Many kits and protocols for nucleic acid isolation - manual and automated - are available today. However, very few of them guarantee a fully automated sample preparation, combined with maximum recovery and sensitivity. Therefore we have developed the new MagNA Pure LC Total Nucleic Acid Isolation Kit - High Performance. The kit is based on the well established magnetic bead technology, that is successfully being used on the different MagNA Pure Systems for many years. This new kit is optimized to guarantee a very high recovery of total viral DNA and RNA from plasma, serum and whole blood, resulting in a high sensitivity in viral NA detection. In addition, it enables the processing of a wide range of sample volumes from 100 to 1,000 µl. Furthermore, optional external lysis protocols are available to enable virus inactivation outside the MagNA Pure LC Instrument, if desired.
Materials and Methods
The new kit was developed using human EDTA plasma, citrate plasma, serum and whole blood as sample material. For this study the samples were spiked with different amounts of DNA viruses (CMV, EBV) and RNA viruses (HAV, Influenza A) and processed on the MagNA Pure LC Instrument and also on competitor platforms. All samples were tested at least in four-fold replicates. The elution volume of the purified nucleic acid was 50 or 100 µl. Analysis was performed on the LightCycler® 480 Instrument, using parameter-specific quantitative real-time PCR and RT-PCR assays for the four virus types listed above. The amplification reaction was set up with 15 µl of the respective master mix and 5 µl of the respective eluate and was run for 45 cycles.
Results and Discussion
All kit components have been optimized, many different reagent compositions were tested and all processing steps and reaction conditions were varied to find the optimal total nucleic acid (TNA) isolation procedure. The new protocols are compatible with both the MagNA Pure LC 1.0 Instrument and the MagNA Pure LC 2.0 Instrument.
It was found that the highest sensitivity was achieved in combination with a very intensive binding step, resulting in a relatively long run time (180 minutes for 32 samples). This “High Sensitivity (Total NA HS) Protocol” enables the detection of less than 100 virus copies per isolation (using 200 µl sample volume), theoretically being equivalent to less than 10 copies per (RT-) PCR. This protocol showed good results in our sensitivity tests (Table 1), comparable to, or even better than the best competitor system (in our hands).
However, for many applications such a maximum sensitivity is not necessary, therefore it was decided to also offer a shorter “High Performance (Total NA HP) Protocol”, that is a little less sensitive, but much faster (90 minutes for 32 samples).
In order to compare the recovery of the new kit with that of the best competitor method, the new “Total NA HP” and “Total NA HS” protocols and competitor B were run in parallel using “medium” virus concentrations of approximately 104 copies per sample. The results were similar to those of the hit rate experiments: The new kit and protocols showed an excellent performance (Figure 1), especially the Total NA HS Protocol was comparable to or even than the best competitor system (in our hands).
Cross Contamination Tests
The new reagents and protocols were checked for cross contamination, using 16 positive and 16 negative plasma samples, arranged in a checkerboard pattern. The positive samples were spiked with plasmids, containing a Parvo B19 specific sequence, in a final concentration of 1x107 and 2.5x108 plasmids/ml. The isolation runs were performed on both the MagNA Pure LC 1.0 Instrument and the MagNA Pure LC 2.0 Instrument. Analysis of the eluates was performed using automated PCR set-up on both MagNA Pure LC Instruments, followed by a sensitive Parvo B19 specific PCR on the LightCycler® 480 System. No cross contamination was found.
Large Volume Protocols
The new kit is able to process sample volumes of 100, 200, 500 and 1,000 µl. The reagent amounts allow 288 isolations of 100 or 200 µl samples or 192 isolations of 500 µl samples or 96 isolations of 1,000 µl samples. The protocols for 500 and 1,000 µl samples should only be used with plasma or serum, not with whole blood, due to the high amount of genomic DNA in the blood cells.
This study shows that the new MagNA Pure LC Total Nucleic Acid Isolation Kit - High Performance enables the fully automated efficient isolation of total viral nucleic acids from various sample materials, with a high recovery and sensitivity. The user can choose between several protocol variants according to the specific needs. In addition, the flexible reagent concept enables the processing of a wide range of sample volumes. Therefore the new kit will be a useful tool for the automated analysis of infectious disease parameters.
This article was originally published in Biochemica 2/2009, pages 19-20. ©Springer Medizin Verlag 2009