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Volume 30, Number 8—August 2024
Dispatch

Multiplex Dual-Target Reverse Transcription PCR for Subtyping Avian Influenza A(H5) Virus

Malaya K. Sahoo, Ingrid E.A. Morante, ChunHong Huang, Daniel Solis, Fumiko Yamamoto, Uzoamaka C. Ohiri, Daniel Romero, and Benjamin A. PinskyComments to Author 
Author affiliations: Stanford Health Care Clinical Virology Laboratory, Stanford, California, USA (M.K. Sahoo, I.E.A. Morante, U.C. Ohiri, D. Romero, B.A. Pinsky); Stanford University School of Medicine, Stanford (C. Huang, D. Solis, F. Yamamoto, B.A. Pinsky)

Main Article

Table 1

Primers and probes used in study of multiplex dual-target reverse transcription PCR for subtyping avian influenza A(H5) virus*

Target Name Sequence, 5′ → 3′ Final concentration
A(H5)
 FluA_H5_v4_1F TACCAGATACTGTCAATTTATTCAAC 400 nM
FluA_H5_v4_1R GTAACGACCCATTGGAGCACATCC 400 nM
FluA_H5_v4_1Prb.FAM FAM-CTGGCAATC/ZEN/ATGRTRGCTGGTCT-3IABkFQ 200 nM
FluA_H5_v4_2F TGGGTACCATCATAGCAATGAGCA 400 nM
FluA_H5_v4_2R AACTCCCTTCCAACTGCCTCAAA 400 nM
FluA_H5_v4_2Prb.FAM
 FAM-TGGGTACGC/ZEN/TGCGGACAAAGAATCCA-3IABkFQ
 200 nM
A (M)
 Pan-FluA-F GACCRATCCTGTCACCTCTGAC 400 nM
Pan-FluA -R AGGGCATTYTGGACAAAKCGTCTA 400 nM
Pan-FluA -prb_Q705
 Q705-TGCAGTCCTCGCTCACTGGGCACG- BHQ-3
 200 nM
Human
RNase P		 RNase P Fwd AGATTTGGACCTGCGAGCG 100 nM
RNase P Rev GAGCGGCTGTCTCCACAAGT 100 nM
RNase P Probe CF560 CF560-TTCTGACCTGAAGGCTCTGCGCG-BHQ-1 50 nM

*H5 probes were purchased from Integrated DNA Technologies (https://www.idtdna.com). The probes contain the internal quencher, ZEN, in addition to the 3′ Iowa BHQ FQ (3IABkFQ) (proprietary to Integrated DNA Technologies). Influenza A (M) and human RNase P probes were purchased from Biosearch Technologies (https://www.biosearchtech.com). Bold type indicates mixed bases. BHQ, Black Hole Quencher; CF560, CalFluor 560; FAM, 6-carboxyfluorescein; M, matrix gene; Q705, Quasar 705.

Main Article

Page created: July 02, 2024
Page updated: July 21, 2024
Page reviewed: July 21, 2024
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