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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method

Published: August 6th, 2016

DOI:

10.3791/54289

1Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, 2Pritzker School of Medicine, The University of Chicago

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

Several reports have discussed the development of paper- or membrane-based extraction methods for use in point-of-care (POC) devices 1-5 with the aim of making the exquisite sensitivity and specificity of molecular diagnostics available to all. The World Health Organization (WHO) Sexually Transmitted Diseases Diagnostics Initiative coined the term ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Delivered to those who need it) to describe the ideal characteristics of a POC test 6. Of these guidelines, the equipment-free characteristic is particularly challenging to achieve for molecular diagnostics. Ho....

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Ethics statement: The whole blood specimens used in this study are not considered to be research involving human subjects. The specimens were obtained for clinical diagnostic purposes, which were satisfied, and the remaining portion of these specimens was provided for the FINA research assay. The specimens were coded such that the investigators were not able to readily ascertain the identity of individuals.

1. Preparation of FINA Sample Preparation Module

  1. Prepare blotting pad by cutting a 35 x 35 mm

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The workflow for extracting proviral DNA from whole blood spiked with 8e5-LAV cells is shown in Figure 1. Figure 2 shows the FINA sample module and prepared qPCR tube. This method allows for efficient amplification of HIV-1 provirus from 8e5-LAV cells at different copy numbers, as shown in the standard curve of contrived specimens (Figure 3). PCR had an efficiency of 103%, as calculated from Efficiency = -1+10(-1/slope). Equati.......

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EID linked to rapid treatment access has been demonstrated to reduce infant mortality due to HIV infection 16. Because of the persistence of maternal antibodies in an infant's blood, rapid HIV antibody tests have limited utility in determining the status of HIV-exposed infants. The WHO recommends that all infants born to HIV-1 positive mothers should be tested at 4-6 weeks of age, using a virological test 17. We have reported the development of an assay for the detection and quantitation of HIV-.......

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This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

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Name Company Catalog Number Comments
Fusion 5 GE Healthcare Life Sciences 8151-9915
707 blotting pad VWR International 28298-014
PARAFILM M VWR International 52858-000
3M Double-Coated Polyester Diagnostic Tape  3M Medical Specialties 9965
200 µl qPCR strip tubes Agilent 401428
optical strip caps Agilent 401425
Mx3005p qPCR System Agilent 401456
sodium hydroxide Sigma-Aldrich 221465 A.C.S. Reagent
Triton X-100 Sigma-Aldrich T9284 BioXtra
dimethyl sulfoxide Sigma-Aldrich D8418 for molecular biology
fetal bovine serum, certified, U.S. origin Thermo Fisher Scientific 16000-044
Hammer driven small hole punch 3/16" hole diameter (5.1 mm) McMaster Carr 3424A16
Hammer driven small hole punch 1/4" hole diameter (7.14 mm) McMaster Carr 3424A19
Hammer driven small hole punch 5/16" hole diameter (8.35 mm) McMaster Carr 3424A23

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