Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR

Dalla-Costa, Líbera Maria; Morello, Luis Gustavo; Conte, Danieli; Pereira, Luciane Aparecida; Palmeiro, Jussara Kasuko; Ambrosio, Altair Rogerio; Cardozo, Dayane; Krieger, Marco Aurélio; Raboni, Sônia Mara

doi:10.1016/j.mimet.2017.06.020

Cited by 21 publications

(29 citation statements)

References 21 publications

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“…The assay efficiency curve was 101.94% showing that the results are highly reliable, and the standard curve can be used for an absolute quantification of the viral genome. In addition, the melting curve showed a unique peak at 79.03 °C that could be used to confirm ChPV in a sample, and therefore avoid false-negative results in tissue samples with interfering DNA, such as thymus, spleen, bursa, and blood [ 33 , 34 ]. Hence, in the present study, ChPV was detected and quantified in the enteric contents of broilers, laying hens, and breeders using the fast-qPCR based on the SYBR ® Green assay that had been reported in previous studies [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

Núñez

Santander-Parra

Chaible

et al. 2018

Veterinary Sciences

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Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR® Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR® Green was employed, and the Fast mode of a thermocycler was used. The assay detects 109 to 101 copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 °C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 × 106 CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 × 106 CG/uL DNA were detected in chickens with RSS.

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Section: Discussionmentioning

confidence: 99%

Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

Núñez

Santander-Parra

Chaible

et al. 2018

Veterinary Sciences

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“…Aforementioned PCR-based detection of fungal DNA sequences can be sensitive, rapid, specific [212,221,222] and it permits both intraspecies differentiation and species identification of yeast isolates [223]. A relevant step for PCR performance is the DNA extraction, which should be universal, which originates pure and high-quality DNA [224]. Because fungal cell walls are strong and difficult to disrupt, DNA isolation requires effort to overcome this barrier.…”

Section: Molecular Methods Used To Detect Yeastsmentioning

confidence: 99%

Advances in Chemical and Biological Methods to Identify Microorganisms—From Past to Present

et al. 2019

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Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive (e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal isolates and current analysis call for broad reports of microorganisms, involving the application of molecular techniques (e.g., 16S ribosomal RNA gene sequencing based on polymerase chain reaction). The goal of this review is to present the past and the present methods of detection and identification of microorganisms, and to discuss their advantages and their limitations.

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“…Lindh and Lehane 2011; Koga and Moran 2014; Dalla‐Costa et al . 2017; Pendleton et al . 2017; Evans et al .…”

Section: Methodsmentioning

confidence: 99%

How the ‘kitome’ influences the characterization of bacterial communities in lepidopteran samples with low bacterial biomass

Voirol

Valsamakis

et al. 2021

Journal of Applied Microbiology

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Aims: We aimed to elucidate whether the DNA extraction kit and bacteria therein affect the characterization of bacterial communities associated with butterfly samples harbouring different bacterial abundancies. Methods and Results: We analysed bacteria associated with eggs of Pieris brassicae and with adults of this butterfly, which were either untreated or treated with antibiotics (ABs). Three DNA extraction kits were used. Regardless of the extraction kit used, PCR amplification of the bacterial 16S rRNA gene detected very low bacterial presence in eggs and AB-treated butterflies. In untreated butterflies, bacterial signal intensity varied according to the kit and primers used. Sequencing (MiSeq) of the bacterial communities in untreated and AB-treated butterflies revealed a low alpha diversity in untreated butterflies because of the dominance of few bacteria genera, which were detectable regardless of the kit. However, a significantly greater alpha diversity was found in AB-treated butterflies, evidencing a true bias of the results due to bacterial contaminants in the kit. Conclusions: The so-called 'kitome' can impact the profiling of Lepidopteraassociated bacteria in samples with low bacterial biomass. Significance and Impact of the Study: Our study highlights the necessity of method testing and analysis of negative controls when investigating Lepidoptera-associated bacterial communities.

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Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR

Cited by 21 publications

References 21 publications

Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

Advances in Chemical and Biological Methods to Identify Microorganisms—From Past to Present

How the ‘kitome’ influences the characterization of bacterial communities in lepidopteran samples with low bacterial biomass

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