The overall goal of this FINA method which stands for filtration isolation of nucleic acids is to isolate genomic DNA for PCR amplification from complex specimens quickly and simply without the use of laboratory instrumentation such as a centrifuge. This rapid paper based DNA extraction method for HIV pro viral DNA was developed for incorporation into a quantitative PCR diagnostic device. The main advantage of this technique is that the sample prep modules and PCR tubes can be prepared in advance so the DNA extraction takes less than two minutes.
We first developed this method to be incorporated into a sample to answer early infant diagnostic test for HIV-1 from heelstick blood. Generally individuals new to this method may struggle because removing the backing of the double sided tape that holds the cell capture membrane to the side of the PCR tube requires practice. Demonstrating the procedure will be Jen Reed, a senior technician from my lab.
Begin this procedure by preparing the materials needed for assembling the FINA sample preparation module. Prepare the blotting pad by cutting a 35 by 35 millimeter square of a 2.6 millimeter thick 100%cotton pad. Prepare plastic paraffin film with a paper backing tape.
Cut a 25 by 50 mm piece of paraffin film and then use a hammer to punch a 7.14 mm diameter hole in the center of the tape. Prepare a bound glass blood separator membrane or capture disk by using a hammer to punch an 8.35 mm diameter circle. To assemble the FINA sample preparation module use forceps to place the capture disc in the center of the blotting pad.
Remove the backing of the paraffin film tape and place it over the capture disc so that the whole of the paraffin tape leaves the center of the capture disc exposed. Ensure maximum contact between the disc 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 disc. A 5.1 mm tape disc is required to anchor the capture membrane to the side of qPCR tube to prevent the membrane from blocking the fluorescence detection of the real time PCR instrument.
Prepare this tape disc by using a hammer to punch a circle of double coated polyester diagnostic tape from a sheet of the tape. Using forceps remove one side of the tape sticker liner. Place the tape into a 200 microliter 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 the prepared disc. The detection of proviral DNA from whole blood spiked with 8e5 LAV cells will be demonstrated in this video.
Thaw out the previously frozen 8e5 LAV cell pellets on ice. Prepare a serial dilution of the cells in freezing medium with concentrations ranging from 1 to 400 cells per microliter. Pipette 10 microliters of cells from each of the dilutions into a micro centrifuge tube.
Add 100 microliters of fresh HIV-1 negative eDTA treated whole blood sample to each tube to create a standard curve. Mix by gently flicking the bottom of the tube five times. To begin the DNA extraction procedure, lyse the blood cells by adding triton X-100 to a final concentration of one percent.
Mix by gently flicking the bottom of the tube five times. The blood should turn a translucent red. Pipette the entire blood specimen onto the capture disc.
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 contact membrane and blotting pad assembly ensures rapid sample wicking. Allow the sample to soak through the filter.
The blood lysate has soaked into the capture disc and the disc appears mat. Add 600 to 1, 000 microliters of 10 mmol sodium hydroxide dropwise onto the capture disc. The filter will change from red to white, indicating clearance of hemoglobin.
Next separate the disc from the blotting pad and apply the disc to the tape in the prepared PCR tube. After the filter is placed in the PCR tube analyze the sample right away. Prepare the qPCR master mix as described in the protocol text and be sure that the filter is completely covered with the master mix and stays fixed to the side of the tube throughout the reaction.
Perform amplification using a real time PCR device as described in the protocol text. Alternatively if qPCR cannot be performed immediately dry the filter overnight in a box containing calcium sulfate dessicant. On the following day, cap and place the PCR tube in a foiled pouch with silica dessicant and store at room temperature until ready for analysis.
This method allows for efficient amplification of HIV-1 provirus from 8e5 LAV cells at different copy numbers. The solid lines are amplification plots obtained from four replicates of HIV-1 negative whole blood spiked with 4, 000, 400, 40 and 10 8e5 LAV cells with 500 copies per reaction of the internal control. The dotted line indicates the threshold.
This next graph shows standard curves of quantitation cycle values calculated from the amplification plot vs. log copy number of 8e5 LAV cells per 100 microliters of whole blood. The calculated PCR efficiency was 103%The HIV proviral DNA standard curve replicates also have highly reproducible amplification.
Additionally the presence of an internal control of 500 copies of hydroxy pyruvate reductase shows that there is no PCR inhibition resulting from extracting blood with this method independent of the number of copies of HIV-1 provirus present. The solid lines represent the amplification plots and the dotted line represents the threshold. Following this procedure PCR targets can be amplified from various specimen types to detect your gene of interest.
For example cheek swabs for pharmacogenetic screening or blood for rapid genotyping of animal models. Don't forget that working with blood borne pathogens can be extremely hazardous and universal precautions should always be taken while performing this procedure.
