A fast and sensitive nucleic acid extraction method for the detection of Cryptosporidium by PCR in environmental water samples

2002 ◽  
Vol 2 (3) ◽  
pp. 95-100 ◽  
Author(s):  
J. Dellundé ◽  
S. Pina ◽  
J. Jofre ◽  
F. Lucena
Keyword(s):  
Nucleic Acid ◽  
Environmental Samples ◽  
Environmental Water ◽  
Acid Extraction ◽  
Nucleic Acid Binding ◽  
Nucleic Acid Extraction ◽  
Naturally Occurring ◽  
Extraction And Purification ◽  
Guanidinium Thiocyanate ◽  
Cryptosporidium Oocysts

A new protocol based on a combination of an excystation process followed by a nucleic acid extraction that combines the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate and the nucleic acid-binding properties of silica particles is described for the extraction and purification of nucleic acids from Cryptosporidium. The application of nested and/or semi-nested PCR using different external and internal primers to DNA extracted by this method from seeded and naturally occurring Cryptosporidium oocysts concentrated and purified from environmental samples detects numbers of oocysts ranging from 20 to 50. The method is feasible, detects mostly excystable oocysts and no problems of inhibition of PCR were observed when applied to environmental samples.

Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction

The Analyst ◽  
2020 ◽  
Vol 145 (6) ◽  
pp. 2412-2419 ◽  
Author(s):  
Rachel N. Deraney ◽  
Lindsay Schneider ◽  
Anubhav Tripathi
Keyword(s):  
Nucleic Acid ◽  
Electric Field ◽  
Microfluidic Chip ◽  
Applied Electric Field ◽  
Electroosmotic Flow ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Magnetic Forces ◽  
Extraction And Purification

NA extraction and purification utilitzing a microfluidic chip with applied electric field to induce electroosmotic flow opposite the magnetic NA-bound bead mix.

scholarly journals Automated TruTip nucleic acid extraction and purification from raw sputum

PLoS ONE ◽  
2018 ◽  
Vol 13 (7) ◽  
pp. e0199869 ◽  
Author(s):  
Nitu Thakore ◽  
Ryan Norville ◽  
Molly Franke ◽  
Roger Calderon ◽  
Leonid Lecca ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Extraction And Purification

scholarly journals A bifurcated continuous field-flow fractionation (BCFFF) chip for high-yield and high-throughput nucleic acid extraction and purification

Lab on a Chip ◽  
2019 ◽  
Vol 19 (22) ◽  
pp. 3853-3861 ◽  
Author(s):  
Chenguang Zhang ◽  
Gongchen Sun ◽  
Satyajyoti Senapati ◽  
Hsueh-Chia Chang
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Yield ◽  
Acid Extraction ◽  
Field Flow Fractionation ◽  
Nucleic Acid Extraction ◽  
Isolation And Purification ◽  
Extraction And Purification ◽  
Flow Fractionation ◽  
Continuous Field

We report a new Bifurcated Continuous Field-Flow Fractionation (BCFFF) microfluidic chip for isolation and purification of nucleic acids from blood plasma with high and concentration-independent yield. The platform is ideal for isolation and quantification of small miRNAs.

scholarly journals Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

Micromachines ◽  
2017 ◽  
Vol 8 (7) ◽  
pp. 228 ◽  
Author(s):  
Darren Branch ◽  
Erika Vreeland ◽  
Jamie McClain ◽  
Jaclyn Murton ◽  
Conrad James ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Acid Extraction ◽  
Ultrasonic Technique ◽  
Nucleic Acid Extraction ◽  
Extraction And Purification

Semi-automatic instrumentation for nucleic acid extraction and purification to quantify pathogens on surfaces

The Analyst ◽  
2019 ◽  
Vol 144 (22) ◽  
pp. 6586-6594 ◽  
Author(s):  
Won-Nyoung Lee ◽  
Hyun Jin Yoo ◽  
Kim Huyen Nguyen ◽  
Changyoon Baek ◽  
Junhong Min
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Automated Detection ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Extraction And Purification ◽  
Short Time

A semi-automated detection system compatible with PCR that can detect infectious pathogens on wide surfaces in a short time.

scholarly journals Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

2011 ◽  
Vol 77 (13) ◽  
pp. 4336-4343 ◽  
Author(s):  
Akihiko Hata ◽  
Hiroyuki Katayama ◽  
Masaaki Kitajima ◽  
Chettiyappan Visvanathan ◽  
Chea Nol ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Water Samples ◽  
Rna Extraction ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Acid Extraction ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Viral Nucleic Acid

ABSTRACTInhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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