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

Summary

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.

Abstract

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.

Introduction

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. However, every innovation in the field will advance the goal of reaching those in most need, and there is hope for near-term improvements in test performance by adapting existing technology ⁷.

Here we describe a simple protocol for extracting DNA from whole blood that does not require complex chemistry or laboratory instrumentation. The FINA (filtration isolation of nucleic acids) sample preparation method was originally developed to extract leukocyte DNA from whole blood in order to detect the HIV-1 provirus as part of a sample-to-answer point-of-care (POC) quantitative PCR (qPCR) early infant diagnostic (EID) platform for use in limited resource settings ^8-11. FINA extraction differs from conventional purification methods which use silica membranes or silica-coated paramagnetic particles to reversibly bind DNA in the presence of chaotropic agents ¹². Instead, FINA uses vertical filtration via a separation membrane to extract cellular DNA from whole blood directly. The membrane containing the entrapped DNA can be placed directly in a PCR tube and either immediately used in a PCR reaction or air dried for later amplification ⁹. No chaotropic agents, phenol, or alcohols are used in the sample extraction, eliminating the extensive washing steps needed to remove potent qPCR inhibitors derived from the extraction process ^13,14.

The FINA membrane can capture either cells ⁹ or genomic DNA liberated by cell lysis ¹¹ before the specimen is added to the membrane. For the cell capture, whole blood is added directly to the membrane. The cells are subsequently lysed in the membrane by adding 10 mM NaOH. The advantage to this method is that it involves only 3 steps: 1) sample addition; 2) cell lysis/wash and 3) filter disk placement in qPCR tube. The drawback to this method is that the membrane can only hold a defined number of cells directly proportional to the diameter of the membrane disk. To reach the limit of detection required for EID, 100 µl of whole blood is required for sample input which entails a filter that is too large to be placed in a qPCR tube. Lysing the blood cells with detergent before adding the sample to the collection membrane adds a processing step, but allows the use of a smaller filter for the same sample size. We were able to demonstrate high reproducibility, single copy detection, and quantification of as little as 10 copies of HIV-1 proviral DNA from 100 µl of blood using this test configuration ¹¹.

In this report, we describe the FINA protocol as originally developed for laboratory use. The membrane/filter sandwich known as the FINA sample preparation module can be prepared in large batches and stockpiled for later use. When specimens are to be extracted this process takes 2 min and can be performed in varying size batches. The qPCR can be run immediately or the filters containing the embedded DNA can be stored until it is convenient to perform qPCR. This method is very convenient for routine analysis of specimens in both low and high resource settings.

Protocol

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 mm² of a 2.6 mm thick 100% cotton pad. Cut one pad per specimen.
2. Prepare plastic paraffin film with a paper backing tape by a cutting piece of paraffin film (25 mm (h) by 50 mm (w)) and then punching a 7.14 mm diameter hole in the center of the tape using a hammer driven hole punch. Prepare one tape per specimen.
3. Prepare a bound glass blood separator membrane (capture membrane) disk by punching a 8.35 mm diameter circle using a hammer driven hole punch. Punch one disk per specimen. The disk has a particle retention capability of 2.3 µm.
4. Assemble the FINA sample preparation module by placing the capture disk in the center of the blotting pad using forceps. Place the paraffin film tape over the capture disk so that the hole of the paraffin tape leaves the center of the capture disk exposed.
5. Ensure maximum contact between the disk and the blotting pad by stretching the paraffin tape slightly to cover the blotting pad and pressing the paraffin tape firmly to stick it to the blotting pad around all edges of the disk.
6. Make as many modules as needed for the experiment or stockpile for future use.

2. Preparation of qPCR Tube

1. Prepare a 5.1 mm tape disk that will anchor the cell capture membrane to the side of the qPCR tube to prevent the membrane from blocking the fluorescence detection by punching a circle of double-coated polyester diagnostic tape with a hammer driven punch from a sheet of the tape. Prepare one tape disk per specimen.
2. Remove one side of the tape's sticker liner. Place the tape into a 200 µl PCR tube, sticking it onto one side and toward the bottom of the tube. Remove the second sticker liner. The tube is now ready to receive prepared disk.
3. Produce as many tubes as needed for the experiment or stockpile for future use.

3. Contrive Blood Specimen

Note: If a genuine test specimen is being prepared, proceed to step 4.

1. Thaw 8e5-LAV cells ¹⁵ harboring a single copy of the HIV-1 provirus obtained from the Virology Quality Assurance Laboratory (VQA; Rush Presbyterian/St. Luke's Medical Center, Chicago, IL).
   Note: They are stored as frozen cell pellets at a concentration of 4,000 cells/µl. Cell count was verified as described previously ⁹.
2. Prepare serial dilution in freezing medium (90% fetal bovine serum, 10% dimethyl sulfoxide) to concentrations ranging from 1 to 400 cells/µl.
3. Pipet 10 µl cells from each of the serial dilutions into a microcentrifuge tube. Add 100 µl of fresh HIV-1 negative EDTA treated whole blood sample to each tube to create a standard curve of 8e5-LAV copy numbers 10-4,000. Mix by gently flicking the bottom of the tube 5 times.

4. Perform FINA Extraction

1. Lyse blood cells by adding Triton-X100) to a final concentration of 1% (11 µl 10% Triton-X100 to 110 µl whole blood and cells from step 3.3).
2. Mix by gently flicking the bottom of the tube 5 times. The blood should turn translucent red.
3. Pipet all of the blood specimen onto the capture disk.
   Note: A water tight seal between the paraffin tape and the capture membrane ensures that the blood flows through the membrane, and good contact between the capture membrane and blotting pad assembly ensures rapid sample wicking.
4. Allow sample to soak through the filter.
   Note: The blood lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the capture disk. The lysate has soaked into the disk when it appears matte.
5. Add 600-1,000 µl 10 mM NaOH drop-wise onto the capture disk.
   Note: The wash buffer can also be added as a bulk addition of 600 µl. The buffer will form a droplet on the hydrophobic paraffin tape and wick through the hole to the disk in approximately 20 sec. The filter will change from red to white indicating clearance of hemoglobin.
6. Separate the disk from the blotting pad with forceps and apply the disk to the tape in the prepared PCR tube. If there is any red color left on the filter add 50-100 µl of wash buffer to clear the filter before placing in the tube.
   Note: Infrequently, excess pressure applied during preparation of the FINA modules results in partitioning of the membrane layers during separation from the blotting pad. Discard these disks and prepare a fresh sample.
7. After the filter is placed in the PCR tube, analyze the samples right away. Alternatively, dry overnight in a box containing calcium sulfate desiccant, then cap and place in a foil pouch with silica desiccant and store until ready for analysis.

5. qPCR Reaction

1. Prepare 100 µl qPCR master mix per reaction using HIV specific primers and probes as described previously ^9,11. Include an internal amplification control to monitor for the presence of amplification inhibitors and to verify that a negative sample is a true negative. Be sure that filter is completely covered with master mix and that the filter stays fixed to side of tube throughout the reaction.
2. Perform amplification using a real-time PCR device as previously described ⁹.

Results

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...

Discussion

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-...

Materials

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