Evaluation of Different Sample Types with the Maxwell ®

Evaluation of Different Sample Types with the Maxwell ®

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