globin gene mutations by polymerase chain reaction.

The polymerase chain reaction and oligonucleotide probe hybridization technique were applied to the detection of two common mutations of the beta-globin gene found in Chinese, namely the 4-base pair deletion at the 41-42 codons and the C to T substitution at nucleotide 654 of IVS-2. The accuracy of the method was established using beta-thalassemia cases with known mutations or haplotypes of the restriction fragment length polymorphism (RFLP). A further 11 cases of thalassemia intermediate and thalassemia minor were then analysed with the same approach. Our results showed that 5 of the 11 cases carried the TCTT-deletion at codons 41-42. Our method is economical both in terms of materials and time needed and in an alternative to the use of the molecular RFLP approach in the prenatal diagnosis of beta-thalassemia.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Detection and differentiation of the gene for toxin co-regulated pili (tcpA) in Vibrio cholerae non-O1 using the polymerase chain reaction

Abstract The polymerase chain reaction has been used to differentiate the gene which encodes the toxin co-regulated pili ( tcpA ) of the El Tor and classical biotypes of Vibrio cholerae O1. The same PCR primers were applied to strains belonging to non-O1 serogroups that produced cholera toxin. The size of fragment amplified was either identical to the tcpA of biotype El Tor (471 bp) or to the tcpA of biotype classical (617 bp). All strains belonging to the novel epidemic serogroup O139 generated a 471-bp fragment identical to El Tor tcpA . The present study suggests that there may be an association between non-O1 serogroup and tcpA type.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Study of the epidemic significance of noncultivated forms of Vibrio cholerae by the polymerase chain reaction]

A specific method of the isolation of the cholera toxin gene by the directional amplification of DNA in the polymerase chain reaction (PCR) has been developed. The product of this reaction has a molecular weight of 440 sequence pairs and is a DNA fragment located on the A-subunit of V. cholerae gene vct. The sensitivity of the method permits the detection of one bacterial cell in the reaction mixture. The method is effective when V. cholerae purified DNA, cell lysates and the DNA of total microflora isolated from the water of natural springs are used. The study of water samples from natural water bodies by the method of PRC has revealed cholera toxin genes of V. cholerae noncultivated forms ni 5 out of 7 water samples taken from natural water bodies at the regions of Azerbaijan endemic for cholera and made it possible to evaluate the number of V. cholerae. The prospects of using PCR for the control of the epidemiological situation in regions endemic for cholera are discussed.     

Detection of Listeria monocytogenes by polymerase chain reaction oriented to inlB gene.

Primers were designed for the detection of Listeria monocytogenes by the polymerase chain reaction oriented to specific sequences of the inlB gene encoding an internalin. At optimized reaction conditions, 100% sensitivity (on a panel of 33 strains of L. monocytogenes) and 100% specificity (on panels of 15 strains of other Listeria spp. and 41 other bacteria), were determined for the inlB-L/R primers. The detection limit of PCR with these primers was 10(4) cfu/ml and was not affected by up to 10(8) cfu/ml of L. innocua.     

Detection of Nitrosomonas spp. by polymerase chain reaction

Abstract A unique genomic DNA fragment was isolated from Nitrosomonas europaea ATCC 19718. Based on the sequence of this fragment, oligonucleotide primers for polymerase chain reaction amplification were prepared which amplify sequences of 775 and 658 bp. The predicted DNA fragments were both amplified from the genome of N. europaea and a Nitrosomonas spp. isolated from a local oxidation pond. The primers failed to amplify DNA from the genomes of the ammonia oxidiser Nitrosolobous multiformis , the nitrite oxidiser Nitrococcus mobilis as well as from the genomes of other unrelated heterotrophic bacteria. These DNA sequences could be amplified from 0.01 ng of N. europaea genomic DNA or from 100 intact cells, and it was possible to detect Nitrosomonas DNA in a DNA mixture extracted from water samples drawn from a local oxidation pond.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

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.     

Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes

The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3×10⁸ molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time.

Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.     

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.