Boiling Extraction Method VS Commercial Kits for Bacterial DNA Isolation from Food Samples

Dimitrakopoulou, Maria-Eleni; Stavrou, Venia; Kotsalou, Chrysoula; Vantarakis, Apostolos

doi:10.26502/jfsnr.2642-11000057

Cited by 16 publications

(13 citation statements)

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“…Despite that, the difference in Ct values did not exceed two cycles. Several past studies concluded no significant difference between the heat extraction and the commercial kit extraction methods similar to that of this study and suggest the applicability of heating bacterial material for DNA extraction, too [35,36,[43][44][45]. One study assessed the ability of the bacterial DNA extracted using heat treatment to perform PCR, Restriction Fragment Length Polymorphism (RFLP), and DNA sequencing and concluded comparable results to the more complicated DNA extraction methods of these processes and suggested its applicability on other biological samples, as well [35].…”

Section: Performance Of Dna Extraction Methodssupporting

confidence: 78%

“…It sometimes requires special equipment, such as a centrifuge, to perform the procedure, which may be unsuitable for application in developing countries. Simplified versions of DNA extraction have been previously performed by heating bacterial material without adding reagents, such as by heating bacterial colonies suspended in distilled water in test tubes on a hot water bath for 10 min; by heating the bacterial colonies suspended in distilled water in a microwave oven for 10 s followed by centrifugation; by incubating the bacteria at 99 • C for 15 min; by culturing the bacteria followed by centrifugation and heating the precipitate at 100 • C for 10 min; and by boiling the bacteria suspended in TE buffer for 15 min followed by centrifugation [35,36,43,44]. The E. coli K-12 samples cultured using the method in this study underwent DNA extraction using a kit and by heating and exhibited comparable results.…”

Section: Performance Of Dna Extraction Methodsmentioning

confidence: 99%

“…One study assessed the ability of the bacterial DNA extracted using heat treatment to perform PCR, Restriction Fragment Length Polymorphism (RFLP), and DNA sequencing and concluded comparable results to the more complicated DNA extraction methods of these processes and suggested its applicability on other biological samples, as well [35]. Similarly, another study compared the cost per sample and the average process duration among six different DNA extraction methods and found that the boiling method cost nothing to perform and averaged about 20 min to process, whereas the cost per sample of the remaining five methods ranged from €3.96 to €5.7 and the durations ranged from 30 min to >1 h [44]. For these reasons, the heat DNA extraction was implemented on the cultured samples to obtain at least the qualitative data from qPCR to verify the presence of viable E. coli in the wastewater samples quickly and efficiently.…”

Section: Performance Of Dna Extraction Methodsmentioning

confidence: 99%

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Occurrence and Reduction of Shiga Toxin-Producing Escherichia coli in Wastewaters in the Kathmandu Valley, Nepal

Sthapit

Malla

Tandukar

et al. 2022

Water

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Inadequately treated effluents discharged from wastewater treatment plants (WWTPs) severely affect the environment and the surrounding population. This study analyzed the presence of the Shiga toxin-producing Escherichia coli (STEC) genes, stx1, and stx2, and the E. coli gene, sfmD, in municipal WWTP A (n = 11) and B (n = 11) where the reductions were also evaluated; hospitals (n = 17), sewage treatment plants (STPs) (n = 4) and non-functional WWTPs (not-working WWTPs) (n = 5) in the Kathmandu Valley, Nepal. The sfmD gene was detected in 100% of the samples in WWTPs, hospitals, and not-working WWTPs and 50% of STP samples. The highest detection of stx1 and stx2 was shown in the WWTP influents, followed by WWTP effluents, not-working WWTP wastewater, hospital wastewater, and STP wastewater. Log10 reduction values of sfmD, stx1, and stx2 in WWTP A were 1.7 log10, 1.7 log10, 1.4 log10, whereas those in WWTP B were 0.5 log10, 0.6 log10, 0.5 log10, respectively, suggesting the ineffective treatment of STEC in the wastewater in the Kathmandu Valley. The high concentrations of the stx genes in the wastewaters suggest the increasing presence of aggressive STEC in the Kathmandu Valley, which should be a major public health concern.

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Section: Performance Of Dna Extraction Methodssupporting

confidence: 78%

Section: Performance Of Dna Extraction Methodsmentioning

confidence: 99%

Section: Performance Of Dna Extraction Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Occurrence and Reduction of Shiga Toxin-Producing Escherichia coli in Wastewaters in the Kathmandu Valley, Nepal

Sthapit

Malla

Tandukar

et al. 2022

Water

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“…They found no significant difference between the PCR output from both techniques and suggested that heating was efficient, simple, cheap and suitable for high-through-put. [36,37] Similar to what was seen in the case of bacteria, heating the mosquito midguts and salivary glands yielded similar qPCR detection rates for sporozoites while higher detection rates with oocysts as compared to DNA extraction.…”

Section: Discussionmentioning

confidence: 57%

Using qPCR to compare the detection ofPlasmodium vivaxoocysts and sporozoites inAnopheles farautimosquitoes between two DNA extraction methods

Timinao

Jamea

Katusele

et al. 2022

Preprint

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Background: Currently, the gold standard to assess parasite developmental stages in mosquitoes is light microscopy. Microscopy can miss low-density infections, is time-consuming and not species-specific. This can place limitations on studies, especially when the infection status of larger mosquito populations is important and studies are done in co-endemic settings with multiple circulating parasite species. Enzyme-linked immunosorbent assay (ELISA) has been the alternative technique to evaluate the infectivity of mosquitoes especially in field studies however it is semi-quantitative. Molecular techniques that have been used to detect the mosquito stages of malaria parasites including P. vivax. Here, we present a quantitative real-time assay (qPCR) assay that can be used to detect low-density P. vivax oocyst and sporozoite infections. Parasite detection via qPCR after performing the conventional DNA extraction versus direct qPCR following heating of the infected mosquito samples was compared. Methods: Colony reared Anopheles farauti mosquitoes were exposed to blood samples collected from infected individuals using a direct membrane feeding assay. The fully fed mosquitoes were kept for 7 and 14 days post-feed before dissection to confirm presence of oocysts and sporozoites. Infected mosquito guts and the salivary glands (with the head and thorax) were stored and DNA was extracted either by heating or by performing conventional column-based DNA extraction. Following DNA extraction the infected samples were subjected to qPCR to detect P. vivax parasites. Results: DNA extraction of 1 or more oocysts by heating resulted in an overall sensitivity of 78% (57/73) and single oocysts infections were detected with a sensitivity of 82% (15/17) in the heating arm as well. We observed a 60% (18/30) sensitivity with sporozoites where DNA was extracted using the conventional DNA extraction method prior to qPCR diagnosis. We show that the heating method significantly improved the detection of oocysts over conventional DNA extraction. There was no significant difference in the DNA copy numbers when comparing the detection of oocysts from the conventional DNA extraction versus heating. There was also no significant difference in the detection rate of sporozoite samples when comparing the two DNA extraction protocols. However, we observed that the DNA copy numbers of the sporozoites detected in the heating arm was significantly higher than in the conventional DNA extraction arm. Conclusion: We have adapted a qPCR assay which, when coupled with heating to release DNA reduces sample processing time and cost. Direct qPCR after heating will be a useful tool when investigating transmission blocking vaccines or antimalarials or when evaluating field caught mosquitoes for the presence of malaria parasites.

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“…study, we undertook the lysis by heating at 98˚C in SDW for 2 min. Cell lysis by heating works well for bacterial cells [38][39][40]. We have implemented the same approach for small, soft-bodied insects.…”

Section: Plos Onementioning

confidence: 99%

Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications

Jangra

Ghosh

2022

PLoS ONE

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Nucleic acid extraction is the first and foremost step in molecular biology studies. Extraction of DNA from small, soft-bodied insects is often time-consuming and costly. A fast, easy, and cost-effective DNA extraction method with greater yield and purity of DNA would aid in the rapid diagnostics, screening of large populations, and other routine PCR-based applications. The present study evaluated and standardized a rapid and zero-cost DNA extraction from soft-bodied small insects for routine molecular studies. Five rapid DNA extraction methods viz. extraction in sterile distilled water (SDW), 1X phosphate-buffered saline (PBS, pH 7.4), 1.4 M sodium chloride (NaCl), 20 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), and elution from blotted nitrocellulose membrane (NCM) were compared with standard CTAB extraction buffer and DNeasy® Blood and Tissue Kit methods. The average yield, purity, storage stability, time, and cost of extraction were assessed for all the methods and compared. A method of DNA extraction by simply crushing the soft-bodied insects in SDW was ideal in terms of yield, purity, storability, and performing routine PCR-based applications including detection of pathogens from vector species. The extraction could be accomplished in 2.5 min only with zero-reagent cost. The method would be useful in rapid molecular diagnostics and screening large populations of soft-bodied insects.

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Boiling Extraction Method VS Commercial Kits for Bacterial DNA Isolation from Food Samples

Cited by 16 publications

References 0 publications

Occurrence and Reduction of Shiga Toxin-Producing Escherichia coli in Wastewaters in the Kathmandu Valley, Nepal

Occurrence and Reduction of Shiga Toxin-Producing Escherichia coli in Wastewaters in the Kathmandu Valley, Nepal

Using qPCR to compare the detection ofPlasmodium vivaxoocysts and sporozoites inAnopheles farautimosquitoes between two DNA extraction methods

Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications

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