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

Chen, Shu; Yee, A. J.; Griffiths, Mansel W.; Larkin, C.; Yamashiro, Carl T.; Behari, Richa; Paszko-Kolva, Christine; Rahn, Kris; Grandis, Stephanie A. De

doi:10.1016/s0168-1605(97)01241-5

Cited by 167 publications

(95 citation statements)

References 18 publications

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“…One technique being evaluated is the 5Ј nuclease assay (5NA). 7 This has been used to detect Listeria, 8 human papillomaviruses, 9 Salmonella, 10 hepatitis C virus, 11 Aspergillus, 12 tuberculosis, 13 plague, 14 and orthopoxviruses. 15 The TaqMan System (Perkin Elmer/Applied Biosystems, Foster City, CA) represents a 5NA in which a reporter dye (6-carboxy-fluorescein [FAM], tetrachloro-6-carboxyfluorescein [TET], 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein [JOE], or VIC, proprietary name; Perkin-Elmer) is covalently linked to the 5Ј end of a selected oligonucleotide probe.…”

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confidence: 99%

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

Higgins¹,

Hubálek²,

Halouzka³

et al. 2000

The American Journal of Tropical Medicine and Hygiene

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Abstract. We investigated the use of a TaqMan 5Ј nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (Ͻ100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.

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confidence: 99%

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

Higgins¹,

Hubálek²,

Halouzka³

et al. 2000

The American Journal of Tropical Medicine and Hygiene

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“…Sensitivity of the PCR assay could be affected by insufficient enrichment or poor quality of the target DNA, or by food ingredients that inhibit the PCR reaction (Chen et al, 1997). An additional centrifugation step just prior to the PCR reaction was suggested by Chen et al (1997) to better remove PCR inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…An additional centrifugation step just prior to the PCR reaction was suggested by Chen et al (1997) to better remove PCR inhibitors. Wilson (1997) recommended a washing step to reduce the fat and other residues in foods which could inhibit the PCR reaction, whereas Josefsen et al (2007) reported lower reproducibility with the washing step.…”

Section: Discussionmentioning

confidence: 99%

“…Chen et al (1997) used a fluorogenic TaqMan probe to detect 2-3 cells in 25-g food samples following 24-h enrichment. Trkov et al (1999) were able to detect 1 cell in a 25-g food sample after 30-h enrichment.…”

Section: Discussionmentioning

confidence: 99%

“…More recently, fluorescence-based PCR systems have become available for detection of PCR products in real time. PCR products can be detected using real-time PCR by SYBR green I (a fluorescent dsDNA binding dye) or fluorescently labelled oligonucleotide probes such as TaqMan, molecular beacon, and FRET probes that bind within the PCR product (Chen et al, 1997;Liming and Bhagwat, 2004;Patel et al, 2006;Szmolka et al, 2006). Furthermore, most commercially available PCR assays detect Salmonella spp.…”

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Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

Patel

Bhagwat

2008

Acta Veterinaria Hungarica

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A real-time PCR assay was evaluated for the rapid detection (10 h) of Salmonella in meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated with Salmonella enterica serovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

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Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay for Detecting Salmonella

Chen

Martinez

Mulchandani

2000

Analytical Biochemistry

139

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

Cited by 167 publications

References 18 publications

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay for Detecting Salmonella

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