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Pathamp flua reagents

Manufactured by Thermo Fisher Scientific
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Sourced in United States

The PathAmp FluA Reagents is a molecular diagnostic test kit designed for the detection of influenza A virus. It utilizes real-time reverse transcription-polymerase chain reaction (RT-PCR) technology to identify the presence of influenza A viral RNA in clinical samples.

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Lab products found in correlation

Ion pgm system, by Thermo Fisher Scientific (2 mentions) Ampure xp purification kit, by Beckman Coulter (1 mentions) Ion pgm sequencing 200 kit v2, by Thermo Fisher Scientific (1 mentions) Ion xpress plus fragment library kit, by Thermo Fisher Scientific (1 mentions) Qiaamp viral rna mini kit, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Hiseq platform, by Illumina (1 mentions) Dna clean concentrator, by Zymo Research (1 mentions)

4 protocols using pathamp flua reagents

1

Comprehensive Influenza Virus Sequencing

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For all selected isolates, all 8 gene segments (HA, neuraminidase [NA], nucleoprotein [NP], polymerase basic 1 [PB1] and 2 [PB2], polymerase acidic [PA], matrix [M], and nonstructural [NS]) were sequenced by using a next-generation sequencing strategy for influenza A virus sequencing with the Ion PGM System and PathAmp FluA Reagents (Life Technologies, Carlsbad, CA, USA). Viral RNA extraction was performed by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription PCR amplification of all 8 gene segments were performed by using PathAmp Flu A Preamplification Reagents (Life Technologies). Amplicons were purified and quantitated by using the Ampure XP purification kit (Beckman, Brea, CA, USA). The genomic libraries were prepared with the Ion Xpress Plus Fragment Library Kit (Life Technologies). Enzymatic fragmentation was used for the 200-bp read protocol with a 10-min incubation time. Samples were assigned barcodes by using the Ion Xpress Barcode Adapters 1–32 Kit (Life Technologies). Automated template preparation was performed by using the Ion OneTouch 2 System (Ion PGM Template OT2 200 Kit; Life Technologies). Final sequencing was performed with the Ion PGM Sequencing 200 Kit v2 (Life Technologies) using the Ion 316 Chip V2.
Ke C., Lu J., Wu J., Guan D., Zou L., Song T., Yi L., Zeng X., Liang L., Ni H., Kang M., Zhang X., Zhong H., He J., Lin J., Smith D., Burke D., Fouchier R.A., Koopmans M, & Zhang Y. (2014). Circulation of Reassortant Influenza A(H7N9) Viruses in Poultry and Humans, Guangdong Province, China, 2013. Emerging Infectious Diseases, 20(12), 2034-2040.
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2

Viral RNA Extraction and Sequencing

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Viral RNA from an isolated virus was extracted using the RNeasy Mini Kit (Qiagen), amplified by RT-PCR [13 (link)] and was subjected to dideoxynucleotide sequencing or next-generation sequencing using the Ion PGM System with PathAmp FluA Reagents (Life Technologies). The sequences were submitted to the Global Initiative on Sharing All Influenza Data (GISAID) [14 (link)] (EPI674320, EPI674374 to EPI674424, EPI676397 to EPI676400, EPI676490, EPI676491, EPI696727 and EPI696728). Phylogenetic analysis was performed with the H9 haemagglutinin (HA) coding sequence (1,093 nt, 115–1,207 nt from ATG) aligned with reference strains from GISAID (Table 1). Phylogenetic trees were constructed by maximum likelihood method with bootstrap analysis (n = 1,000) by MEGA (version 6.0).
Zhou J., Wu J., Zeng X., Huang G., Zou L., Song Y., Gopinath D., Zhang X., Kang M., Lin J., Cowling B.J., Lindsley W.G., Ke C., Peiris J.S, & Yen H.L. (2016). Isolation of H5N6, H7N9 and H9N2 avian influenza A viruses from air sampled at live poultry markets in China, 2014 and 2015. Eurosurveillance, 21(35), 30331.
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3

Full Genome Amplification and Ion Torrent Sequencing

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PathAmp FluA Reagents (ThermoFisher, Waltham, USA) were used to amplify the full genome. The primer sequences were as follows: 5′-CTGGATACGCCAGCRAAAGCAGG-3′ (sense) and 5′-GACCTGATGCGGAGTAGAAACAAGG-3′ (antisense). Whole-genome sequencing was then performed on an Ion Torrent™ Personal Genome Machine™ platform (Thermo Fisher Scientific) with a read length of 200 base pairs following the instructions, and the data were analysed using CLC Genomics Workbench 7.5.1 software.
Zhang Y., Dong J., Bo H., Dong L., Zou S., Li X., Shu Y, & Wang D. (2019). Genetic and biological characteristics of avian influenza virus subtype H1N8 in environments related to live poultry markets in China. BMC Infectious Diseases, 19, 458.
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4

Whole Genome Sequencing of Influenza Virus

Cited 1 time
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RNAs were extracted from the A/Perth/16/2009 WT and resistant viruses and subjected to reverse transcription and PCR amplification using the PathAmp FluA Reagents (Thermo Fisher). A fraction of the PCR products were resolved and visualized on 1% agarose gel to ensure the presence of all 8 influenza genome segments (PB2, PB1, PA, HA, NP, NA, M, NS) at the expected sizes (2.31, 2.27, 2.12, 1.70, 1.54, 1.41, 0.98, 0.84 kb). The remaining PCR products were purified using the DNA Clean & Concentrator (Zymo Research), and contracted to SeqWright (GE Healthcare) for library preparation and whole genome sequencing with Illumina paired-end technology (2x100bp) on the Illumina Hiseq platform (Illumina). The genomic alignment software GSNAP was used for all FASTQ sequence alignment [48 (link)]. GenBank sequences of A/Perth/16/2009 (Accession No. KJ609203—KJ609210) were used as the reference sequences for alignment (S1 Table). Sequence analysis was carried out by Genentech in-house viral bioinformatics pipeline based on R and Bioconductor packages: GenomicRanges [49 (link)], GenomicAlignments [49 (link)], VariantTools [50 ], gmapR [51 ]. Only base-calls with Q-score ≥ 30 were tallied for variant calling. Amino acid substitutions with respect to the reference sequences were determined at a frequency threshold of 50%.
Chai N., Swem L.R., Reichelt M., Chen-Harris H., Luis E., Park S., Fouts A., Lupardus P., Wu T.D., Li O., McBride J., Lawrence M., Xu M, & Tan M.W. (2016). Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody. PLoS Pathogens, 12(6), e1005702.
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