Library of prefabricated locked nucleic acid hydrolysis probes facilitates rapid development of reverse-transcription quantitative real-time PCR assays for detection of novel influenza A/H1N1/09 virus.
BACKGROUND
The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods.


METHODS
We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)--a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes--specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin.


RESULTS
Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100-1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent.


CONCLUSIONS
Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.
Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany.
Jürgen J
JJ
Wenzel
Heiko
H
Walch
Markus
M
Bollwein
Hans Helmut
HH
Niller
Waltraud
W
Ankenbauer
Ralf
R
Mauritz
Hans-Joachim
HJ
Höltke
Héctor Manuel
HM
Zepeda
Hans
H
Wolf
Wolfgang
W
Jilg
Udo
U
Reischl
D000818:Animals; D003429:Cross Reactions; D005240:Feasibility Studies; D005456:Fluorescent Dyes / Q000737:chemistry; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology; D009439:Neuraminidase / Q000235:genetics; D015341:Nucleic Acid Probes / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
D005456:Fluorescent Dyes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D015341:Nucleic Acid Probes; D009841:Oligonucleotides; C477371:locked nucleic acid; D009439:Neuraminidase