Droplet digital PCR, the new tool in HIV reservoir quantification?

Background: Digital PCR is a relatively old concept for absolute quantification of DNA using PCR, but recent technological developments allowed its wide use. The current state of art technique for performing digital PCR is based on microdroplet technology. Direct absolute quantification relieves the necessity of standard curves and increases assay accuracy. In addition, the end point PCR set-up allows higher assay flexibility and decreases quantitative bias due to variations in PCR efficiency. In the present work, these theoretical advantages were assessed on the QX100 droplet digital PCR (Bio-rad) on various virological markers to assess the possible use of ddPCR in HIV research.Methods: First, ddPCR was compared to a highly sensitive method of real-time PCR based quantification of cellular associated spliced and unspliced HIV RNA. Second, different methods of DNA extractions in combination with ddPCR were compared for quantification of total and episomal HIV DNA. Hereby, the maximal amount of restriction digested DNA was assessed in the ddPCR. Third, a touchdown procedure was optimized for an HIV specific primer probe set with a low melting temperature using touchdown ddPCR.Results: The comparsion of the nested real-time quantitative PCR to ddPCR indicated that ddPCR is at least equally sensitive to qPCR but also that false positive negative control samples may interfere with quantification at the level of single copies. Episomal 2LTR quantification was compared on ddPCR between total DNA extracted DNA and plasmid purified DNA, revealing a higher accuracy of 2LTR measurements in the total DNA extracts. Assessment of total DNA load in digital PCR reactions revealed a higher tolerance for inhibition compared to qPCR, but a strong influence of the concentration of restriction digestion mix on ddPCR efficiency. Finally, a touchdown procedure revealed that digital PCR can combine a higher flexibility in assay design while retaining accurate quantitative power compared to qPCRConclusions: We transferred 4 assays used in HIV reservoir research to the ddPCR platform. Although ddPCR has some major advantages for low level quantification in HIV reservoir research, some technical hurdles, including the occurrence of false negative control samples need still be addressed to ameliorate the current technology.Background: Digital PCR is a relatively old concept for absolute quantification of DNA using PCR, but recent technological developments allowed its wide use. The current state of art technique for performing digital PCR is based on microdroplet technology. Direct absolute quantification relieves the necessity of standard curves and increases assay accuracy. In addition, the end point PCR set-up allows higher assay flexibility and decreases quantitative bias due to variations in PCR efficiency. In the present work, these theoretical advantages were assessed on the QX100 droplet digital PCR (Bio-rad) on various virological markers to assess the possible use of ddPCR in HIV research.Methods: First, ddPCR was compared to a highly sensitive method of real-time PCR based quantification of cellular associated spliced and unspliced HIV RNA. Second, different methods of DNA extractions in combination with ddPCR were compared for quantification of total and episomal HIV DNA. Hereby, the maximal amount of restriction digested DNA was assessed in the ddPCR. Third, a touchdown procedure was optimized for an HIV specific primer probe set with a low melting temperature using touchdown ddPCR.Results: The comparsion of the nested real-time quantitative PCR to ddPCR indicated that ddPCR is at least equally sensitive to qPCR but also that false positive negative control samples may interfere with quantification at the level of single copies. Episomal 2LTR quantification was compared on ddPCR between total DNA extracted DNA and plasmid purified DNA, revealing a higher accuracy of 2LTR measurements in the total DNA extracts. Assessment of total DNA load in digital PCR reactions revealed a higher tolerance for inhibition compared to qPCR, but a strong influence of the concentration of restriction digestion mix on ddPCR efficiency. Finally, a touchdown procedure revealed that digital PCR can combine a higher flexibility in assay design while retaining accurate quantitative power compared to qPCRConclusions: We transferred 4 assays used in HIV reservoir research to the ddPCR platform. Although ddPCR has some major advantages for low level quantification in HIV reservoir research, some technical hurdles, including the occurrence of false negative control samples need still be addressed to ameliorate the current technology.C