Development and evaluation of a new Real-Time RT-PCR assay for detection of proposed influenza D virus.
The occurrence of virus belonging to the putative genus Influenzavirus D, has been demonstrated all-around the world arousing interest within the scientific community. Most of the published virological surveys are based on the first described Real-Time PCR method, designed on the PB1 gene of the first isolate. The necessity of extending investigation to different animal species and geographic areas, requires a continuous update of molecular tests, considering newly sequenced strains. Moreover, the availability of an alternative assay, is essential either to confirm data, or for ensuring the detection of the widest number of strains. A new Real-Time PCR, specific for influenza D virus (IDV), was developed and evaluated. The target sequences of primers and probe are highly conserved among IDV strains currently known. The specificity of the method was demonstrated in silico by BLAST, and in vitro with a huge panel of common swine and bovine respiratory pathogens. The analytical sensitivity of the Real-Time PCR was estimated through synthetic RNA molecules and the limit of detection was about 20 copies/μL. The assay was assessed in field and proved to be a valuable tool for the detection of IDV strains.
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Circonvallazione Sud 21/a, Mantova 46100, Italy. Electronic address: silvia.faccini@izsler.it.
Silvia
S
Faccini
Bologna University, Italy.
Aurora
A
De Mattia
OIE Reference Laboratory for Swine Influenza, Parma, Italy; IZSLER Parma, Italy.
Chiara
C
Chiapponi
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Brescia, Italy.
Ilaria
I
Barbieri
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Brescia, Italy.
Maria Beatrice
MB
Boniotti
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Circonvallazione Sud 21/a, Mantova 46100, Italy.
Carlo
C
Rosignoli
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Circonvallazione Sud 21/a, Mantova 46100, Italy.
Giuliana
G
Franzini
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Brescia, Italy.
Ana
A
Moreno
OIE Reference Laboratory for Swine Influenza, Parma, Italy; IZSLER Parma, Italy.
Emanuela
E
Foni
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Circonvallazione Sud 21/a, Mantova 46100, Italy.
Arrigo Daniele
AD
Nigrelli
D000818:Animals; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007251:Influenza, Human / Q000821:virology; D015345:Oligonucleotide Probes / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000662:veterinary / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D018117:Thogotovirus / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D015345:Oligonucleotide Probes
Cattle; IDV; Influenzavirus D; Real-Time RT-PCR; Swine