Comparison of <scp>DNA</scp> extraction techniques for the recovery of bovine <scp>DNA</scp> from fly larvae crops

Cantu, Cesar; Bucheli, Sibyl R.; Houston, Rachel

doi:10.1111/1556-4029.15010

Cited by 4 publications

(3 citation statements)

References 39 publications

(86 reference statements)

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“…The qPCR assay implemented for pre-screening includes a competitive IAC to reduce the hands-on work and less pipetting of reagents, while increasing the confidence in the results by assuring lack of reaction inhibition (Rip and Gouws, 2009 ). It is worth noting that no qPCR inhibition was observed in any of the samples analyzed, most likely due to the two-step enrichment method which reduces the food debris transferred to the final sample, along with the implementation of the DNeasy PowerSoil Pro kit, which has been reported to be highly efficient in removing PCR inhibitors, such as humic acid, from a wide variety of samples (Pearman et al, 2020 ; Magnani, 2021 ; Cantu et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

Azinheiro

Roumani²,

Costa-Ribeiro³

et al. 2022

Front. Microbiol.

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Microbial pathogens may be present in different types of foods, and hence the development of novel methods to assure consumers' safeness is of great interest. Molecular methods are known to provide sensitive and rapid results; however, they are typically targeted approaches. In recent years, the advent of non-targeted approaches based on next-generation sequencing (NGS) has emerged as a rational way to proceed. This technology allows for the detection of several pathogens simultaneously. Furthermore, with the same set of data, it is possible to characterize the microorganisms in terms of serotype, virulence, and/ or resistance genes, among other molecular features. In the current study, a novel method for the detection of Listeria monocytogenes based on the “quasimetagenomics” approach was developed. Different enrichment media and immunomagnetic separation (IMS) strategies were compared to determine the best approach in terms of L. monocytogenes sequences generated from smoked salmon samples. Finally, the data generated were analyzed with a user-friendly workflow that simultaneously provided the species identification, serotype, and antimicrobial resistance genes. The new method was thoroughly evaluated against a culture-based approach, using smoked salmon inoculated with L. monocytogenes as the matrix of choice. The sequencing method reached a very low limit of detection (LOD50, 1.2 CFU/ 25 g) along with high diagnostic sensitivity and specificity (100%), and a perfect correlation with the culture-based method (Cohen's k = 1.00). Overall, the proposed method overcomes all the major limitations reported for the implementation of NGS as a routine food testing technology and paves the way for future developments taking its advantage into consideration.

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Section: Discussionmentioning

confidence: 99%

Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

Azinheiro

Roumani²,

Costa-Ribeiro³

et al. 2022

Front. Microbiol.

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“…Bacterial DNA was first extracted using the EZ1 DNeasy Blood Tissue Kit (Qiagen GmbH, Hilden, Germany) [ 32 ]. The absorbance measurements for DNA purity ranged from 260 to 280 nm (Spectrophotometer ND-100, Nanodrop Thermo Fisher Scientific, Wilmington, DE, USA).…”

Section: Methodsmentioning

confidence: 99%

Screening of colistin-resistant bacteria in livestock animals from France

Hamame

Davoust

Hasnaoui

et al. 2022

Vet Res

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Colistin is frequently used as a growth factor or treatment against infectious bacterial diseases in animals. The Veterinary Division of the European Medicines Agency (EMA) restricted colistin use as a second-line treatment to reduce colistin resistance. In 2020, 282 faecal samples were collected from chickens, cattle, sheep, goats, and pigs in the south of France. In order to track the emergence of mobilized colistin resistant (mcr) genes in pigs, 111 samples were re-collected in 2021 and included pig faeces, food, and water from the same location. All samples were cultured in a selective Lucie Bardet Jean-Marc Rolain (LBJMR) medium and colonies were identified using MALDI-TOF mass spectrometry and then antibiotic susceptibility tests were performed. PCR and Sanger sequencing were performed to screen for the presence of mcr genes. The selective culture revealed the presence of 397 bacteria corresponding to 35 different bacterial species including Gram-negative and Gram-positive. Pigs had the highest prevalence of colistin-resistant bacteria with an abundance of intrinsically colistin-resistant bacteria and from these samples one strain harbouring both mcr-1 and mcr-3 has been isolated. The second collection allowed us to identify 304 bacteria and revealed the spread of mcr-1 and mcr-3 in pigs. In the other samples, naturally, colistin-resistant bacteria were more frequent, nevertheless the mcr-1 variant was the most abundant gene found in chicken, sheep, and goat samples and one cattle sample was positive for the mcr-3 gene. Animals are potential reservoir of colistin-resistant bacteria which varies from one animal to another. Interventions and alternative options are required to reduce the emergence of colistin resistance and to avoid zoonotic transmissions.

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“…The extracted DNA was preserved at −20 • C. Before the DNA extraction of insect meals, removal of fat and oils, independent of their processing method, was carried out. This step was added because the lipid concentration of insects [44] could interfere in DNA extraction process [45] and inhibit PCR [46]. The fat and oil extraction process involved resuspending 10 mg of sample in methanol-chloroform-water (2:1:0.8) for one day and then washing in distilled water and PBS 1× buffer to eliminate the remains of the solution previously used.…”

Section: Sample Processing and Dna Extractionsmentioning

confidence: 99%

Genetic Identification and Traceability of Insect Meals

Moulistanos,

Karaiskou,

Gkagkavouzis

et al. 2023

Insects

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Insects have been proposed as a rich alternative source of protein for the partial or total replacement of fishmeal in aquaculture. For maximum safety and effectiveness of insect meals, control of the quality composition of these products is considered mandatory. The aim of this study was the genetic analysis of the composition of commercially available insect meals at the species level. Commercially available Hermetia illucens, Tenebrio molitor and Musca domestica individuals, as well as nine insect meals produced from these species, were analyzed. The genetic identification of insects at the species level was based on a COI fragment, and analysis of the insect meals’ composition was performed with the processes of cloning and colony PCR. Genetic analysis indicated that the commercially available larvae morphologically identified as Musca domestica belonged to the species Muscina stabulans. In the commercially available insect meals, no other animal species was identified beyond the expected one. However, in the insect meal produced for research purposes, fungal growth was detected. The used methodology, herein, allows for the qualitative genetic identification of insect meals and could be included in the methods of traceability of products containing insects and other animal species.

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Comparison of DNA extraction techniques for the recovery of bovine DNA from fly larvae crops

Cited by 4 publications

References 39 publications

Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

Screening of colistin-resistant bacteria in livestock animals from France

Genetic Identification and Traceability of Insect Meals

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