A Widely Applicable Protocol for DNA Isolation from Fecal Samples

Zhang, Baowei; Li, Ming; Ma, Lichao; Wei, Fuwen

doi:10.1007/s10528-006-9050-1

Cited by 96 publications

(91 citation statements)

References 24 publications

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“…In our laboratory the stool kit extracted samples cost over NZ $ 11, labour costs excluded, and the extraction cost of the swabbed samples was approximately NZ $ 5.7. Zhang et al (2006) found the stool-kit to fail for some herbivorous animals, in agreement with our fi nding of the three species of herbivore showing the greatest diff erence in terms of target DNA concentration and PCR inhibition. Although the stool-kit procedure could be partially automated by performing the second half of the extraction in a QIAcube (Qiagen), the fi rst half (sample processing and inhibitor removal) had to be done manually.…”

Section: Dna Extraction Methodssupporting

confidence: 88%

“…Problems caused by coextracted inhibitors, together with DNA degradation and low template DNA concentrations, range from amplifi cation failure to allelic dropout and peak imbalance (Opel et al 2010). Removal of inhibitors normally involves an extra step of incubation with materials such as InhibitEX tablets (Qiagen, Hilden, Germany) or starch (Zhang et al 2006, Kawamoto et al 2013. Alternatively, partial inhibition can be circumvented by diluting the extract and increasing the number of cycles in the PCR, although this can result in higher rates of amplifi cation failure and genotyping errors (Fernando et al 2003).…”

mentioning

confidence: 99%

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An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

et al. 2009

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Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field‐swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.

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Section: Dna Extraction Methodssupporting

confidence: 88%

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confidence: 99%

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

et al. 2009

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“…This could be attributed to the quantity of original sample, as in some cases the cloacal swabs contained only small quantities of faeces. Other studies have also reported a correlation between the amount of the faecal material and DNA yield (Ariefdjohan et al, 2010;Zhang et al, 2006).…”

Section: Page | 106mentioning

confidence: 88%

Studies on avian pathogenic Escherichia coli in commercial broiler chicken in South East Queensland

Awawdeh¹,

Turni²,

Henning³

et al.

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Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis, a localised or systemic infection resulting in clinical diseases such as colisepticemia, chronic respiratory disease and swollen-head syndrome. Globally, avian colibacillosis is the leading cause of morbidity and mortality in poultry, and it has been associated with massive economic losses and welfare problems.This organism is of public health significance as APEC is communicable to humans. The diagnosis of avian colibacillosis relies on clinical signs, typical pathological lesions and culture of E. coli from affected tissue(s). Antimicrobial therapy is often used both for treatment and control. Previous overseas studies have characterised APEC and identified virulence genes (VGs) that can be used as molecular markers for the identification of APEC. Little is known about APEC in broiler chickens in Australia.The aim of this thesis was to gain a better understanding of the epidemiology of APEC in Australian broiler flocks and how factors including presence of VGs and phylogenetic group can improve the identification of this pathotype in Australia. Firstly, three faecal DNA extraction methods were evaluated. Faeces were collected from healthy chickens and chickens with colibacillosis from commercial broiler farms in South East Queensland (SEQ). The extracted DNA was screened by a pentaplex-PCR for five APEC-associated VGs (iroN, iutA, iss, hlyF and ompT). DNA extracted from E. coli isolates cultured from the cloaca and organs of the birds were screened using the same PCR.Repeated bead beating plus column elution was the preferred DNA extraction method, as it yielded good PCR quality and adequate quantity DNA. However, identifying APEC by direct detection of the five VGs from the faecal material was not feasible as all of these genes were also detected in all of the birds. However, the VGs were more commonly detected in E. coli isolates cultured from birds with colibacillosis.A cross-sectional study was performed to estimate APEC farm-level prevalence in healthy broiler chickens in SEQ and to identify potential risk factors associated with the carriage of APEC. At the farm-level, all of the 40 farms sampled were positive for APEC, that is at least one bird per farm carried APEC, while the within-farm prevalence was 63% (95% Confidence Interval: 55.8, 70.2).Higher APEC within-farm bird-level prevalence was significantly associated with the usage of well water as a source of drinking water, failure to disinfect the water line after each flock, farm visitors not showering before entering the shed, distances greater than 20 metres between the car park and the poultry shed and the presence of wild birds within 50 metres of the shed. Chlorinating the drinking water combined with automatic water filtration reduced within-farm bird-level APEC prevalence. Page | 3Therefore, based on the results concluded from the multivariable model, improving biosecurity and water treatments might reduce APEC prevalence, decrease the risk of co...

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“…В первой серии экспериментов мы исследовали, влияет ли количество взятого в анализ кала на регистрируемое в последствии относительное содержание геномной ДНК человека. В различных работах по метагеномному анализу микробиоты кишечника человека исследуют навески от десятков миллиграммов до нескольких граммов [4,14,19,20]. В настоящей работе мы отобрали пять навесок по 100 мг и выделяли ДНК из каждой независимо, согласно методу 1, ранее используемому в нашей практике [15].…”

Section: кострюкова и дрunclassified

Variability in the relative quantity of human DNA resulted from metagenomic analysis of gut microbiota

et al. 2014

BIOMED KHIM

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We conducted the comparative study of seven different methods of total DNA extraction from human feces. All these methods are recommended in protocols for metagenomic analysis of human gut microbiota. We studied the relative quantity of human DNA calculated from shotgun sequencing on a SOLiD 4 genetic analyzer of metagenomic samples. It was shown that either initial amount of feces or a method applied for total DNA extraction do not affect on final relative human DNA abundance, which is less than 1% in healthy people. Invariance of this parameter allows to consider increased abundance of human DNA in metagenomic samples as a potential marker of inflammatory bowel diseases.

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A Widely Applicable Protocol for DNA Isolation from Fecal Samples

Cited by 96 publications

References 24 publications

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

Studies on avian pathogenic Escherichia coli in commercial broiler chicken in South East Queensland

Variability in the relative quantity of human DNA resulted from metagenomic analysis of gut microbiota

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