DATA SHEET ViralSEQ™ Vesivirus Detection Assay

ViralSEQ™ Vesivirus Detection Assay Integrated sample preparation and real-time PCR assay for the detection of Vesivirus in cell culture samples

• Detection of known Vesivirus strains

• Rapid time-to-results in less than 5 hours

• TaqMan®-based real-time PCR assay

• Demonstrated sensitivity and specificity

• Proprietary optimized sample preparation for high RNA recovery

Viral contamination in mammalian product manufacturing presents a serious risk to the manufacturing process, the manufacturing facility, and the integrity of the product. Vesivirus 2117 is an RNA that has reemerged as a potential threat to mammalian cell Table 1. Exclusion Panel. The ViralSEQ™ Vesivirus Detection Assay has been culture production. To meet the challenge of rapid, specific, and demonstrated to be specific for Vesivirus and does not detect RNA purified sensitive detection of Vesivirus, Applied Biosystems has developed from related and unrelated species and organisms. the first commercially available integrated kit for isolation and Bacillus 2.5 ng Chicken 5 ng detection of Vesivirus from mammalian cell culture samples. The Clostridium 2.5 ng 5 ng ViralSEQ™ Vesivirus Detection Assay is a PCR-based test that enables users to quickly isolate and detect Vesivirus 2117 and all other known E. coli 2.5 ng Human 5 ng ™ strains with high sensitivity and specificity. The test utilizes PrepSEQ Lactococcus 2.5 ng MDCK 5 ng sample preparation magnetic beads to isolate viral RNA from a wide range of sample types, combined with TaqMan®-based technology Shigella 2.5 ng Rat 5 ng for sensitive and specific detection of the viral RNA. The kit also Yeast 5 ng CHO 500 ng includes a proprietary positive control template that helps eliminate Human RNA 1 µg the possibility of false positive results arising from inadvertent contamination of test samples with the positive control.

FAM FAM Vesivirus targets

FAM FAM Discriminatory RT-PCR positive control VIC VIC

NED Internal positive control

Figure 1. Multiplex Assay and Control Design. The ViralSEQ™ Vesivirus Assay’s unique design includes two viral targets and discriminatory positive controls. This allows detection of known Vesivirus . The discriminatory positive control helps monitor the real-time PCR reaction and cross-contamination of samples to reduce false positive results. -6 10 10 dilution (280 TCID50)

40 -7 10 dilution (28 TCID50)

-8 10 dilution (2.8 TCID50) 35 40 1

30 35 t C Delta Rxn

t 30

25 C 0.1 25 20 20

15 0.01 15 4 1 3 5 7 9 511 13 15 176 19 21 23 25 27 29 31 33 35 37 39 -6 -7 -8 10 10 10 supernatant 10 10 10 NTC Cycle Number Cell Number TCID50 280 28 2.8

Figure 3. High Sensitivity and Broad Dynamic Range. The ViralSEQ™ Vesivirus Assay can detect from 106 down to 10 copies of in vitro–transcribed Vesivirus RNA -6 per reaction. 10 dilution (280 TCID50)

40 -7 10 dilution (28 TCID50)

-8 10 dilution (2.8 TCID50) 35 40 40 y = 1.4663Ln(x) + 39.433 R2 = 0.9987 35 30 35 t C 30

t 30 t

25 C C 25 25 20 20 20 RT-PCR efficiency: 98% 15 15 15 4 5 6 2 3 4 5 6 7 10-6 10-7 10-8 NTC 10 10 10 supernatant 1 10 10 10 10 10 10 10 Cell Number Copy Number TCID50 280 28 2.8

Figure 2. PrepSEQ™ 1-2-3 Sample Preparation Kit. Optimized for use with the Figure 4. High PCR Efficiency.Using the viral RNA template, the ViralSEQ™ ViralSEQ™ Vesivirus Assay, the sample preparation procedure allows detection Vesivirus Assay demonstrated high real-time PCR efficiency and a broad linear -6 -8 of 2.8 TCID50 in 100 µL of cell culture and serum. In the above example, 10 to 10 detection range. dilutions of (used as a surrogate for Vesivirus) were spiked into 100 µL of fetal bovine serum (top panel) and cell culture (bottom panel). The serial

dilutions of the virus correspond to TCID50 of 280, 28, and 2.8.

ORDERING INFORMATION

Description Size Part Number ViralSEQ™ Vesivirus Detection Assay 100 rxns 4448398C Contains real-time PCR assay, buffer and enzyme mix, positive control & negative control PrepSEQ™ 1-2-3 Nucleic Acid Extraction Kit 100 rxns 4452222 Buffers, magnetic beads, and enzymes

For Research Use Only. Not for use in diagnostic procedures. Practice of the patented 5’ Nuclease Process requires a license from Applied Biosystems. The purchase of TaqMan® Non-coding Assays includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research when used with the separate purchase of an Authorized 5’ Nuclease Core Kit. No other patent rights are conveyed expressly, by implication, or by estoppel. For further information on purchasing licenses contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Trademarks of Life Technologies Corporation and its affiliated companies: AB Logo™, Applied Biosystems®, FAM™, Life Technologies™ (Stylized), NED™, PrepSEQ™, ViralSEQ™, VIC®. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. © 2010 Life Technologies Corporation. All rights reserved. Printed in the USA. 06/2010 CO13952

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