8/6/2019 TechNote_Noro_october1305
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PSS Bio Instruments, Inc. Technical bulletin-101305-1
MagtrationSystem 12GC: Application data-----Microbiology
RNA extraction of Norovirus from stool using
Magtration-MagaZorbRNA Common Kit-200Theodore Chiu and Linda Chui.
Provincial Laboratory for Public Health (Microbiology), Edmonton,
Alberta, Canada
PSS Bio Instruments, Inc.
IntroductionMagtration
System 12GC is a fully automated
DNA/RNA isolation system coupled with an extractionkit,
Magtration
-MagaZorb
DNA or RNA. To
determine the applicability of this instrument in adiagnostic
Microbiology laboratory for virus detection instool samples,
noroviruswas selected. The objectiveof this study was to evaluate
the efficiency of extractionas compared with the established
routine extractionprotocol.
Materials and Methods
Preparation of stool samples for RNA extraction
An aliquot of approximately 100 L of fecal samplewas added to
500L DEPC water and mixed by vortexfor 30 sec. The suspension was
centrifuged @ 7000xgfor 5 min. The supernatant was used for RNA
isolation.
RNA IsolationThe routine protocol for RNA extraction in
thislaboratory for norovirus from stool sample is a silica-based
method as described by Boom et al (1990). A100L volume of the above
supernatant was mixedwith 500 L of lysis buffer, 20 L of silica was
addedand mixed for 10 sec. The mixture was kept at roomtemperature
for 15 min on a rotator and followed bycentrifugation for 20 sec at
13,000xg. The supernatantwas discarded and 1000L of wash buffer was
addedto the pellet, mixed by vortex and centrifuged for 20sec at
13000xg. Supernatant was discarded and thewashing procedure was
repeated. A second wash with1000L of 70% ethanol (in DEPC water)
was added tothe pellet, vortexed and mixed on the rotator for 5
minand followed by centrifugation at 13000xg for 20 sec.The
supernatant was discarded and the wash step wasrepeated. A third
wash step with 1000L of acetonewas added to the pellet, vortexed
and centrifuged at13000xg for 20 sec. Supernatant was discarded
andthe pellet was dried for 10 min at 60C in heatingblock. The
pellet was resuspended in 50L of DEPCwater and incubated for 10 min
at 60 C for 10 min.The mixture was centrifuged for 5 min at 13000xg
andthe supernatant containing RNA was transferred to anew tube.
RNA Isolation using Magtration
System 12GCRNA was extracted with Magtration
-MagaZorb
RNA
Common Kit-200 by Magtration
System 12GC. Therewas no DNase treatment. A 100L supernatant
asdescribed in the section of preparation of stool
samples was applied to the instrument and RNA waseluted with 50L
volume.
RT-PCRReverse transcription was performed to generatecDNA for
Real-time PCR with ABI Prism detectionsystem.
ResultsComparison Study with clinical samplesForty nine randomly
selected samples extracted byboth methods were compared and the
results werepresented in the following table.
RoutineMagtration
Positive Negative
Pos. 31 1Neg. 1 16
There were 2 samples that showed discordant results.By repeating
the extraction and amplification, these 2samples turned positive
but with very late crossingpoints. The previous negative results
were most likelydue to sampling error.
Reproducibility of RNA Isolation using Magtration
System 12GCConfirming the precision and reproducibility of
theMagtration
12GC, one sample was extracted 5 times
and run simultaneously on the ABI Prism Detectionsystem.
Accession # CrossingPoint
05MD2510 29.0105MD2510 28.3005MD2510 28.3805MD2510 28.4505MD2510
29.26
All Crossing points are within 1 cycle showing the
reproducibility of the extraction by Magtration
12GCon stool specimens.
ConclusionThe RT-PCR results using RNA extracted from
stoolspecimens by Magtration
System 12GC with
Magtration
-MagaZorb
RNA kit-200 for Norovirusshowed comparable results with our
routine extractionprotocol.
Boom et al (1990) J. Clin Microbiol 2: 495-503
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