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PubMed1999

Simian virus 40 (SV40)-like DNA sequences not detectable in finnish mesothelioma patients not exposed to SV40-contaminated polio vaccines.

Occupational asbestos exposure can be demonstrated in 80% of mesothelioma cases. A possible role of simian virus 40 (SV40) in the etiology of mesothelioma was raised because several studies reported the presence and expression of SV40-like DNA sequences in human mesotheliomas. It is also known that expression of SV40 large T antigen inhibits cellular Rb and p53. This suggests that SV40 might render infected cells more susceptible to asbestos carcinogenicity. The SV40-like sequences are suggested to have arisen from contaminated polio vaccines. Millions of people in the United States and most European countries were inoculated with SV40-contaminated polio vaccine in 1955-1963. However, in Finland, where polio vaccination started in 1957, no SV40-contaminated vaccine was used. We used a polymerase chain reaction-based method to test for the presence of SV40-like sequences in DNA extracted from the frozen tumor tissues of 49 Finnish mesothelioma patients, most of whom had been occupationally exposed to asbestos. All of the Finnish tumor tissues tested negative for SV40-like sequences. The results suggest that the SV40-like sequences detected in mesothelioma tissue in some previous studies may indeed originate from SV40-contaminated polio vaccines. It is a matter of speculation whether the absence of SV40 infection has contributed to the relatively low incidence of mesothelioma in Finland (1/10(5) in 1990-1995).

Adult↗

PubMed2000

Rapid detection of porcine reproductive and respiratory syndrome viral nucleic acid in blood using a fluorimeter based PCR method.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus recognised world wide as an important cause of reproductive failure and pneumonia in pigs. American and European strains of PRRSV, differentiated antigenically and genomically, have been reported. PRRSV infections are currently diagnosed using serology, virus isolation and/or immunocytochemistry. In order to overcome various drawbacks associated with these techniques, conventional, block-based RT-PCR methods for the detection of PRRSV nucleic acid in clinical samples have been described. These methods require gel electrophoresis for analysis of PCR products and present high risk of DNA carry-over contamination between the samples tested. We describe the detection of PRRSV RNA in serum samples and in blood impregnated filter disks (FDs), obtained from experimentally inoculated pigs, using a closed-tube, fluorimeter-based PCR assay. The assay eliminates the use of gel electrophoresis, and is as sensitive and specific as the conventional block-based PCR assay, detecting positive samples as early as 1 day post-inoculation. We also report a rapid fluorimeter based PCR method for differentiating American and European strains of PRRSV.

Animals↗

PubMed1994

Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (< 1-62% of the unmodified 3'-nucleotides being dephosphorylated) were used to hydrolyze the DNA. The exonuclease with the lowest phosphatase activity produced a recovery of up to 9.60 mumol of benzo[a]pyrene adducts per mole of DNA. Recovery of benzo[a]pyrene adducts was reduced to 0.56 mumol of adduct per mole of DNA using the exonuclease with the highest phosphatase activity. Phosphatase in the exonucleases also dephosphorylated N-hydroxy-2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling.

2-Naphthylamine↗

PubMed2004

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR.

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular 'batch-stamps' that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes.

Base Sequence↗

PubMed1986

The metabolic activation and DNA adducts of dinitropyrenes.

Dinitropyrenes are contaminants in diesel emissions that are mutagenic in bacteria and mammalian cells, and tumorigenic in laboratory animals. In this project, we investigated the factors that contributed to the extreme genotoxicity of dinitropyrenes in bacteria and determined if these factors were important in mammalian cells. Xanthine oxidase, a mammalian nitroreductase, catalyzed the conversion of the dinitropyrenes to DNA-bound products, but the level of binding did not exceed that observed with 1-nitropyrene. This suggested that factors in addition to nitroreduction were important in the metabolic activation of dinitropyrenes. 1-Nitro-6-nitrosopyrene and 1-nitro-8-nitrosopyrene were synthesized and reacted with DNA under reducing conditions. The same C8-substituted deoxyguanosine adducts were formed that were found in the xanthine oxidase-catalyzed reactions, which confirmed that incubation with this nitroreductase generated reactive N-hydroxy arylamine intermediates. In incubations with rat and human liver microsomes and cytosol, 1-nitropyrene and 1,3-dinitropyrene were reduced to a lesser extent than 1,6- and 1,8-dinitropyrene, which was in accord with their relative mutagenicities. Each of the cytosolic incubations were similar in that oxygen decreased aminopyrene, but not nitrosopyrene, formation. The data indicated that reduced derivatives of the nitrosopyrenes were redox cycling with oxygen, which decreased cytosolic aminopyrene formation. In cytosolic incubations, oxygen inhibited the reduction of 1-nitropyrene and 1,3-dinitropyrene to a greater extent than 1,6- and 1,8-dinitropyrene. By comparison, in microsomal investigations, the nitroreduction of each nitrated pyrene was equally oxygen-sensitive. This apparently was caused by the initial nitroanion radicals reacting with oxygen to decrease nitrosopyrene formation. Although more extensive nitroreduction of each compound was detected in anaerobic incubations, aerobic reduction of these compounds did occur and may be important during in vivo exposure to nitrated pyrenes. When rat liver cytosol was incubated with the nitrated pyrenes, very low levels of DNA binding were detected. Addition of acetyl coenzyme A (AcCoA) to these incubations increased the binding of the dinitropyrenes 20- to 40-fold, while the binding of 1-nitropyrene was not affected. The extent of AcCoA- dependent binding of the dinitropyrenes reflected the amount of nitroreduction; however, the increase in binding did not occur with dog liver cytosol, which was known to be deficient in N-acetylases. These results indicated that cytosolic nitroreductases catalyzed the formation of N-hydroxy arylamine intermediates, which in the case of dinitropyrenes were converted to reactive N-acetoxy arylamines by cytosolic AcCoA-dependent acetylases.

Animals↗

PubMed1995

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp.

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a "comet with tail" formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants.

Animals↗

PubMed1996

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.

Base Sequence↗

PubMed1995

Alternative methods of preparing whole-cell DNA from fungi for dot-blot, restriction analysis, and colony filter hybridization.

There is a large and increasing number of methods for preparing whole-cell DNA from fungi. Modifications have evolved for two reasons. This first is to simplify the protocol as much as possible to allow processing of large sample numbers, in some cases for very specific uses, e.g., dot-blots. The second is to increase the quality of the DNA. Most preparations are contaminated with varying amounts of polysaccharides and unknown wall contaminants that can inhibit subsequent restriction or ligation. The extent of contamination varies with the species, the individual isolate, and at least in Neurospora, with the method or extent of growth. This paper offers three new methods. The first is a simplified procedure for isolating denatured DNAs from filamentous fungi for dot-blot analysis. The second is a rapid method for isolating DNAs from large numbers of small- to medium-scale cultures of filamentous fungi. These preparations are sufficiently pure for a variety of enzymatic reactions. The third is a nonenzymatic method for yeast colony filter hybridization that is simple, inexpensive, and efficient and results in uniform signals for a variety of species.

Aspergillus↗

PubMed2002

ED(27) trophoblast-like cells isolated from first-trimester chorionic villi are genetically identical to HeLa cells yet exhibit a distinct phenotype.

ED(27) trophoblast-like cells were prepared from human chorionic villus samples obtained at 9 weeks gestation and have been grown continuously in vitro without phenotypic drift for nearly a decade. These cells express many trophoblast markers, including cytokeratin, placental alkaline phosphatase (PLAP), secretion of 17beta-estradiol, and a microvillous apical surface. The ED(27) cell line is a useful model system for studies of placental cell biology and has been distributed to laboratories world-wide. However, experiments to investigate their relationship to primary villous cytotrophoblast have shown that these cells do not secrete detectable amounts of human chorionic gonadotropin in culture and, when digested with trypsin, disperse into individual cells. Furthermore, immunocytochemical studies demonstrated that, unlike villous cytotrophoblasts, ED(27) cells were immunoreactive with monoclonal antibodies recognizing some HLA Class I antigens. This was not HLA-G, however, as would be expected if these cells originated from extravillous cytotrophoblasts, but rather classical HLA-A, B which is thought not to be expressed by any trophoblast subpopulations. These inconsistencies prompted us to question the authenticity of the continuous cell line as it now exists. Genetic haplotype analysis using the polymerase chain reaction (PCR) revealed that ED(27) was genetically identically to the HeLa cell line. Inasmuch as HeLa cells have never been grown in the laboratory (DAK), the only possible origin of HeLa cell contamination of ED(27) cells was the WISH cell line, and further PCR analysis revealed that this cell line was also genetically identical to HeLa. Like ED(27) cells, HeLa cells and WISH cells synthesized small amounts of estrogen and were found to express PLAP and antigens recognized by the monoclonal antibodies ED822, directed against the syncytiotrophoblast, and J1B5 directed against villous cytotrophoblast. These results point out the need for adherence to rigorous and consistent quality control measures to assure the authenticity of cell lines used as in vitro model systems.

Adult↗

PubMed1993

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.

Amino Acid Sequence↗

PubMed2006

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi.

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.

Animals↗

PubMed2005

Multicenter performance evaluation of a transcription-mediated amplification assay for screening of human immunodeficiency virus-1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA in blood donations.

BACKGROUND: The performance of the recently launched Procleix Ultrio (Chiron/Gen-Probe) human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) blood screening assay was evaluated in a European multicenter study. STUDY DESIGN AND METHODS: Serial dilutions of reference materials were tested to determine the detection limits. Robustness and specificity were assessed by testing alternating high-load HCV RNA-positive and -negative samples, and 2912 test pools of eight donations. The added value of minipool and single-donation HBV nucleic acid testing protocols was compared to the currently used Prism (Abbott GmbH & Co. KG) hepatitis B surface antigen (HBsAg) and Auszyme (Abbott GmbH & Co. KG) dynamic HBsAg tests in 15 HBV seroconversion panels. RESULTS: The 95 percent detection limits (and 95% confidence interval [CI]) on the WHO International Standards was 26 (16-58) IU per mL for HIV-1 RNA, 4.6 (3.7-6.5) IU per mL for HCV RNA, and 11 (7.3-22) IU per mL for HBV DNA. No cross-contamination was observed. Testing 2912 pools of eight donations revealed 16 initial reactive samples; 11 were confirmed. The specificity after initial testing and percentage of invalid results were 99.83 and 0.48 percent, respectively. The HBV window-period (WP) reductions relative to HBsAg seroconversion in Prism and Auszyme dynamic HBsAg were, respectively, 6 days (95% CI, 3-8) and 9 days (95% CI, 7-12) in 1:8 minipool (MP) testing. CONCLUSION: The performance characteristics of Procleix Ultrio assay and the Procleix HIV-1 and HCV assay are comparable. The sensitivity for HIV-1 and HCV met the directives of the Paul-Ehrlich Institute and the FDA. The assay can reduce the WP for HBV by 6 days to 2 weeks when used in small MP (<1:8) or single-donation screening protocols.

Blood Donors↗

PubMed1995

Improved simple generation of GTG-band specific painting probes.

We developed an improved, simple method of generating chromosome-region-specific probes from only a few microdissected chromosomes. One to five dissected fragments from a defined chromosomal region were processed with a PEG/proteinase K cycling deproteinization step and directly amplified with a two-step amplification system using a degenerate oligonucleotide primed shuttle polymerase chain reaction (DOP-Shuttle-PCR). This modified method offered three advantages over previously reported methods: relaxation of the highly condensed chromosomal DNA, reduction of the risk of endogenous and exogenous contamination, and high efficiency amplification of template DNA. High intensity in the fluorescence in situ hybridization (FISH) signals from normal metaphase chromosomes, as well as regional specificity of these probes, corresponding to regions on R-banded chromosomes, were observed.

Base Sequence↗

PubMed1981

Incorporation of [3H]thymidine into mtDNA of ram spermatozoa.

Freshly ejaculated spermatozoa from 6 rams incorporated [3H]-thymidine only after a lag period of 4 h. The thymidine incorporation was dependent on the time after ejaculation regardless of the rate of fructolysis, which was controlled by cold shock. The DNA synthesis period lasted for 3 h followed by a degradation period of 2 h. This synchronized level of labelling of DNA with tritiated thymidine was not due to bacterial contamination, sperm cell mitosis, mitochondrial division, or DNA repair. The fact that the maximal level of the DNA synthesis was dependent upon the external pH of the seminal plasma, in a parabolic fashion with a minimum around pH 6.3, indicated that this activity was of a 'metabolic' type. The sensitivity of the sperm DNA synthesis to 10 micrograms ethidium bromide/ml indicated that it was of a mitochondrial origin.

Animals↗

PubMed2003

Nitrogenase genes in PCR and RT-PCR reagents: implications for studies of diversity of functional genes.

Studies of the diversity of microorganisms in the environment have been facilitated by use of PCR and reverse transcription PCR (RT-PCR). Inhibition of the PCR by complex sample matrices and low abundance of some target microorganisms require the use of high-sensitivity amplification procedures, involving a large number of cycles or nested PCR methods. Using these methods, we frequently observed contamination of the amplification reagents, including polymerases, by genomic DNA containing nitrogenase (nifH) and rRNA genes. Contaminating genes were sequenced and found to belong to a variety of rRNA clades, but only three major nifH clades. These sequence types included a few nifH sequences reported in previous studies of the environment. Contamination could be reduced by restriction digestion and ultrafiltration of PCR reagents, but efficiency of amplification was also reduced. Our results suggest that studies relying on large numbers of PCR amplification cycles to assess environmental gene diversity should take precautions to assure that clone libraries generated from amplified PCR products are not the result of contaminated PCR reagents.

Artifacts↗

PubMed2002

Use of heat labile UNG in an RT-PCR assay for enterovirus detection.

A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to replace the Roche AMPLICOR Enterovirus Test used in our laboratory from 1996 to 1999. The new assay design was optimized to match or exceed the performance of the Roche AMPLICOR Enterovirus test kit with respect to analytical sensitivity and specificity, contamination control, ease of use and availability of reagents. This new assay uses a heat labile form of the enzyme uracil DNA glycosylase (UNG) for amplicon contamination control and an RT-PCR enzyme mixture, enabling a one tube/one step amplification. RNA preparation was undertaken using a commercial extraction kit. End detection was accomplished using a probe-capture enzyme immuno assay (EIA) plate format. This EV RT-PCR assay exceeds the performance of the Roche AMPLICOR Enterovirus assay in a direct comparison. The combined enzymological approach has potential application to a wide variety of assays requiring sensitive RNA detection and stringent contamination control, including those utilizing real time detection methods.

Artifacts↗

PubMed1991

DNA strand breaks induced in cultured human and rodent cells by chlorohydroxyfuranones--mutagens isolated from drinking water.

Chlorohydroxyfuranones, by-products of chlorine disinfection and drinking water contaminants, are shown to produce DNA strand breaks in human and rodent cells. One chlorohydroxyfuranone, 3-chloro-4-dichloromethyl-5-hydroxy-2[5H]-furanone (MX), a potent bacterial mutagen, induces 232 +/- 89 DNA strand breaks.(cell-microM)-1 in human CCRF-CEM cells over a concentration range of 4.4 to 220 microM. This constitutes a DNA damage potency comparable to dimethylsulfate (DMS). By comparison, 3,4-dichloro-5-hydroxy-2[5H]-furanone (MA), another chlorohydroxyfuranone which is approximately four orders of magnitude less mutagenic than MX in Salmonella typhimurium strain TA100, is only about tenfold less potent as an inducer of DNA strand breaks in these cells, i.e., 18.2 +/- 3.1 strand breaks.(cell-microM)-1. The DNA strand-breaking potential of MX is inactivated by prior incubation with a rat liver S9 homogenate. In addition, both chlorohydroxyfuranones are ineffective at producing DNA strand breaks in primary rate hepatocytes (PRH) at concentrations below those which produce cytotoxicity as assessed by release of the cellular enzyme lactate dehydrogenase (LDH). Prior treatment of the PRH with 750 microM diethyl maleate, a glutathione-depleting agent, did not enhance the cytotoxicity nor the DNA strand-breaking potential of either chlorohydroxyfuranone. This could indicate that glutathione-glutathione-S-transferase is not an important mechanism for the detoxification of these compounds in PRH.

Animals↗

PubMed1997

The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities.

The TaqMan LS-50B PCR Detection System facilitates the automated and direct detection of polymerase chain reaction (PCR) products. The system employs the 5' nuclease activity of Taq DNA polymerase to hydrolyse a Salmonella specific internal fluorogenic probe for monitoring the amplification of a 287-bp region of the Salmonella invA gene. Using the fluorogenic 5' nuclease assay, 164 Salmonella strains representing all the subspecies of Salmonella enterica were detected while over 50 non-Salmonella strains were not detected. The detection limit of the assay was two colony forming units (cfu) per PCR reaction when a pure culture of S. typhimurium was used. Six protocols for the isolation of PCR-amplifiable DNA were evaluated using chicken carcass rinses, ground beef, ground pork and raw milk contaminated with Salmonella. Of the six DNA isolation protocols, a modified sample preparation protocol using the EnviroAmp kit was chosen for subsequent studies because it was reliable, easy to use and efficient for the isolation of PCR-amplifiable DNA from foods. A detection limit of 3-7 cfu per PCR reaction was obtained using food samples that were pre-enriched overnight and then inoculated with Salmonella. The detection limit was below 3 cfu/25 g or 25 ml when foods inoculated with Salmonella were pre-enriched overnight. Naturally contaminated foods (50 chicken carcass rinses and 60 raw milk samples) were examined using both the fluorogenic 5' nuclease assay and a modified semi-solid rappaport vassiliadis (MSRV) culture method. Thirty four of the 110 samples tested were Salmonella-positive and 74 were Salmonella-negative by both the 5' nuclease assay and the MSRV method. Two samples were Salmonella-positive by the 5' nuclease assay, but negative by the MSRV method. The correlation between the 5' nuclease assay and the MSRV method was over 98%.

5'-Nucleotidase↗