Transcript artus HCV RG RT-PCR

HCV RNA purification optimisation results in higher analytical
sensitivity for the artus HCV RG RT-PCR assay
Micalessi MI., Cuypers A., Peeters B., Smismans A., Van Meensel B., Frans J.
Laboratory of Clinical Microbiology, Imelda Hospital, Bonheiden, Belgium.
8th EMMD 2013
Scheveningen
BACKGROUND
The artus HCV RG RT-PCR kit (Qiagen) combined with the QIAamp MinElute Virus Spin kit (Qiagen) for viral RNA purification is used in our daily routine for the
detection and quantification of HCV RNA in plasma. The assay can quantify HCV RNA over the range of 400 to 1 x 106 IU/ml and is also able to pick up 50 IU/ml
with 95% probability. Moreover, this approach is accredited under the ISO 15189 standard.
However, the current guidelines for therapy of chronic hepatitis C genotype 1 with protease inhibitors require the use of quantitative HCV RNA assays with a lower
limit of quantification (LLOQ) of less than or equal to 25 IU/ml and a lower limit of detection (LLOD) of approximately 10-15 IU/ml.
AIM
The HCV RNA purification protocol was adapted to increase the analytical sensitivity of the artus HCV RG RT-PCR assay.
MATERIAL AND METHODS
• Dilution series of the 4th WHO international standard (NIBSC): 200 IU/ml to 6.25 IU/ml.
HCV RNA
- 1000 µl instead of 400 µl HCV RNA
sample volume
- 30 µl instead of 60 µl detection
elution volume
purification
QIAamp MinElute Virus Spin
Vacuum protocol
artus HCV RG RT-PCR assay
RESULTS
Figure 1 Determination of the LLOD for the artus
HCV RT-PCR assay with the adapted purification
protocol. Minimum 4 replicates at each concentration
(200, 50, 25, 12.5, 6.25 IU/ml) were tested on 2
different days. Probit analysis was performed using
xlstat (version 2013.1).
Probability
0.05
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
0.95
0.99
IU/ml
2.270
4.176
6.483
8.147
9.569
10.898
12.226
13.648
15.312
17.619
19.525
23.099
Table 2 Probit analysis showed a detection rate of
95% and 80% at 19.5 IU/ml and 15.3 IU/ml,
respectively.
Table 1 Results of the artus HCV RT-PCR assay for the
HCV RNA extracts of several dilutions. Pos, positive;
Neg, negative.
CONCLUSION
• The optimised QIAamp MinElute Virus Vacuum protocol combined with the artus HCV RG RT-PCR kit resulted in a 95% LLOD of 19.5 IU/ml, which is slightly
higher than the targeted LLOD.
• In future experiments, the sample volume will be increased to obtain an LLOD of 10-15 IU/ml and the lower limit of linearity will be assessed to determine the
LLOQ.
ACKNOWLEDGEMENT
We would like to thank Qiagen for providing the QIAvac 24 Plus vacuum manifold and all reagents to conduct this study.