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Figure 2. Analysis of clinical specimens. RNA extracts from clinical
specimens containing known pathogens were reverse transcribed into cDNA
(Superscript RT system, Invitrogen, Carlsbad, CA; 20-µL volume).
Five microliters of the reaction were subjected to Mass Tag PCR by using
primers coupled to Masscode tags (Qiagen Masscode technology, Qiagen,
Hilden, Germany). Detection of (A) influenza virus A (H1N1), (B) respiratory
syncytial virus (RSV) group B, (C) human coronavirus SARS (HCoV-SARS),
(D) human parainfluenza virus (HPIV) types 1 and (E) 3, and (F) enterovirus
(EV) by using a 30-plex assay, including 60 primers targeting influenza
A virus matrix gene (FLUAV-M), and for typing N1, N2, H1, H2, H3, and
H5 sequences, as well as influenza B virus (FLUBV), RSV groups A and B,
HCoV-229E, -OC43, and -SARS, HPIV types 1, 2, 3, and 4 (groups A and B
combined; 4 primers), human metapneumovirus (HMPV, 4 primers), measles
virus (MEV), EV (degenerate primer pair targeting all serogroups), human
adenoviruses (HAdV, degenerate primer pair targeting all serogroups),
human herpesvirus 1 (HHV-1, herpes simplex virus), human herpesvirus 3
(HHV-3; varicella-zoster virus), Mycoplasma pneumoniae, Chlamydia
pneumoniae, Legionella pneumophila, Streptococcus pneumoniae,
Haemophilus influenzae. The bar indicates an arbitrary cut-off
threshold of 2.7 (4 times average background determined with random human
DNA).
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