Dataset Information

A Simple and Cost-Effective Freeze-Thaw Based Method for Plasmodium DNA Extraction from Dried Blood Spot.

ABSTRACT: Background:Available DNA isolation methods for Plasmodium involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction of Plasmodium DNA from dried filter paper spot (DBS), appropriate for resource-limited setups. Methods:The protocol involves simple freezing and thawing of DBS, neither involves any purification step nor any chemical reagent. The method was assessed in terms of DNA quantity, PCR detection sensitivity, time requirement, cost effectiveness, labor intensiveness and degree of shearing. The reliability of this method was confirmed by comparing it with other in use methods for Plasmodium DNA isolation. Results:Pure DNA was obtained with this method, as exemplified by the absorbance ratio (260nm /280nm) of 1.2. The protocol produced digestible, PCR-grade genomic DNA, also found to be suitable for sequencing. DNA isolated remained stable and retained its integrity after storage for one month at 4 °C. Conclusion:Our process substantiated as efficient, reproducible, simple, fast, and inexpensive. Development of this optimized freeze-thaw based DNA extraction method for malaria parasite may provide a valuable tool for molecular analysis in resource-limited setups. This is the first report of DNA extraction from DBS of Plasmodium utilizing freeze-thaw.

SUBMITTER: Sharma S

PROVIDER: S-EPMC6511605 | biostudies-literature | 2019 Jan-Mar

REPOSITORIES: biostudies-literature

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Publications

A Simple and Cost-Effective Freeze-Thaw Based Method for <i>Plasmodium</i> DNA Extraction from Dried Blood Spot.

Sharma Supriya S Mann Riti R Kumar Sandeep S Mishra Neelima N Srivastava Bina B Valecha Neena N Anvikar Anupkumar R AR

Iranian journal of parasitology 20190101 1

<h4>Background</h4>Available DNA isolation methods for <i>Plasmodium</i> involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction of <i>Plasmodium</i> DNA from dried filter paper spot (DBS), appropriate for resource-limited setups.<h4>Methods</h4>The protocol involves simple freezing and thawing of DBS, neither involves any purification step nor any chemical reagent. The method was assessed ...[more]

PMID: 31123466

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Project description:Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at -80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1-3 times at -20°C, -80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at -20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at -20°C, -80°C, and in liquid nitrogen. (4) Among the conditions of -20°C, -80°C, and liquid nitrogen, -20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

Dataset Information

A Simple and Cost-Effective Freeze-Thaw Based Method for Plasmodium DNA Extraction from Dried Blood Spot.

Publications

A Simple and Cost-Effective Freeze-Thaw Based Method for <i>Plasmodium</i> DNA Extraction from Dried Blood Spot.

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