A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

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Pea Detection in Food and Feed Samples by a Real-Time PCR Method Based on a Specific Legumin Gene That Allows Diversity Analysis

Journal of Agricultural and Food Chemistry ◽

10.1021/jf8023968 ◽

2008 ◽

Vol 56 (23) ◽

pp. 11098-11104 ◽

Cited By ~ 4

Author(s):

Sonia Ramos-Gómez ◽

Lorena López-Enríquez ◽

Constantino Caminero ◽

Marta Hernández

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Diversity Analysis ◽

Food And Feed ◽

Pcr Method ◽

Feed Samples

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Testing animal feed for the presence of ruminant DNA using the official real-time PCR method

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/333/1/012086 ◽

2019 ◽

Vol 333 ◽

pp. 012086

Author(s):

K Nesic ◽

N Pavlovic ◽

M Pavlovic ◽

A Tasic ◽

J Kureljusic ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Animal Feed ◽

Pcr Method

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Real-Time PCR Detection and Identification of Prohibited Mammalian and Avian Material in Animal Feeds

Journal of Food Protection ◽

10.4315/0362-028x-72.5.1055 ◽

2009 ◽

Vol 72 (5) ◽

pp. 1055-1062 ◽

Cited By ~ 19

Author(s):

SAIRA CAWTHRAW ◽

GINNY C. SAUNDERS ◽

TREVOR C. MARTIN ◽

JASON SAWYER ◽

OTTO WINDL ◽

...

Keyword(s):

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

Animal Feed ◽

Meat And Bone Meal ◽

Bone Meal ◽

Detection And Identification ◽

Pcr Assays ◽

Species Specific ◽

Feed Samples

A method for the detection and identification of “prohibited” mammalian or avian material in animal feed was developed and assessed through the analysis of DNA. A generic real-time PCR assay was designed to detect the presence of mammalian and avian mitochondrial DNA 16S rRNA genes in animal feed samples. Samples positive with this screening method were further investigated using identification assays to detect the 16S rRNA gene from bovine, ovine, porcine, and avian species and to determine whether the DNA originated from species whose material is prohibited from inclusion in farmed animal feed. An internal positive control was coamplified in the 16S real-time PCR assays to monitor PCR amplification efficiency and avoid potential false-negative results. Using vegetable-based feed standards spiked with meat and bone meal generated with a commercial rendering process, 0.1% meat and bone meal could be detected using the general and species-specific 16S assays. The species-specific assays had 100% specificity for the homologous target species. The 16S real-time PCR assays were evaluated alongside existing tests based on protein evaluation or microscopic examination for a wide range of commercial animal feed samples. In total, 111 (0.76%) of 14,678 samples examined contained prohibited material based on the results from at least one of these tests. However, most positive results did not represent noncompliance because they were associated with samples of pet food, which can legitimately contain material prohibited for use in food for farmed animals. The species-specific 16S assays confirmed the presence of prohibited material in 75% of the 111 samples, whereas the existing protein and microscope tests confirmed the presence of this material in 25 and 54% of the samples, respectively.

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Screening the Presence of Amflora Transgenic Potato in Food or Feed Products

Acta Medica Marisiensis ◽

10.1515/amma-2016-0051 ◽

2016 ◽

Vol 62 (4) ◽

pp. 392-394

Author(s):

Manuela-Claudia Curticăpean

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Recombinant Dna ◽

Animal Feed ◽

Transgenic Potato ◽

Food Samples ◽

Potato Tubers ◽

Pcr Method ◽

Starch Composition ◽

Qualitative Event

AbstractObjective. Amflora potato, event EH92-527-1 was genetically modified to produce only the amylopectin component from starch composition. The presence of the transgenic potato in the food on Romanian market should be verified although the Amflora potato uses for industrial purposes and animal feed. The aim of this study was to detect the presence of the Amflora potato in the food or feed products.Methods. For this purpose, five samples of potato tubers and four samples of chips were analyzed. DNA isolation was performed with E.Z.N.A. ® MicroElute Genomic DNA kit (Omega Bio-Tek, USA). For identification a potential presence of the recombinant DNA in the food samples it was used GMOIdent RT Event EH92-527-1 Potato kit (Eurofins GeneScan, Germany), that is a real-time PCR kit for qualitative event-specific detection of AmfloraTM EH92-527-1 potato.Results. Amflora transgenic potato was not detected in any of analyzed samples.Conclusions. The results of real-time PCR method confirm the absence of Amflora event EH92-527-1 in all potato tubers and chips analyzed samples.

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Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

Anti-Infective Agents ◽

10.2174/2211352518999200629165108 ◽

2020 ◽

Vol 18 ◽

Author(s):

Pegah Shakib ◽

Mohammad Reza Zolfaghari

Keyword(s):

Streptococcus Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Cross Sectional Study ◽

Laboratory Culture ◽

Cross Sectional ◽

Negative Results ◽

False Negative Results ◽

Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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