Evaluation of Different Sample Types with the Maxwell® 16 Viral Total Nucleic Acid Purification Kit Michelle Mandrekar1, Susan Koller1 and John Fulmer2 1Research and Development, Promega Corp. Madison, WI. 2Avero Diagnostics, Dallas, TX 1. 4. Introduction Purifying viral nucleic acid from a variety of human sample sources is an important step in identifying many viruses and determining viral load. The Maxwell® 16 LEV Viral Total Nucleic Acid Purification Kit was designed to purify nucleic acid from viruses in plasma or serum. 5. Sample 200ul NATrol Control Serum 50ul swab-transport media 40ul swab-transport media 20ul swab-transport media 10ul swab-transport media 5ul swab-transport media Binax Positive Control FluA&B Binax Negative Control FluA&B Instrument in Low Elution Volume (LEV) format •Maxwell® 16 cat# AS1000 or AS2000 (need firmware 4.61 or higher) OR •Maxwell®16 MDx cat# AS3000 (firmware for MDx has Viral method) 6. The Standard Method Add Lysis Buffer and Proteinase K to tube Virus in 100, 200 or 300ul plasma or serum The cartridge is loaded into a Maxwell® 16 instrument where RNA and DNA are bound to a paramagnetic particle, washed, then eluted Vortex lysate plasma samples incubated 10 minutes at 56ºC Serum samples incubated 10 minutes at room temp then 10 minutes at 56°C Lysate is transferred into the LEV cartridge Nasal Swabs The nasal swab was vortexed for 30 seconds in M4-RT. 300ul of liquid was removed and processed with the standard plasma method. As a control, 200ul of Influenza A NATtrol was also processed. The eluates were assayed with Universal Flu A TaqMan® primers and probe in AgPath-ID One-Step RT-PCR. Reagent •Maxwell® 16 Viral Total Nucleic Acid Purification Kit (Cat# AS1150) Add plunger and elution tube with 50ul water Ct Value 18.3 21.6 23.1 23.8 24.3 30.2 25.4 No Ct 7. Cervical Swab Processing The cervical swab specimens were centrifuged and the supernatant removed. The cervical cells were resuspended in 200ul TE and vortexed. •46 SurePathTM Pap Test specimens were boiled 15 minutes, and returned to room temperature before processing with the standard Maxwell Viral plasma method or Agencourt® Genfind (Agencourt Biosciences). •50 ThinPrep Pap Test specimens were processed with the standard Maxwell Viral plasma method or Agencourt Genfind. Preserved Cervical Swab Specimens: SurePathTM Pap Test specimens (BD) ThinPrep Pap Test specimens (Hologic TM) Reagents and Instrument 3. Nasal Swabs: 5-50ul of Influenza A NATtrolTM (Zeptometrix) was applied to a nasal swab with dried mucus from a non-infected donor, and immediately placed in MicrotestTM Transport Media M4RT (Remel). Flu A&B Control Swabs (Binax) were also placed in M4RT media. Bodily Fluids: 5-50ul BK Virus NATtrolTM (Zeptometrix) Control was added to a urine specimen or plasma. We are currently developing research applications for this kit to purify viral nucleic acid from other sample sources: nasal swabs in transport media, bodily fluids (e.g. urine) and preserved cervical swab specimens. 2. Test Sample Preparation Result: FluA was detected on all swabs with FluA, and not detected on the negative control. Bodily Fluids 8. Cervical Swab Results The eluate was assayed with CervistaTM HPV HR assay (Hologic). n = 96 Agencourt Genfind Extraction Maxwell 16 Viral TNA Extraction Positive 24 25 Negative 72 71 Result: 99% of the samples gave the same result (positive or negative). Maxwell extraction was comparable to Agencourt Genfind extraction. 9. Conclusions 200ul of urine, with 5-50ul BKV, was processed with the standard plasma method. The eluates were assayed with TaqMan® primers and probe in AgPath-ID One-Step RT-PCR. BK NATtrol was used as a reference standard curve (R2= 0.987). The Maxwell® 16 LEV Viral Total Nucleic Acid Purification Kit purified nucleic acid from a variety of sample types (nasal swabs, urine, and cervical swabs) with little or no additional processing before the standard plasma purification method. Result: BK virus was detected in all urine and plasma samples. The RNA or DNA purified from viruses was suitable for use in downstream applications, such as real-time PCR. www.promega.com