Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

Abstract We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

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Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

Water Science & Technology Water Supply ◽

10.2166/ws.2021.197 ◽

2021 ◽

Author(s):

Eun-Sook Lee ◽

So-Yang Cha ◽

Jong-Soon Jung

Keyword(s):

Helicobacter Pylori ◽

Real Time ◽

Dna Extraction ◽

Water Samples ◽

Real Time Pcr ◽

Heavy Rain ◽

Extraction Methods ◽

Pcr Inhibitors ◽

H Pylori ◽

Dna Extraction Methods

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

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Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

Journal of Veterinary Science ◽

10.4142/jvs.2010.11.2.143 ◽

2010 ◽

Vol 11 (2) ◽

pp. 143 ◽

Cited By ~ 17

Author(s):

Ji-Yeon Hyeon ◽

In-Gyun Hwang ◽

Hyo-Sun Kwak ◽

Chankyu Park ◽

In-Soo Choi ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Salmonella Enteritidis ◽

Extraction Methods ◽

Inhibitory Effect ◽

Pcr Assay ◽

Dna Extraction Methods

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Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.46510-0 ◽

2006 ◽

Vol 55 (9) ◽

pp. 1187-1191 ◽

Cited By ~ 51

Author(s):

Lisa J. Griffiths ◽

Martin Anyim ◽

Sarah R. Doffman ◽

Mark Wilks ◽

Michael R. Millar ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Diagnostic Technique ◽

Extraction Methods ◽

Simple Method ◽

Bead Beating ◽

Fungal Dna ◽

Dna Extraction Methods ◽

High Level

Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze–thaw method, a freeze–boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.

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