ABSTRACT
A polymerase chain reaction (PCR) method for direct detection of Salmonella spp. in whole-shell eggs is described. The method does not require isolating strains. Preenrichment, rapid DNA isolation, and a nested PCR system targeting the invA gene enabled detection of the genus Salmonella. The specific nested PCR product of 283 base pairs was formed from all 21 serovars, including 43 Salmonella strains tested. No PCR product was formed from 56 non-Salmonella enterobacteriacea and other bacterial strains tested. Experiments with artificially contaminated eggs showed a detection limit of about 10(3)-10(4) colony-forming units (cfu)/egg before and about 1-10 cfu/egg after preenrichment. In analysis of 180 single eggs from 4 flocks and 36 pools of 5 eggs each from another 4 flocks from the same producer, Salmonella spp. were detected in 5 of 90 eggs from 2 different flocks. Determination of anti-Salmonella antibodies in eggs yielded positive results for 2 additional and the 2 PCR-positive flocks. In contrast, classical selective culture detected Salmonella spp. in only 1 of 100 eggs in one flock when 100 eggs from each flock were analyzed.
Subject(s)
DNA/analysis , Eggs/microbiology , Food Microbiology , Salmonella/isolation & purification , Bacteriological Techniques , Base Sequence , Food Analysis , Molecular Sequence Data , Polymerase Chain Reaction , Sensitivity and Specificity , Time FactorsABSTRACT
We have developed a simple, fast, and efficient procedure to detect enteroviruses in water samples. Aliquots of water are subjected to two-step filtration, with the second filter containing a positively charged nylon membrane that holds back virus particles. Viruses thus adsorbed are directly lysed, and RNA is isolated by hybridization to specific oligonucleotides bound to magnetic beads. The solution used contains guanidine thiocyanate, which lyses virus particles, inactivates enzymes, e.g., RNases, allows mild hybridization conditions, and does not influence biotin-streptavidin interaction on magnetic beads. Detection and specific identification are accomplished by reverse transcription PCR of the highly conserved noncoding region at the 5' end of virus RNA combined with Southern hybridization. The system was tested with tap water artificially spiked with poliovirus vaccine and yielded a detection limit of 20 50% tissue culture infective doses per liter. We used the same procedure to investigate the water quality of surface water at public beaches by rivers and lakes. Of 40 samples tested, 7 were positive for enteroviruses. A comparison with enterobacterial contamination determined by PCR and classical microbiological methods in parallel showed that enteroviruses were found only in samples also positive for Escherichia coli. In conclusion, this procedure can easily be adapted to test large water samples and is simple enough to be used for routine determinations of water quality in terms of virus contamination.
Subject(s)
Enterovirus/genetics , Enterovirus/isolation & purification , Polymerase Chain Reaction/methods , Water Microbiology , Base Sequence , DNA Primers/genetics , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Viral/genetics , DNA, Viral/isolation & purification , Enterovirus Infections/prevention & control , Escherichia coli/genetics , Escherichia coli/isolation & purification , Evaluation Studies as Topic , Fresh Water , Humans , Molecular Sequence Data , Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Transcription, Genetic , Water Supply/standardsABSTRACT
The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.
Subject(s)
DNA, Bacterial/isolation & purification , Food Microbiology , Listeria monocytogenes/isolation & purification , Meat Products/microbiology , Polymerase Chain Reaction/methods , Bacterial Typing Techniques , Base Sequence , DNA, Bacterial/genetics , Enterobacteriaceae/classification , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Evaluation Studies as Topic , Food Contamination , Food Handling , Listeria monocytogenes/classification , Listeria monocytogenes/genetics , Molecular Sequence Data , Oligonucleotides , Polymorphism, GeneticABSTRACT
Plasma produced from ultraviolet-transmitting solid targets undergoing intense (>10(16) W/cm(2)) subpicosecond (~600 fs) ultraviolet (248 nm) irradiation have been studied under conditions for which no interfering prepulse plasma is generated. Time and spatially integrated measurements of the x-ray emission for H-like and He-like Mg and Si were found to be consistent with LASNEX calculations that model the laser-target interaction.
