Akonni TruTip(®) and Qiagen(®) methods for extraction of fetal circulating DNA--evaluation by real-time and digital PCR.

Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed--a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.

Automated, simple, and efficient influenza RNA extraction from clinical respiratory swabs using TruTip and epMotion.

BACKGROUND: Rapid, simple and efficient influenza RNA purification from clinical samples is essential for sensitive molecular detection of influenza infection. Automation of the TruTip extraction method can increase sample throughput while maintaining performance. OBJECTIVES: To automate TruTip influenza RNA extraction using an Eppendorf epMotion robotic liquid handler, and to compare its performance to the bioMerieux easyMAG and Qiagen QIAcube instruments. STUDY DESIGN: Extraction efficacy and reproducibility of the automated TruTip/epMotion protocol was assessed from influenza-negative respiratory samples spiked with influenza A and B viruses. Clinical extraction performance from 170 influenza A and B-positive respiratory swabs was also evaluated and compared using influenza A and B real-time RT-PCR assays. RESULTS: TruTip/epMotion extraction efficacy was 100% in influenza virus-spiked samples with at least 745 influenza A and 370 influenza B input gene copies per extraction, and exhibited high reproducibility over four log10 concentrations of virus (<1% CV). RNA yields between the three automated methods differed by less than 0.5 log10 gene copies. 99% of clinical specimens that were PCR-positive after easyMAG or QIAcube extraction were also positive following TruTip extraction. Overall Ct value differences obtained between TruTip/epMotion and easyMAG/QIAcube clinical extracts ranged from 1.24 to 1.91. Pairwise comparisons of Ct values showed a high correlation of the TruTip/epMotion protocol to the other methods (R2>0.90). CONCLUSION: The automated TruTip/epMotion protocol is a simple and rapid extraction method that reproducibly purifies influenza RNA from respiratory swabs, with comparable efficacy and efficiency to both the easyMAG and QIAcube instruments.

High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots.

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).