Food matrix standards for the quantification of allergenic food ingredients using real-time PCR

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead–based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix–associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

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Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines

Molecules ◽

10.3390/molecules23081932 ◽

2018 ◽

Vol 23 (8) ◽

pp. 1932 ◽

Cited By ~ 4

Author(s):

Byeong Moon ◽

Wook Kim ◽

Inkyu Park ◽

Gi-Ho Sung ◽

Pureum Noh

Keyword(s):

Dietary Supplements ◽

Real Time ◽

Real Time Pcr ◽

Herbal Medicines ◽

Fungal Species ◽

Scar Markers ◽

Pcr Assay ◽

Food Ingredients ◽

The Real ◽

Species Specific

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine “Cordyceps”, and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

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Effect of inulin on the human gut microbiota: stimulation ofBifidobacterium adolescentisandFaecalibacterium prausnitzii

British Journal Of Nutrition ◽

10.1017/s0007114508019880 ◽

2008 ◽

Vol 101 (4) ◽

pp. 541-550 ◽

Cited By ~ 440

Author(s):

Carlett Ramirez-Farias ◽

Kathleen Slezak ◽

Zoë Fuller ◽

Alan Duncan ◽

Grietje Holtrop ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bifidobacterium Longum ◽

Control Period ◽

Bifidobacterium Bifidum ◽

Bifidobacterium Adolescentis ◽

Food Ingredients ◽

Bifidobacterium Animalis ◽

Bacterial Groups

Prebiotics are food ingredients that improve health by modulating the colonic microbiota. The bifidogenic effect of the prebiotic inulin is well established; however, it remains unclear which species ofBifidobacteriumare stimulatedin vivoand whether bacterial groups other than lactic acid bacteria are affected by inulin consumption. Changes in the faecal microbiota composition were examined by real-time PCR in twelve human volunteers after ingestion of inulin (10 g/d) for a 16-d period in comparison with a control period without any supplement intake. The prevalence of most bacterial groups examined did not change after inulin intake, although the low G+C % Gram-positive speciesFaecalibacterium prausnitziiexhibited a significant increase (10·3 % for control periodv.14·5 % during inulin intake,P = 0·019). The composition of the genusBifidobacteriumwas studied in four of the volunteers by clone library analysis. Between three and fiveBifidobacteriumspp. were found in each volunteer.Bifidobacterium adolescentisandBifidobacterium longumwere present in all volunteers, andBifidobacterium pseudocatenulatum,Bifidobacterium animalis,Bifidobacterium bifidumandBifidobacterium dentiumwere also detected. Real-time PCR was employed to quantify the four most prevalentBifidobacteriumspp.,B. adolescentis,B. longum,B. pseudocatenulatumandB. bifidum, in ten volunteers carrying detectable levels of bifidobacteria.B. adolescentisshowed the strongest response to inulin consumption, increasing from 0·89 to 3·9 % of the total microbiota (P = 0·001).B. bifidumwas increased from 0·22 to 0·63 % (P < 0·001) for the five volunteers for whom this species was present.

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Rapid, Specific, and Sensitive Detection of Spoilage Molds in Orange Juice Using a Real-Time Taqman PCR Assay

Journal of Food Protection ◽

10.4315/0362-028x-69.2.385 ◽

2006 ◽

Vol 69 (2) ◽

pp. 385-390 ◽

Cited By ~ 7

Author(s):

KAI WAN ◽

AHMED E. YOUSEF ◽

STEVE J. SCHWARTZ ◽

HUA H. WANG

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Orange Juice ◽

Raw Materials ◽

Pcr Primers ◽

Cross Reactivity ◽

Rrna Gene ◽

Control Analysis ◽

Financial Loss ◽

Food Ingredients

The outgrowth of spoilage organisms, including molds and yeasts, results in significant financial loss to the food industry and wastes natural resources. The objective of this study was to develop a rapid, specific, and sensitive real-time PCR method for detecting spoilage molds during screening of raw materials and final product quality control analysis. The 18S rRNA gene was used to develop PCR primers and probe. With this set of primers and probe, less than 1,000 mold cells per milliliter of orange juice (10 cells per reaction) were detected with the real-time PCR system within 6 to 7 h. No cross-reactivity was found with other common foodborne bacteria, yeasts, or food ingredients. This technique is significantly faster than current detection and identification procedures, which take from days to weeks.

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