scholarly journals Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

2000 ◽  
Vol 66 (9) ◽  
pp. 4131-4135 ◽  
Author(s):  
A. Vishnubhatla ◽  
D. Y. C. Fung ◽  
R. D. Oberst ◽  
M. P. Hays ◽  
T. G. Nagaraja ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Pcr Amplification ◽  
Enteric Bacteria ◽  
Taqman Assay ◽  
Virulent Strains ◽  
Field Samples ◽  
Ground Pork ◽  
Standard Culture ◽  
Pure Cultures ◽  
Nuclease Assay

ABSTRACT We have developed a rapid procedure for the detection of virulentYersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species ofYersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri,Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia,Salmonella, Citrobacter, andFlavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥102 CFU/ml in pure cultures and ≥103 CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked withY. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocoliticawith both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

Evaluation of a 5′-Nuclease (TaqMan) Assay for the Detection of Virulent Strains of Yersinia enterocolitica in Raw Meat and Tofu Samples†

2001 ◽  
Vol 64 (3) ◽  
pp. 355-360 ◽  
Author(s):  
A. VISHNUBHATLA ◽  
R. D. OBERST ◽  
D. Y. C. FUNG ◽  
W. WONGLUMSOM ◽  
M. P. HAYS ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Detection System ◽  
Detection Methods ◽  
Taqman Assay ◽  
Contamination Rate ◽  
Virulence Plasmid ◽  
Recovery Method ◽  
Ground Pork ◽  
Pure Cultures ◽  
Nuclease Assay

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5′-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with “gold standard” methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5′-nuclease assay was able to identify the organism in 100% of the repetitions when 102 CFU/ml or more organisms were present in pure cultures and 103 CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 105 CFU/ml or more organisms were present in pure culture and 106 CFU/g or more organisms were present in inoculated ground pork. The 5′-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5′-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5′-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Evaluation of a 5′ -Nuclease (TaqMan) Assay with the Thin Agar Layer Oxyrase Method for the Detection of Yersinia enterocolitica in Ground Pork Samples†

2004 ◽  
Vol 67 (2) ◽  
pp. 271-277 ◽  
Author(s):  
V. C. H. WU ◽  
D. Y. C. FUNG ◽  
R. D. OBERST
Keyword(s):  
Detection Limit ◽  
Yersinia Enterocolitica ◽  
Food Samples ◽  
Selective Medium ◽  
Taqman Assay ◽  
Ground Pork ◽  
Sensitivity Studies ◽  
Agar Layer ◽  
Pork Samples ◽  
Nuclease Assay

A 5′-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5′-nuclease assay for detecting Y. enterocolitica O:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5′-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of “Oxyrase® for Agar” onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (−15°C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5′-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

Comparison of Enrichment and Plating Media for Recovery of Virulent Strains of Yersinia enterocolitica from Inoculated Beef Stew

1983 ◽  
Vol 46 (11) ◽  
pp. 957-964 ◽  
Author(s):  
D. A. SCHIEMANN
Keyword(s):  
Yersinia Enterocolitica ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Selective Enrichment ◽  
Serotype O ◽  
Virulent Strains ◽  
Different Temperatures ◽  
Pure Cultures ◽  
Agar Media ◽  
Quantitative Recovery

Five plating agar media were evaluated for their ability to recover pure cultures of virulent strains of Yersinia enterocolitica serotypes O:3, O:8 and O:5,27. Cellobiose-arginine-lysine and bismuth sulfite agars were unproductive at 32°C but gave quantitative recovery with 48 h of incubation at 22°C. Adjustment of the pH of bismuth sulfite agar to 7.4 made the medium inhibitory. MacConkey, DHL and cefsulodin-irgasan-novobiocin agars gave quantitative recovery with 24 h of incubation at 32°C. Four preenrichment media incubated at four different temperatures, three selective enrichment media incubated at 22°C, and the five plating media were evaluated for their ability to recovery Y. enterocolitica from beef stew seeded with a background of ten other gram-negative bacteria. None of the plating media was superior for recovery; however, cefsulodin-irgasan-novobiocin agar showed the highest confirmation rate for presumptive colonies. Buffered-sorbitol-bile broth was inferior to richer media such as trypticase soy broth for preenrichment. Of the three selective enrichment media examined, only bile-oxalate-sorbose broth was found useful, especially for strains of serotype O:8 which could be recovered after 1 d of preenrichment and 3 d of selective enrichment at 22°C. Strains of serotypes O:8 and O:3 were recovered when two cells with 107 cells of ten other gram-negative bacteria were added to 10 g of beef stew following preenrichment in trypticase soy broth at 2°C for 7 d and selective enrichment in bile oxalate-sorbose broth at 22°C for 3 to 5 d. Strains of serotype O:5,27 were more difficult to recover even with longer enrichment times. These studies indicated that the most comprehensive enrichment system for recovery of Y. enterocolitica from foods is preenrichment in trypticase soy broth at 22°C for 1 d and 2 to 4°C for 4 to 7 d followed by selective enrichment in bile-oxalate-sorbose broth at 22°C for 3 to 5 d and isolation on cefsulodin-irgasan-novobiocin agar.

scholarly journals Identification of Pantoea stewartii subsp. stewartii by PCR and Strain Differentiation by PFGE

Plant Disease ◽  
2002 ◽  
Vol 86 (3) ◽  
pp. 304-311 ◽  
Author(s):  
David L. Coplin ◽  
Doris R. Majerczak ◽  
Yongxiang Zhang ◽  
Won-Sik Kim ◽  
Susanne Jock ◽  
...  
Keyword(s):  
Sweet Corn ◽  
Low Frequency ◽  
Its Region ◽  
Enteric Bacteria ◽  
Pantoea Stewartii ◽  
Seed Health ◽  
Field Samples ◽  
Pure Cultures ◽  
Primer Sets ◽  
Pigmented Bacteria

Stewart's bacterial wilt and leaf blight of sweet corn and maize is caused by Pantoea stewartii subsp. stewartii. This bacterium can be seed transmitted at a low frequency, so it is subject to quarantine restrictions by many countries. To develop a polymerase chain reaction assay for the identification of this pathogen from field samples and for use in seed health tests, four primer pairs were tested. These were selected from the sequences of hrpS, cpsDE, and the 16S rRNA intergenic transcribed spacer (ITS) region. Under optimal reaction conditions, about 20 and 200 cells of P. stewartii could be detected in pure cultures and leaf lesions, respectively. Other plant-associated enteric bacteria (e.g., P. agglomerans pv. herbicola, P. ananas, Erwinia amylovora, and E. carotovora) either did not produce amplicons or they were not the correct size for P. stewartii. To test further for possible false positives, 29 yellow-pigmented bacteria, mainly other Pantoea spp., were isolated from lesions on old corn leaves and assayed with the ITS primer sets. Except for weak, variable reactions with three P. ananas strains, the bacteria did not test positive. Pulsed field gel electrophoresis (PFGE) was evaluated as an additional test to confirm the identity of P. stewartii. After digestion with SpeI and XbaI, P. stewartii strains could be easily distinguished from related Erwinia and Pantoea spp. and each other.

Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2630-2640 ◽  
Author(s):  
J. T. Tambong ◽  
R. Xu ◽  
E. S. P. Bromfield
Keyword(s):  
Root Zone ◽  
Phylogenetic Analyses ◽  
Melting Curve ◽  
Pcr Amplification ◽  
23S Rrna ◽  
Melting Curve Analysis ◽  
Its1 Region ◽  
A Genome ◽  
Pure Cultures ◽  
Intercalating Dye

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNAIle and tRNAAla in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

scholarly journals Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1042-1048 ◽  
Author(s):  
C. L. Trout ◽  
J. B. Ristaino ◽  
M. Madritch ◽  
T. Wangsomboondee
Keyword(s):  
Late Blight ◽  
Phytophthora Infestans ◽  
Pcr Amplification ◽  
Accurate Method ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Direct Pcr ◽  
Field Samples ◽  
Pcr Products ◽  
Oomycete Pathogen

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

scholarly journals Association of AFF3 Gene Polymorphism rs10865035 with Rheumatoid Arthritis: A Population-Based Case-Control Study on a Pakistani Cohort

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Yasir Ali ◽  
Suleman Khan ◽  
Yangchao Chen ◽  
Nadia Farooqi ◽  
Zia-Ul Islam ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Additive Model ◽  
Pcr Amplification ◽  
Population Based ◽  
Recessive Model ◽  
Taqman Assay ◽  
Amplification Refractory Mutation System ◽  
Multiple Loci ◽  
Geographical Variations ◽  
Mutation System

Rheumatoid arthritis (RA) is one of the complex diseases with the involvement of the genetic as well as environmental factors in its onset and severity. Different genome-wide association and candidate gene studies have shown the role of several genetic variants in multiple loci/genes with ethnical and geographical variations. This study was designed to detect the association of a single-nucleotide polymorphism (SNP) rs10865035 in the AFF3 gene with the genetic background of rheumatoid arthritis (RA) in the Pakistani cohort. A total of 703 individuals, including 409 RA patients and 294 healthy controls, were genotyped using TaqMan assay and Tri primer ARMS-PCR (amplification-refractory mutation system-polymerase chain reaction) methods. The association of rs10865035 with the RA was statistically determined using different models. Interestingly, besides the homozygous recessive model (G/G vs. A/G + A/A) (OR = 1.693(1.06–2.648); P  = 0.025), all other models, which included the codominant (χ2 = 5.169; P  = 0.075), homozygous dominant (A/A vs. G/G + A/G) (OR = 0.867 (0.636–1.187); P  = 0.41), heterozygous (A/G vs. A/A + GG) (OR = 0.491 (0.667–1.215); P  = 0.49), and additive model (OR = 0.826 (0.665–1.027); P  = 0.08) showed insignificant distribution of the genotypes among the cases and controls. These findings suggest that the AFF3 gene (rs10865035) has no significant role in the onset of RA in the Pakistani population.

scholarly journals Monitoring Methanotrophic Bacteria in Hybrid Anaerobic-Aerobic Reactors with PCR and a Catabolic Gene Probe

1999 ◽  
Vol 65 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Carlos B. Miguez ◽  
Chun F. Shen ◽  
Denis Bourque ◽  
Serge R. Guiot ◽  
Denis Groleau
Keyword(s):  
Pcr Amplification ◽  
Granular Sludge ◽  
Anaerobic Sludge ◽  
Initial Population ◽  
Gene Probe ◽  
Soluble Methane Monooxygenase ◽  
Chlorinated Pollutants ◽  
Pure Cultures ◽  
Amplification Assay ◽  
Aerobic Bioreactors

ABSTRACT We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on themmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants.

AnDiaTec Salmonella sp. PCR-ELISA for Analysis of Food Samples

2004 ◽  
Vol 67 (8) ◽  
pp. 1585-1590 ◽  
Author(s):  
CHRISTOPH METZGER-BODDIEN ◽  
ANJA BOSTEL ◽  
JOHANNES KEHLE
Keyword(s):  
Pcr Amplification ◽  
Reference Method ◽  
Evaluation Study ◽  
Microtiter Plate ◽  
Food Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacteriological Method ◽  
Contaminated Food ◽  
Pure Cultures ◽  
Sensitivity Specificity

A commercially available PCR kit (AnDiaTec Salmonella sp. PCR-ELISA) was developed and evaluated for the detection of Salmonella sp. in food samples. The test is based on PCR amplification and hybridization of the amplified DNA to a microtiter plate followed by the detection of PCR product in the manner of an enzyme-linked immunosorbent assay test. The sensitivity and specificity were evaluated first with Salmonella pure cultures and artificially contaminated food samples, including food types for which an inhibition of the PCR reaction was expected. Both experiments proved a very good sensitivity, specificity, and reliability of the test with a very broad range of food types. In a second evaluation study, more than 1,100 food samples of different types were tested in parallel with the PCR method and with the International Standardization Organization 6579 bacteriological reference method. The results of this evaluation study and the results from other experiments on dilutions of artificially contaminated food samples led to the establishment of a positive-negative cutoff value (optical density at 450 nm of more than 0.9) with respect to the conventional bacteriological method. Using this positive-negative cutoff, 98% agreement to the bacteriological method was obtained.

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