Detection of Campylobacter in air samples from poultry houses using shot-gun metagenomics – a pilot study

Background: Foodborne pathogens such as Campylobacter jejuni are responsible for a large fraction of the gastrointestinal infections worldwide associated with poultry meat. Campylobacter spp. can be found in the chicken fecal microbiome and can contaminate poultry meat during the slaughter process. The current standard methods to detect these pathogens at poultry farms use fecal dropping or boot swaps in combination with cultivation / PCR. In this study, we have used air filters in combination with shotgun metagenomics for the detection of Campylobacter in poultry houses and MOCK communities to test the applicability of this approach for the detection of foodborne pathogens. Results: The spiked MOCK communities showed that we could detect as little as 200 CFU Campylobacter per sample using our protocols. Since we were interested in detecting Campylobacter, a DNA extraction protocol for Gram negative bacteria was chosen, and as expected, we found that the DNA extraction protocol created a substantial bias affecting the community composition of the MOCK communities. It can be expected that the same bias is present for poultry house samples analyzed. We observed significant amounts of Campylobacter on the air filters using both real-time PCR as well as shotgun metagenomics, irrespective of the amount of spiked in Campylobacter cells, suggesting that the flocks in both houses harboured Campylobacter spp.. Interestingly, in both houses we find diverse microbial communities present in the indoor air. In addition, have we tested the Campylobacter detection rate using shotgun metagenomics by spiking with different levels of C. jejuni cells in both the mock and the house samples. This showed that even with limited sequencing Campylobacter is detectable in samples with low abundance. Conclusions: These results show that air sampling of poultry houses in combination with shotgun metagenomics can detect and identify Campylobacter spp. present at low levels. This is important since early detection of Campylobacter in food production can help to decrease the number of food-borne infections.

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OPTIMATION OF DNA EXTRACTION PROTOCOL OF Elaeidobius kamerunicus

Jurnal Penelitian Kelapa Sawit ◽

10.22302/10.22302/iopri.jur.jpks.v24i2.10 ◽

2018 ◽

Vol 24 (2) ◽

pp. 77-86

Author(s):

Sri Wening ◽

Agus Eko Prasetyo ◽

Tjut Ahmad Perdana Rozziansha ◽

Agus Susanto

Keyword(s):

Dna Fingerprinting ◽

Dna Extraction ◽

Oil Palm ◽

Fruit Set ◽

Fragment Length Polymorphism ◽

Basic Knowledge ◽

Biological Study ◽

Palm Fruit ◽

Extraction Protocol ◽

Elaeidobius Kamerunicus

African pollination weevil (Elaeidobius kamerunicus Faust) has an important role in the productivity of Indonesian oil palm plantation. Up to now, there has not been a comprehensive biological study of the species at molecular level. The basic knowledge is very useful for exploitation of the weevil for effective oil palm fruit set development. This research aimed to obtain DNA extraction protocol of E. kamerunicus for DNA fingerprinting of the species. Results showed that using a DNA extraction kit,material disruption by using micro pestle resulted the highest quantity of DNA, while there were no significant differences of resulted DNA quantity among treatments using tissue lyser for material disruption. DNA extracted by using micro pestle or tissue lyser for material disruption is adequate for DNA fingerprinting using AFLP (Amplified Fragment Length Polymorphism) and sequencing techniques.

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Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements

Microbiome ◽

10.1186/s40168-021-01048-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Dieter M. Tourlousse ◽

Koji Narita ◽

Takamasa Miura ◽

Mitsuo Sakamoto ◽

Akiko Ohashi ◽

...

Keyword(s):

Dna Extraction ◽

High Throughput Sequencing ◽

Performance Metrics ◽

Collaborative Study ◽

Human Microbiome ◽

Second Phase ◽

Intermediate Precision ◽

Fecal Microbiome ◽

Library Construction ◽

Wide Range

Abstract Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products.

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A modified DNA extraction protocol and its utility in seed genetic purity assessment

Seed Science and Technology ◽

10.15258/sst.2011.39.1.24 ◽

2011 ◽

Vol 39 (1) ◽

pp. 236-242 ◽

Cited By ~ 3

Author(s):

S.N. Sharma ◽

V. Kumar ◽

G. Singh ◽

R. Sharma

Keyword(s):

Dna Extraction ◽

Genetic Purity ◽

Purity Assessment ◽

Extraction Protocol ◽

Modified Dna

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Small volume fungal genomic DNA extraction protocol for Illumina genome v2 (protocols.io.3w9gph6)

protocols.io ◽

10.17504/protocols.io.3w9gph6 ◽

2019 ◽

Author(s):

Jana M ◽

Lilly Moore

Keyword(s):

Dna Extraction ◽

Genomic Dna ◽

Small Volume ◽

Genomic Dna Extraction ◽

Extraction Protocol

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Methodology Simple and inexpensive DNA extraction protocol for studying the bacterial composition of sludges used in microbial fuel cells

Genetics and Molecular Research ◽

10.4238/2013.february.4.2 ◽

2013 ◽

Vol 12 (1) ◽

pp. 282-292 ◽

Cited By ~ 3

Author(s):

B. Canto-Canché ◽

M. Tzec-Simá ◽

J.I. Vázquez-Loría ◽

H. Espadas-Álvarez ◽

B.H. Chí-Manzanero ◽

...

Keyword(s):

Fuel Cells ◽

Dna Extraction ◽

Microbial Fuel Cells ◽

Bacterial Composition ◽

Extraction Protocol

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ORAL HIV-ASSOCIATED KAPOSI SARCOMA: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY AND qPCR TECHNIQUES FOR DETECTION OF HHV8

Clinical and Laboratorial Research in Dentistry ◽

10.11606/issn.2357-8041.clrd.2015.97224 ◽

2015 ◽

Vol 21 (1) ◽

pp. 29

Author(s):

Priscila Lie Tobouti ◽

Juliana Seo ◽

Michella Bezerra Lima ◽

Bruno Tavares Sedassari ◽

Norberto Nobuo Sugaya ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Kaposi Sarcoma ◽

Positive Staining ◽

Extraction Protocol ◽

Simple Phenol ◽

Two Samples ◽

Hematoxylin And Eosin ◽

The Mean

Objective: To compare the diagnostic accuracy of immunohistochemistry compared to real-time PCR (using a simple phenol-chloroform DNA extraction protocol) in the detection of HHV8 in small oral biopsies of Kaposi sarcoma. Also to validate the use of this DNA extraction protocol to extract HHV8 DNA.Material and methods: Seventeen cases of oral KS were examined. Data including gender, age, and anatomic location were obtained from patient´s records and histological sections stained with hematoxylin and eosin (H&E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.Results: All the patients were HIV positive, the mean age was 35.5 years old, and the affected oral sites were palate (47%), gingiva (29.4%), tongue (11.8%), lip (5.9%), and oral floor (5.9%). Fifteen samples showed positive staining for HHV8 and only two samples were negative. The same results were observed using real-time PCR HHV8-DNA detection.Relevance: Our findings suggest that immunohistochemistry is faster and cheaper to perform than real-time PCR and was shown to have similar levels of sensitivity and accuracy for detection of HHV8 even in small biopsies. Additionally DNA extraction using a non-commercial kit, as done in this study can further reduce the costs to a pathology service.

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