Bioline, your partner for virology research

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1 Bioline, your partner for virology research Discover why scientists at the world s leading virological research institutes and reference laboratories*, have come to trust and depend upon the reliability and superior results demonstrated by Bioline high performance products. Whether studying viral genomics, molecular evolution and phylogenetics, interactions of virus and host genotypes, or the epidemiology of an emerging viral pathogen which all require rapid and highly sensitive analysis of viral nucleic acids, let Bioline be your trusted partner for molecular virology research. *Selected Viral Research and Surveillance Centres WHO Collaborating Center for Reference and Research on Influenza, Melbourne, Australia Chinese Center for Disease Control and Prevention, Beijing, China U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Maryland, USA Institute for Virology, Marburg, Germany Influenza Norovirus Hepatitis Rotavirus HIV HPV HSV EBV RSV For further information on how Bioline can help you with your virology research, speak to your local distributor or go to

2 Key Bioline solutions for virology research SensiFAST Probe One-Step real-time reagents Unrivalled sensitivity for multiplex one-step probe detection of viral RNA SensiFAST SYBR real-time reagents Unparalleled speed and sensitivity for viral quantification and gene expression studies MyTaq One-Step RT-PCR Kit Extremely sensitive and highly reproducible firststrand cdna synthesis and PCR in a single tube MyTaq HS DNA Polymerase New generation enzyme for fast and highly specific amplification of viral DNA VELOCITY DNA Polymerase Ultra-high fidelity amplification of viral DNA Unrivalled stability and highly efficient transcription of viral RNA SensiFAST Probe One-Step Kits Rapid - Optimized for fast reverse transcription and real-time PCR detection in a single tube Accurate quantification - From ultra low-copy viral RNA samples High sensitivity and specificity - Unique buffer chemistry for superior results in both single and multiplex assays Flexible - Compatible with all fast cycling instruments and probe chemistries Viral gene expression analysis Viral RNA quantification Viral pathogen detection Multiplexing assays SensiFAST Probe One-Step Kit have been selected by many of the leading viral research laboratories around the world, including many who are working on influenza screening. Developed for highly reproducible first-strand cdna synthesis and subsequent real-time PCR in a single tube, the kits are formulated for use with probe-detection technology, including TaqMan, Scorpions and molecular beacon probes. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system ensures that SensiFAST Probe One-Step Kits produces fast, highly-specific and ultra-sensitive one-step RT-qPCR results (fig. 1). SensiFAST Probe One-Step can therefore be used for highly accurate quantification such as looking at the number of copies of a viral genome (viral loading) in a tissue and the progress of the infection and its severity. The SensiFAST Probe One-Step Kits consists of a 2x SensiFAST Probe One-Step mix, separate reverse transcriptase and RiboSafe RNase Inhibitor. SensiFAST Probe Hi-ROX and Lo-ROX One-Step Kits also contain premixed ROX for optional use. Fluorescence ( ) SensiFAST Probe Hi-ROX One-Step Kit SensiFAST Probe Lo-ROX One-Step Kit SensiFAST Probe No-ROX One-Step Kit Amplification Plot Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Cycles Fig. 1. Comparison of SensiFAST Probe One-Step (red line) against a leading supplier using fast cycling conditions. A fragment of ß-actin amplified in triplicate using gene specific primers and TaqMan probe according to each manufacturer s protocol, from 10-fold serial dilution of RNA with either SensiFAST Probe One-Step (red) and competitor mix A (green). The results illustrate that SensiFAST Probe One-Step Kit is faster by four Cts and more sensitive than supplier A (more than 10 fold). SensiFAST Probe One Step Kit Bara, J. & Muturi, E.J. Effect of mixed infections of Sindbis and La Crosse viruses on replication of each virus in vitro. Acta Tropica 130: (2014). Suzuki, K. et al., Promoter targeting shrna suppresses HIV-1 infection in vivo through transcriptional gene silencing. Mol. Ther. Nucl. Acids 2: e137 Wang, C.Y. et al., A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during J. Clin. Virol. 54(1): (2012). Semaan, M. et al., Screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in Göttingen minipigs. Xenotransplant. 20(3): Nelms, B.M. et al., Experimental and natural vertical transmission of West Nile virus by California Culex (Diptera: Culicidae) mosquitoes. J. Med. Entomol. 50(2): SensiFAST Probe Kit Cornall, J. et al., Anal and perianal squamous carcinomas and high-grade intraepithelial lesions exclusively associated with low-risk HPV genotypes 6 and 11. Int. J. Cancer 133(9): Clark, A. et al., A trivial role of STAT4 variant in chronic hepatitis B induced hepatocellular carcinoma. Infect. Genet. Evol. 18: Product Information

3 Bioline, your partner for virology research SensiFAST SYBR Kits MyTaq One Step RT-PCR Kit Rapid and accurate - Hot-start capability saves time and reduces primer-dimer formation Unique buffer chemistry - For highest specificity Broad dynamic range - For high sensitivity Simple and reproducible - Just add primers and template Viral gene-expression analysis Viral DNA quantification Viral pathogen detection SensiFAST SYBR No-ROX Kit SensiFAST SYBR Hi-ROX Kit SensiFAST SYBR Lo-ROX Kit SensiFAST SYBR & Fluorescein Kit 200 Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Extremely sensitive - Highly optimized for detection of low-copy genes High yield - Blend of ultra-stable reverse transcriptase and novel hot-start MyTaq Superior performance - Overcomes secondary structure in difficult and GC-rich targets Fast PCR reactions - Ideal for high throughput applications Simple to use - All-in-one mix for highly-specific and ultrasensitive products for downstream applications MyTaq One-Step RT-PCR Kit 10 Reactions BIO Reactions BIO Reactions BIO SensiFAST SYBR Kits for use with viral DNA samples have been developed for fast, highly reproducible real-time PCR and are validated on commonly used real-time instruments. The latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, ensure that the SensiFAST SYBR Kits produces fast, highlyspecific and ultra-sensitive real-time PCR (fig. 1). SensiFAST SYBR Kits are an ideal choice for gene expression studies and viral load determination from a wide variety of viral DNA samples. The SensiFAST SYBR Hi-ROX and Lo-ROX Kit contain premixed ROX and the SensiFAST SYBR & Fluorescein Kit contain premixed fluorescein, for optional use as a passive reference. Amplification Plot Fig. 1 Comparison of SensiFAST SYBR (red line) against another leading supplier Q (blue line) using fast cycling conditions. A fragment of ubiquitin gene was amplified using SensiFAST SYBR (red) and the results were compared with amplifications using a Kit from supplier Q (blue). The process used a 10 fold serial dilution of RNA (in triplicate) over 5 orders of magnitude. The results exhibit that SensiFAST SYBR was faster (earlier Ct) and more sensitive than supplier Q. Viral gene-expression analysis Viral transcription analysis Gene cloning Multiplex RT-PCR MyTaq One-Step RT-PCR Kit has been designed for extremely sensitive and highly reproducible first-strand cdna synthesis and subsequent PCR in a single tube, from viral RNA using gene-specific primers. The kit contains the latest advances in buffer chemistry, including Bioline ultra-pure dntps, together with reverse transcriptase (RT) and our new generation of very high performance, antibody-mediated hot-start DNA polymerase (MyTaq HS). This ensures that MyTaq One-Step RT-PCR Kit produces fast, highly-specific and ultrasensitive products (fig. 1) for downstream applications, such as the molecular epidemiology and evolution of a virus infection. M 0pg 3pg 30pg 300pg 3ng 30ng Fig. 1. Sensitivity of MyTaq One-Step RT-PCR Kit A serial dilution of total RNA (30ng, 3ng, 300pg, 30pg and 3pg respectively) in duplicate was used. Reverse transcription reaction at 45 C for 40min, followed by 95 C for 5min and 40 cycles at 95 C for 30s, 58 C for 30s and 72 C for 60s, using RN18S-1000 primers (to produce a 1kb fragment), that was analysed on a 1% agarose gel (BIO-41025). HyperLadder 50bp (M) (BIO-33039). SensiFAST SYBR Kits Reuter, A. et al., Antiviral activity of interferon-l in chickens. J. Virol. doi: /JVI Witteveldt, J. et al., The influence of viral RNA secondary structure on interactions with innate host cell defences. Nucl. Acids Res. doi: /nar/gkt1291 Lyons, S. et al., Nonprimate Hepaciviruses in domestic horses, United Kingdom. Emerg. Inf. Dis. 18(12): (2012). Bartholomeeusen, K. et al., Histone deacetylase inhibitors (HDACis) that release the positive transcription elongation factor b (P-TEFb) from its inhibitory complex also activate HIV transcription. J. Biol. Chem. 288(20): Veillette, M. et al., The V86M mutation in HIV-1 capsid confers resistance to TRIM5a by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import. Retrovirol. 10:25 Arzumanyan, A. et al., Hedgehog signaling blockade delays hepatocarcinogenesis induced by Hepatitis B Virus X protein. Cancer Res. 72:5912 (2012). SensiFAST SYBR One Step Kit Harun, M.S. et al., Transcriptional profiling of feline infectious peritonitis virus infection in CRFK cells and in PBMCs from FIP diagnosed cats. Virol. J. 10:329 McFadden, N. et al., Influence of genome-scale RNA structure disruption on the replication of murine norovirus-similar replication kinetics in cell culture but attenuation of viral fitness in vivo. Nucl. Acids Res. 41(12): Reuter, J.D. et al., Assessment of hazard risk associated with the intravenous use of viral vectors in rodents. Comp Med. 62(5): (2012). The outstanding sensitivity of MyTaq One-Step RT-PCR Kit for viral research has been demonstrated in recent publications from four leading WHO Influenza Center research groups studying the molecular epidemiology and evolution of human and avian viral influenza infections (Deng et al., 2013; Vijaykrishna et al., 2013, Job et al., 2013) as well as influenza vaccine effectiveness (Sullivan et al. 2013). from WHO Collaborating Center for Reference and Research on Influenza Sullivan, S. G. et al., Influenza vaccine effectiveness during the 2012 influenza season in Victoria, Australia: Influences of waning immunity and vaccine match. J. Med. Virol. doi: /jmv Deng, Y.M. et al., The use of pyrosequencer-generated sequence-signatures to identify the influenza B-lineage and the subclade of the B/Yamataga-lineage viruses from currently circulating human influenza B viruses. J. Clin. Virol. 58(1):94-99 Vijaykrishna, D., et al., The recent establishment of north american h10 lineage influenza viruses in Australian wild waterfowl and the evolution of Australian avian influenza viruses. J. Virol. 87(18): Job, E.R. et al., Addition of glycosylation to influenza A virus hemagglutinin modulates antibody-mediated recognition of H1N pandemic viruses. J. Immunol. 190(5): Product Information Application Notes

4 Bioline, your partner for virology research MyTaq HS DNA Polymerase VELOCITY DNA Polymerase New - Hot-start polymerase delivers industry leading performance Highest performance and specificity - Resulting from proprietary buffer system and MyTaq enzyme Fast activation times - Powered by new generation antibody-based hot-start polymerase Flexible - Fast or standard PCR reactions High-throughput PCR Multiplex PCR Colony PCR Two-Step RT-PCR Assays with prolonged reaction setup on the bench or liquid handling Amplification of challenging targets susceptible to mispriming Specific amplification of difficult templates (GC rich) Genotyping TA cloning MyTaq HS DNA Polymerase is a high performance PCR product powered by antibody mediated hot-start, specifically designed for fast, highly-specific, hot-start PCR (fig. 1). The product also has the added convenience of room temperature reaction assembly without non-specific amplification or primer-dimer formation. MyTaq HS is ideal for a wide range of downstream viral research applications from multiplex PCR, colony PCR to genotyping. The highly robust performance of MyTaq in viral research has been demonstrated in recent viral studies. In a study of the genome plasticity of Ebola, a biosafety level four pathogen, researchers at the Genomics Division, United States Army Medical Research Institute of Infectious Diseases used MyTaq to amplify cdna for Illumina next-generation sequencing library preparation (Kugelman et al., 2013). In a study of the genetic variation of the HA1 hemagluttinin gene from the H1N1 swine flu virus, responsible for a 2009 pandemic, WHO Influenza researchers used MyTaq HS to amplify HA1 genes for cloning and pyrosequencing assays (Guarnaccia et al., 2013). MyTaq HS DNA Polymerase MyTaq HS Red DNA Polymerase MyTaq HS Mix, 2x MyTaq HS Red Mix, 2x A B C D MyTaq HS MyTaq Supplier F M Units BIO Units BIO Units BIO Units BIO Units BIO Units BIO Reactions BIO Reactions BIO Reactions BIO Reactions BIO Supplier Q M Supplier S Fig. 1 Fast amplification (26.3 minutes) was carried out on a range of genes from genomic DNA A) A 340bp and B) a 450bp fragment of the myc gene, C) a 525bp fragment of the EGFR gene and D) a 530bp fragment of the AGRI1 gene were amplified with MyTaq HS and the results were compared with amplifications with hot-start DNA polymerases from other suppliers. The process used a serial dilution of genomic DNA (100ng, 33ng, 10ng, 4ng, 1ng, 33pg, 10pg and 3pg genomic DNA, lanes 1-8 respectively), incubated for 3 mins at 95ºC followed by 35 cycles of 15s at 95ºC, 55ºC and 72ºC. Marker is HyperLadder 1kb (M) (Cat No. BIO-33025). MyTaq HS performed well across all four genes. Guarnaccia, T. et al., Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in a ferret model. PLoS Pathog. 9(5):e Greewood, A.D. et al., A potentially fatal mix of herpes in zoos. Curr. Biol. 22(18): (2012). Chappell, K.J. et al., Respiratory Syncytial Virus infection is associated with increased bacterial load in the upper respiratory tract in young children. J. Med. Microbiol. S1:005 MyTaq Kugelman, J.R. et al., Ebola virus genome plasticity as a marker of its passaging history: a comparison of in vitro passaging to non-human primate infection. PLoS One 7(11):e50316 (2012). Cságola A. et al., Detection, prevalence and analysis of emerging porcine parvovirus infections. Arch. Virol. 157(6): (2012). Ndze V.N. et al., Detection of novel porcine bocaviruses in fecal samples of asymptomatic pigs in Cameroon. Infect. Genet. Evol. 17: Cadar, D. et al., Capsid protein evolution and comparative phylogeny of novel porcine parvoviruses. Mol. Phylogenet. Evol. 66(1): High-speed, ultra high-fidelity DNA polymerase High processivity Robust, requires minimal optimization Cloning techniques High-fidelity PCR Site-directed mutagenesis VELOCITY is a fast thermostable enzyme possessing 5-3 DNA polymerase and 3-5 proofreading exonuclease activities. VELOCITY provides very high-fidelity with an error-rate of 4.4 x 10-7 combined with high speed DNA synthesis due to the enhanced processivity of the polymerase. VELOCITY is easy to use and requires minimal optimization. The polymerase produces higher yields than equivalent commercially available enzymes and generates blunt-ended amplicons. Due to its robust performance and ultra high-fidelity, VELOCITY is ideal for viral applications including cloning, difficult to amplify templates, site-directed mutagenesis, and sequencing. Highly sensitive - For high-quality, full length cdna from as little as 10pg of total RNA Broad dynamic range - 10pg to 2μg of input RNA Ultra-stable - For long genes and rare transcripts High yield - Produces high quality cdna ideal for realtime PCR First and second strand cdna synthesis Preparation of cdna libraries is a highly sensitive, ultra-stable MMLV reverse transcriptase. Tetro is highly optimized for reverse transcription reactions using a wide range of input viral RNA amounts, such that long and low abundance cdnas can be detected by amplification after cdna synthesis. Due to its highly robust performance and highly active enzymatic activity, is suitable for the most demanding of viral genetic studies. Tetro is ideal for high yield, first-strand cdna synthesis, cdna library construction and the production of full length templates for RT-PCR analysis of viral gene expression. VELOCITY DNA Polymerase Guarnaccia, T. et al., Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in a ferret model. PLoS Pathog. 9(5):e Gáll T. et al., Genomic differences in the background of different severity in juvenile-onset respiratory papillomatoses associated with human papillomavirus type 11. Med. Microbiol. Immunol. 202(5): Müller E.E., et al., Genetic characterization of Herpes Simplex Virus Type 2 UL23 thymidine kinase in Johannesburg, South Africa. J. Antivir. Antiretrovir. 5:80-84 Renaud C. et al., Diagnostic accuracy of an allele-specific reverse transcriptase-pcr assay targeting the H275Y oseltamivir resistant mutation in 2009 pandemic influenza A/H1N1 virus. J. Clin. Virol. 49(1):21-25 (2010). 250 Units BIO Units BIO Units BIO x 10,000 Units BIO Anderson D.E., et al., Elements in the Canine Distemper Virus M 3 UTR Contribute to Control of Replication Efficiency and Virulence. PLoS ONE 7(2): e31561 (2012). Application Notes Application Notes

5 Here is what other virology researchers say about our products We were looking for a master mix to work on the LC480 platform. We use the SensiFAST Probe No-ROX Kit as it offers very good performance (in terms of sensitivity and fluorescence intensity) along with reduced costs. - Institute for Microbiology, German Federal Defence Munich, Germany For viral load measurement we have changed to the SensiFAST SYBR One-Step Kit and gained increased sensitivity down to 10 IU/ml. -Micropathology Ltd, Coventry, UK CPA Accredited, Rapid Diagnosis & Biomedical Research Company For further information on how Bioline can help you with your viral research, speak to your local distributor or go to P4PLA0114V1.2