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® Avian & Swine Influenza Virus V2 enables sensitive amplification and detection of type A influenza 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® Avian & Swine Influenza Virus V2 on the MIC qPCR cycler with a faster run.
METHODS
Samples
In order to evaluate the use of the Bio-T kit® Avian & Swine Influenza Virus V2 on the MIC qPCR Cycler, swab samples, organs, and environmental wipes have been analyzed. Those different sample types were spiked, with an inactivated type A influenza virus strain 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) or ten times lower. Then, the spiked samples were extracted with the BioExtract® SuperBall® extraction kit.
Standard RNA
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® Avian & Swine Influenza Virus V2 allows the detection of type A avian and swine influenza viruses (ASIV) in FAM channel, of an exogenous internal positive control (IC) in VIC channel and of an endogenous positive control (IPC) in CY5 channel.
Reaction set up
5µl of RNA were mixed with 15µl of Bio-T kit® Avian & Swine Influenza Virus V2 Master Mix in a plate for AriaMxTM PCR system and in a MIC tube for MIC qPCR Cycler. This mixing step was performed using Myra Liquid Handling System.
Cycling parameters
All samples and no-template controls were amplified on AriaMxTM and on BMS MIC qPCR Cycler. Cycling parameters of AriaMxTM were set up according to the Bio-T kit® Avian & Swine Influenza Virus V2 handbook. For the MIC qPCR Cycler program, please contact BioSellal.
RESULTS
To evaluate the possibility of using Bio-T kit® Avian & Swine Influenza Virus 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 type A influenza virus between AriaMxTM PCR System and MIC qPCR Cycler with Bio-T kit® Avian & Swine Influenza Virus V2
=> Curves shapes and Ct values for ASIV target on standards RNA are equivalent between AriaMxTM and MIC qPCR cycler.
Swab samples
Figure 2 : Comparaison des formes des courbes et des valeurs Ct pour les échantillons d'écouvillons faiblement positifs pour le virus de la grippe de type A entre les thermocycleurs AriaMxTM et MIC qPCR avec le Bio-T kit® Avian & Swine Influenza Virus V2
=> Curves shapes and Ct values for ASIV, Exogenous IC, and Endogenous IPC targets on type A influenza viruses low positive swab samples are equivalent or better in MIC qPCR cycler compared to AriaMxTM.
Organs
Figure 3: Curves shapes and Ct values comparison for type A influenza viruses low positive encephalon samples between AriaMxTM PCR System and MIC qPCR Cycler with Bio-T kit® Avian & Swine Influenza Virus V2
=> Curves shapes and Ct values for ASIV target and exogenous IC are better and much more reliable in MIC qPCR cycler. That seems to indicate that the MIC qPCR cycler allows a better inhibitor resistance. Endogenous IPC targets detection is equivalent between AriaMxTM and MIC qPCR cycler. Similar results have been obtained for guts samples. These data are available on demand (please contact: contact@biosellal.com)
Environmental wipes
Figure 4: Curves shapes and Ct values comparison for type A influenza virus low positive environmental wipes between AriaMxTM PCR System and MIC qPCR Cycler with Bio-T kit® Avian & Swine Influenza Virus V2
=> Curves shapes and Ct values for ASIV and Exogenous IC targets are better and much more reliable on environmental wipes in MIC qPCR cycler. That seems to indicate that the MIC qPCR cycler allows a better inhibitor resistance.
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 and rapid method using the Bio-T kit® Avian & Swine Influenza Virus V2 on MIC qPCR Cycler. This method can be used for the detection of type A avian and swine influenza viruses from swabs, organs, and environmental wipes. The MIC qPCR Cycler can test up to 48 samples at once in just 65 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 with a gain of speed, portability, and reliability that seems to indicate a better inhibitor resistance with the MIC qPCR cycler. Moreover, it eliminates the need for calibration, annual service, and preventative maintenance. All these data allow an easier and faster avian and swine influenza virus diagnosis. The Bio-T kit® AIV genotypes H5 & H7 V2 allow the genotyping of avian influenza H5 and H7 viruses and it is also compatible with the 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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