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D. Polo et al.
Viral Recovery and RNA Extraction
For virological analysis, molluscs were shucked and the digestive tissue (DT)
removed and homogenized as previously described (Wang et al. 2008). Viral RNA
was extracted in duplicate from DTusing Nucleospin RNA Virus Kit (MachereyNagel; Germany) according to the manufacturer’s protocol.
Extraction and qRT-PCR Controls
Prior to the viral RNA extraction, known amounts of mengovirus clone vMC 0 were
spiked in sample homogenates (10 l of mengovirus stock, 10
3 pfu) to be employed
as a control for the process of nucleic acid extraction (Costafreda et al. 2006). To
test the presence of RT-PCR inhibitors and calculate the qRT-PCR efficiency, an
external control (MNV-1 RNA) was included in each reaction. The cycle threshold
(C t ) value of a sample extracted RNA (2.5 l) mixed with MNV-1 external control
(2.5 l) was compared to the C t value of the external control mixed only with RNAfree sterile water. These steps allowed the identification of samples that required
re-extraction and provided assurance that the samples with no amplification signal
were negative (values below the limit of detection for the assays) and not simply
inhibited. Negative controls containing no nucleic acid as well as positive controls,
containing RNA of MNV-1, were introduced in each run.
Primers and Probes
qRT-PCR for viral detection and quantification was carried out with TaqMan
probes using the Platinum Quantitative RT-PCR Thermoscript One-step System
(Invitrogen, France) following the manufacturer’s instructions in 25 l of a reaction
mixture containing 5 l of extracted RNA. For MNV-1 detection 0.2 M of
reverse and forward primers as well as probe were added. Primer sets and probes
employed were Fw-ORF1/ORF2, Rv-ORF1/ORF2 and probeMGB-ORF1/ORF2
(Baert et al. 2008).
Amplification Conditions
A Mx3005P QPCR System (Stratagene, USA) thermocycler was employed following amplification conditions described by Baert et al. (2008) with minor
modifications. Briefly, after a RT step at 45
ı C for 1 h, PCR amplification was
carried out employing the following cycling conditions: an initial denaturation at
95
ı C for 5 min, and 50 cycles of amplification with a denaturation at 95
ı C for 15 s
and annealing-extension step at 60
ı C for 1 min.
D. Polo et al.
Viral Recovery and RNA Extraction
For virological analysis, molluscs were shucked and the digestive tissue (DT)
removed and homogenized as previously described (Wang et al. 2008). Viral RNA
was extracted in duplicate from DTusing Nucleospin RNA Virus Kit (MachereyNagel; Germany) according to the manufacturer’s protocol.
Extraction and qRT-PCR Controls
Prior to the viral RNA extraction, known amounts of mengovirus clone vMC 0 were
spiked in sample homogenates (10 l of mengovirus stock, 10
3 pfu) to be employed
as a control for the process of nucleic acid extraction (Costafreda et al. 2006). To
test the presence of RT-PCR inhibitors and calculate the qRT-PCR efficiency, an
external control (MNV-1 RNA) was included in each reaction. The cycle threshold
(C t ) value of a sample extracted RNA (2.5 l) mixed with MNV-1 external control
(2.5 l) was compared to the C t value of the external control mixed only with RNAfree sterile water. These steps allowed the identification of samples that required
re-extraction and provided assurance that the samples with no amplification signal
were negative (values below the limit of detection for the assays) and not simply
inhibited. Negative controls containing no nucleic acid as well as positive controls,
containing RNA of MNV-1, were introduced in each run.
Primers and Probes
qRT-PCR for viral detection and quantification was carried out with TaqMan
probes using the Platinum Quantitative RT-PCR Thermoscript One-step System
(Invitrogen, France) following the manufacturer’s instructions in 25 l of a reaction
mixture containing 5 l of extracted RNA. For MNV-1 detection 0.2 M of
reverse and forward primers as well as probe were added. Primer sets and probes
employed were Fw-ORF1/ORF2, Rv-ORF1/ORF2 and probeMGB-ORF1/ORF2
(Baert et al. 2008).
Amplification Conditions
A Mx3005P QPCR System (Stratagene, USA) thermocycler was employed following amplification conditions described by Baert et al. (2008) with minor
modifications. Briefly, after a RT step at 45
ı C for 1 h, PCR amplification was
carried out employing the following cycling conditions: an initial denaturation at
95
ı C for 5 min, and 50 cycles of amplification with a denaturation at 95
ı C for 15 s
and annealing-extension step at 60
ı C for 1 min.