<title>Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples</title>

AbstractEuropean manufacturers of plasma products and German blood transfusion services were the first to introduce nucleic acid amplification testing (NAT) of blood products in the mid-1990s. Their primary goal was to increase the safety of blood by closing as far as possible the diagnostic window, which exists after the onset of viral infection until the appearance of the first detectable antibodies. Sample preparation, transport and storage are crucial steps in a quality-controlled PCR. Sensitivity and contamination rates highly depend on the sample preparation and storage techniques. Anticoagulants must be selected carefully because some may inhibit the PCR. Dilution of samples by pooling needs to be considered and should be compensated for by subsequent virus enrichment procedures, e.g. centrifugation. The whole process of sample preparation, pooling and virus enrichment must be validated and quality control measures must be implemented. Reagents for the extraction of viral nucleic acids should not pose any risk to the laboratory staff. Nevertheless, the reagents should be highly efficient in liberating viral nucleic acids at high yield and purity for the following amplification reactions. At this critical stage, quality control measures should guarantee an efficient extraction process and contain potential sources of contaminations. Several methods are available for the amplification of nucleic acids. PCR is the most common, especially in in-house assays. The amplification of nucleic acids should be performed as far as possible in a closed system, which may be guaranteed best by real-time PCR approaches. Reaction tubes need never be opened during the amplification because detection can be performed through the closed tube. Amplicons that could contaminate the following PCR reactions will not be released. It is of great importance to blood transfusion services to guarantee that negative results un-equivocally indicate virus negative blood donations. Therefore, internal control sequences should be implemented in each individual PCR reaction in order to monitor that the individual PCR has worked correctly. Besides internal control sequences, external negative and positive controls should be implemented in each PCR run to demonstrate false positive reactions as well as to monitor pre-PCR processes like virus enrichment and extraction. The whole process needs to be validated according to the criteria set in national guidelines or by national authorities. External quality assessment programs are highly recommended.

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Filter paper-based spin column for low throughput nucleic acid purification

10.1101/392696 ◽

2018 ◽

Author(s):

Rui Shi ◽

Ramsey S. Lewis ◽

Dilip R. Panthee

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Filter Paper ◽

Genomic Dna ◽

Total Rna ◽

Binding Material ◽

Pcr Product ◽

Spin Column ◽

Nucleic Acid Purification ◽

Commercial Kits

AbstractWe describe a method of recharging used spin column or assembling homemade spin column using filter paper as binding material for low throughput nucleic acid purification. We evaluated the efficiency of filter paper based spin columns in the purification of different type of nucleic acids. For instance, by following protocols of respective commercial kits, we found that filter paper to be a useful binding material for purification of many types of nucleic acids, including plant genomic DNA, plant total RNA, PCR product, and DNA from agarose gels. We also found that filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Also, we present the protocols of using filter paper recharged spin column or homemade spin column for low throughput purification of plant genomic DNA and plant total RNA with commercial kit buffer leftover and less expensive homemade buffer.

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Microfluidic sample preparation: cell lysis and nucleic acid purification

Integrative Biology ◽

10.1039/b905844c ◽

2009 ◽

Vol 1 (10) ◽

pp. 574 ◽

Cited By ~ 186

Author(s):

Jungkyu Kim ◽

Michael Johnson ◽

Parker Hill ◽

Bruce K. Gale

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Cell Lysis ◽

Nucleic Acid Purification

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Spatially isolated reactions in a complex array: using magnetic beads to purify and quantify nucleic acids with digital and quantitative real-time PCR in thousands of parallel microwells

Lab on a Chip ◽

10.1039/d0lc00069h ◽

2020 ◽

Vol 20 (10) ◽

pp. 1771-1779 ◽

Cited By ~ 1

Author(s):

W. Hampton Henley ◽

Nathan A. Siegfried ◽

J. Michael Ramsey

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Nucleic Acids ◽

Real Time ◽

Real Time Pcr ◽

Magnetic Beads ◽

Digital Pcr ◽

Quantitative Real Time Pcr

Encoded beads carrying primer pairs for nucleic acid targets are used for sample preparation and multiplexed-in-space digital PCR quantification.

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Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis

Analytical Chemistry ◽

10.1021/acs.analchem.0c02454 ◽

2020 ◽

Vol 92 (19) ◽

pp. 13254-13261

Author(s):

David E. Gaddes ◽

Pei-wei Lee ◽

Alexander Y. Trick ◽

Pornpat Athamanolap ◽

Christine M. O’Keefe ◽

...

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Nucleic Acid Amplification ◽

Nucleic Acid Amplification Testing ◽

Automated Sample Preparation

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The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa

10.1101/2020.08.06.20169276 ◽

2020 ◽

Author(s):

Gert Johannes Kruger Marais ◽

Michelle Naidoo ◽

Nei-yuan Hsiao ◽

Ziyaad Valley-Omar ◽

Heidi Smuts ◽

...

Keyword(s):

South Africa ◽

Nucleic Acid ◽

Sample Preparation ◽

Preparation Method ◽

Preparation Technique ◽

Rt Pcr ◽

Sample Preparation Method ◽

Diagnostic Laboratory ◽

Percent Agreement ◽

Nucleic Acid Purification

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

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A Completely Automated Sample Preparation Instrument and Consumable Device for Isolation and Purification of Nucleic Acids

Journal of Laboratory Automation ◽

10.1016/j.jala.2010.08.004 ◽

2011 ◽

Vol 16 (5) ◽

pp. 355-365 ◽

Cited By ~ 4

Author(s):

Kyle W. Hukari ◽

Michael Shultz ◽

Nikolas Isely ◽

Richard Milson ◽

Jay A.A. West

Keyword(s):

Sample Preparation ◽

Nucleic Acids ◽

Isolation And Purification ◽

Automated Sample Preparation

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Nucleic Acid Molecules Prepared by Monolayer Techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094140 ◽

1972 ◽

Vol 30 ◽

pp. 178-179

Author(s):

Dimitrij Lang

Keyword(s):

Electron Microscopy ◽

Standard Deviation ◽

Nucleic Acid ◽

Cytochrome C ◽

Nucleic Acids ◽

Contour Length ◽

Sample Standard Deviation ◽

Molecular Weights ◽

Length Measurements ◽

Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

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