A rapid and direct real time PCR-based method for identification of Salmonella spp

The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated through a four times repeated blind experiment performed in two different laboratories including 50 Salmonella spp. with representative strains from each of the 5 different Salmonella subgenera and 30 non-Salmonella strains. Both parameters DeltaR(n) (fluorescence intensity of template through a normalized reporter value) and C(T) (cycle at which the fluorescence intensity achieved a pre-established threshold) were analyzed. Overall mean DeltaR(n) and C(T) values for Salmonella strains (2.14+/-0.87 and 15.30+/-0.90, respectively) were statistically different from values for non-Salmonella strains, allowing the establishment of cut-off DeltaR(n) and C(T) values based on 95% confidence intervals that allowed the correct identification of all strains tested in each independent experiment. The accuracy of this assay in terms of inclusivity and exclusivity was 100%. Moreover, the PCR system proved to be especially convenient because the pre-mix containing all PCR reagents except for the bacterial cells could be kept at -20 degrees C for at least 1 month before its use. The optimized TaqMan real-time PCR assay is a useful, simple and rapid method for routine identification of Salmonella spp., irrespective of the particular subgenus.     

A rapid test for the presumptive identification of Clostridium perfringens

A novel rapid method for the identification of colonies of Clostridium perfringens (key iD Lab M Ltd. Bury, UK) was evaluated. The method consists of a test strip containing substrates for pre-formed enzymes selected for optimum differentiation of C. perfringens from other clostridia. One hundred and forty-six strains of clostridia were tested using the key iD strip. The strip successfully confirmed the identity of all 73 strains of C. perfringens tested, and differentiated these from 73 strains of 20 other clostridial species. C. absonum and C. baratii, spedes which are very similar to C. perfringens, could also be differentiated by this method. The key iD strip is recommended for laboratories as a rapid alternative to more conventional tests for presumptive identification of C. perfringens.     

A rapid urease test for presumptive identification of Cryptococcus neoformans

A rapid method to evidence urease activity is described. Urea hydrolysis and consequent production ammonia are detected by a chemical reaction producing a blue phenol compound (indophenol blue). Three hundred and three yeast were tested. Out of 107 urease-positive organisms detected by Christensen's Urea Agar Test (CUAT) 102 were positive by our method. No false negatives were observed by this method when testing 87 Cryptococcus strains. Ths practical screening test for presumptive identification of Cryptococcus neoformans is simple, unaffected by pH changes and requires 15 minutes to be performed.     

A novel simple and rapid PCR-based site-directed mutagenesis method

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5′ end of one of the primers, whereas the other primer starts with the nucleotide at position (−1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat α integrin CD11b A-domain and the human CD8β cloned in pPICZαB, pGEX-2T, and CDM8 expression vectors, respectively.     

A rapid PCR-based approach for molecular identification of filamentous fungi

In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates     

A rapid PCR-based method for the identification of ob mutant mice

With increasing incidence of obesity, there is greater demand for suitable research and therapeutic models. The ob/ob mouse model develops obesity by 5 weeks of age. Previously, a method using DNA purification, PCR, and restriction digestion of products was devised to identify mice bearing the ob allele. Here, we describe a direct PCR method that requires no DNA purification. Wild-type and ob-specific primers are used under the same conditions in two separate and simultaneously run three-primer PCRs. Standard PCR using the wild-type primer mix produces 191 bp and 104 bp bands in +/+ and ob/+ and only the control 191 bp band in ob/ob animals. The ob-specific primer reaction produces 191 bp and 123 bp bands in ob/+ and ob/ob and only the control 191 bp band in +/+ animals. Phenotypic weight gain in offspring of heterozygous intercrosses was used to validate genotypes. This primer-specific PCR method allows simultaneous identification of +/+, ob/+, and ob/ob genotypes prior to breeding age to facilitate breeding and research studies in an important model of clinical obesity.     

A rapid PCR-based method for identification of four important Candida species

The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidosis. Currently available culture and biochemical methods for detection and identification of Candida species are time-consuming. This study describes the use of a simple and rapid PCR method using species-specific oligonucleotides for the detection of clinical isolates of Candida species. These species-specific oligonucleotides are complementary to unique sequences within the intergenic transcribed spacer 2, located in between the 5.8S and 28S ribosomal DNA, and generated DNA fragments by both the conventional and hemi-nested PCR reactions. Conventional PCR produced a single DNA fragment of variable size in all isolates, while the hemi-nested PCR produced two discrete DNA fragments, both with the expected sizes of 111bp/57bp (C. albicans), 84bp/42bp (C. glabrata), 94bp/45bp (C. krusei) and 95bp/49bp (C. parapsilosis). In conclusion, the PCR-based method described in this study is fast and specific for the identification of clinically important Candida species.     

Improved strategy for presumptive identification of methanogens using 16S riboprinting

The predicted 16S riboprint patterns of 10 restriction endonucleases for 26 diverse methanogens were compared to actual patterns produced on agarose gels. The observed patterns corroborated the expected riboprints. Our analyses confirmed that the endonuclease HaeIII gave the best results generating 15 different riboprint sets. Six of these 15 riboprints represented more than one strain. Of these, three riboprint sets were further differentiated: Methanomicrobium mobile, Methanolacinia paynteri, and Methanoplanus petrolearius were differentiated from each other by the endonuclease AluI; Methanofollis liminatans, Methanospirillum hungatei, and Methanoculleus bourgensis were differentiated from each other by HpaII; and the combination of FokI and MluNI was used to differentiate Methanobrevibacter sp. ZA-10, and Methanobrevibacter arboriphilicus strains DH-1, AZ, and DC from each other. We could not differentiate the following pairs of strains from each other: Methanosarcina mazeii S-6 and C16, Methanobacterium bryantii MoH and MoH-G, Methanobacterium thermoautotrophicum GC-1 and DeltaH, and Methanobrevibacter arborophillicus DC and A2. This riboprint strategy provided a simple and rapid method to presumptively identify 22 of the 26 diverse strains of methanogens belonging to 13 genera from a range of environments.     

A new method for rapid identification of influenza virus isolates.

With the use of bacteria sensitized by influenza virus strain-specific antisera, virus isolates can be identified rapidly. One drop of virus suspension is mixed with one drop of sensitized bacteria on a slide that is then agitated; reaction occurs within 10 minutes. The test is subtype-specific. The mehod is based on the fact that the cell wall of the Cowan type 1 strain of Staphylococcus aureus contains abundant quantities of an antigen, known as protein A, that reacts with the IgG molecule by binding it in such a manner that the antibody-combining sites remain free. If an antigen homologous to the antibody coated on the surface of the bacteria is added to the suspension of sensitized staphylococci, agglutination occurs.     

Application of pyrosequencing for Salmonella enterica rapid identification

Application of pyrosequencing of six Salmonella-specific genes as a rapid Salmonella identification method was tested. Primers for hns, hisJ and hilA had non-specific reactions with non-Salmonella strains. Primers for invA, iroB and fimY had specific PCR products and pyrosequences of Salmonella, suggesting that they were suitable for Salmonella rapid identification.     

Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

A simple and rapid method for the differentiation and identification of thermophilic bacteria

In total, 170 strains of thermophilic bacteria were isolated from deep-sea hydrothermal fields in the Pacific Ocean and a hot spring in Xiamen of China. To facilitate the identification of thermophilic strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins of these strains was first performed. The results showed that there exist four different protein patterns, indicating that the 170 strains might belong to four species or genera. The RAPD (random amplified polymorphic DNA) profiles of nine representative strains were consistent with those of SDS-PAGE. To further identify the species of the nine strains, their 16S rDNA sequences were analyzed. The results showed that the nine strains fell into four species of three genera, which was the same as revealed by SDS-PAGE. Therefore, SDS-PAGE of whole-cell proteins could be used as a rapid and simple method for the discrimination of thermophilic bacteria as the first step of species identification.     

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are ~500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5–7 days) and suitable for synthesizing long segments of DNA (5–6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.     

A rapid PCR-based method for genetically mapping ESTs

A simple, semi-automatable procedure was developed for converting expressed sequence tags (ESTs) into mappable genetic markers. The polymerase chain reaction is used to amplify regions immediately 5′ or 3′ to the coding regions of genes in order to maximise sequence variability between alleles. Fragment length and nucleotide substitution polymorphisms among amplified alleles can be detected using either ethidium bromide staining or automated laser-based fluorescence. A 6% non-denaturing acrylamide gel, analysed with an ABI 377 DNA sequencer, proved capable of resolving homoduplexes and heteroduplexes formed between amplified alleles containing nucleotide substitutions as well as resolving allelic length differences. With this approach 75% of 60 ESTs from a range of Pinus species could be genetically mapped in each of three pedigrees from P. radiata and P. taeda. Furthermore, three or four alleles were detected in each pedigree for 42% of the EST markers. Received: 4 January 2000 / Accepted: 26 May 2000