AIM
Influenza viruses are well known to infect a large spectrum of animal species such as avian, swine, or Humans. Some strains have the capacity to achieve cross-species contamination and may become pandemic. This is what happen with the H1N1 pandemic strain in 2009. In order to prevent the spreading and contamination of humans, animals, and the environment, it is crucial to early detect the influenza virus's circulation. Real-time PCR method such as Bio-T kit® AIV genotypes H5 & H7 V2 enables sensitive detection and genotyping of influenza H5 and H7 viruses. New devices appeared on the market like the MIC qPCR cycler (BioMolecular System, Australia). This magnetic Induction Cycler is portable and allows fast ramping and reliable results. In a context where the time to result is important, BioSellal has evaluated the use of the Bio-T kit® AIV genotypes H5 & H7 V2 on the MIC qPCR cycler.
METHODS
Samples
In order to evaluate the use of the Bio-T kit® AIV genotypes H5 & H7 V2 on the MIC qPCR Cycler, swab samples, organs, and environmental wipes have been analyzed. Those different sample types were spiked, with inactivated type A influenza virus strains provided by the French national reference laboratory (NRL), at a low viral load corresponding to the French required level of detection (lowest concentration of viruses that has to be detected, NED). Then, the spiked samples were extracted with the BioExtract® SuperBall® extraction kit.
RNA standard
Synthetic type A influenza RNA has been serially 10-fold diluted to achieve a last level at 3 times the limit of detection (3xLDRT-qPCR). These dilution steps were performed using the Myra Liquid Handling system.
Master Mix
The Master Mix contained in the Bio-T kit® AIV genotypes H5 & H7 V2 allows the detection of type A avian influenza viruses subtype H5 (H5) in FAM channel, of type A avian influenza viruses subtype H7 (H7) in VIC channel, of an exogenous internal positive control (IC) in Texas Red channel and of an endogenous positive control (IPC) in CY5 channel.
Reaction set Up
5μl of RNA were mixed with 15 μl of Bio-T kit® AIV genotypes H5 & H7 V2 Master Mix in a plate for AriaMxTM PCR System and in Mic tube for MIC qPCR Cycler. This mixing step was performed using the Myra Liquid Handling system.
Cycling parameters
All samples and no-template controls were amplified on an AriaMxTM and on a BMS MIC qPCR Cycler. Cycling parameters of AriaMxTM were set up according to the Bio-T kit® AIV genotypes H5 & H7 V2 handbook. For the MIC qPCR Cycler program, please contact BioSellal.
RESULTS
To evaluate the possibility of using the Bio-T kit® AIV genotypes H5 & H7 V2 on MIC qPCR Cycler, spiked samples and RNA standards were analyzed and compared with AriaMxTM Real-Time PCR System.
Standard RNA
Figure 1: Curves shapes and Ct values comparison for synthetic standards RNA of avian influenza viruses Subtype H5 and H7 between AriaMxTM PCR System and MIC qPCR Cycler with Bio-T kit® AIV genotypes H5 & H7 V2
=> Curves shapes and Ct values for H5 and H7 targets on standards RNA are similar between AriaMxTM and MIC qPCR cycler.
Figure 2: Curves shapes and Ct values comparison for subtype H5 and H7 of influenza virus low positive swab samples between AriaMxTM PCR System and MIC qPCR Cycler with Bio T kit® AIV genotypes H5 & H7 V2
=> Curves shapes and Ct values for H5, H7, exogenous IC, and endogenous IPC targets on spiked swab samples are similar between AriaMxTM and MIC qPCR cycler.
Organs
Figure 3: Curves shapes and Ct values comparison for subtype H5 and H7 of influenza virus low positive encephalon samples between AriaMxTM PCR System and MIC qPCR Cycler with Bio-T kit® AIV genotypes H5 & H7 V2
=> Curves shapes and Ct values for H5, H7, exogenous IC, and endogenous IPC targets on spiked encephalon samples are similar between AriaMxTM and MIC qPCR cycler.
Environmental wipes
Figure 4: Curves shapes and Ct values comparison for subtype H5 and H7 of influenza virus low positive environmental wipes between AriaMxTM PCR System and MIC qPCR Cycler with Bio-T kit® AIV genotypes H5 & H7 V2
=> Curves shapes and Ct values for H5 and H7 are better in MIC qPCR Cycler. For exogenous IC and endogenous IPC targets on spiked environmental wipes, curve shapes and Ct values are similar between AriaMxTM and MIC qPCR cycler.
NB: The Ct values and curve shapes of endogenous IPC target are not relevant in this matrix due to the low number of cells present in this sample type.
CONCLUSIONS
BioSellal has evaluated a new sensitive method using the Bio-T kit® AIV genotypes H5 & H7 V2 on MIC qPCR Cycler. This method can be used for the detection of avian influenza viruses from swabs, organs, and environmental wipes. The MIC qPCR Cycler can test up to 48 samples at once in 1 hour and 45 minutes. This run gave Ct values and shapes of curves similar or better, depending on the sample type, in the MIC qPCR Cycler compared to classical real-time PCR thermal cyclers that are bulkier. The performances are equivalent to a gain in portability and reliability. Another application note about using the Bio-T kit® Avian & Swine Influenza Virus V2 cycler seems to indicate a better inhibitor resistance on the MIC qPCR cycler (please contact BioSellal for more information). Moreover, it eliminates the need for calibration, annual service, and preventative maintenance. All these data allow an easier and faster avian influenza virus diagnosis and genotyping. The Bio-T kit® AIV genotypes H5 & H7 V2 allows the genotyping of avian influenza H5 and H7 viruses and can be used after an avian influenza virus detection with the Bio-T kit® Avian & Swine Influenza Virus V2 that is also compatible on MIC qPCR Cycler. An application note is available for this application (please contact BioSellal for more information: contact@biosellal.com).
ORDERING INFORMATION
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