Abi prism 3730xl

First strand cDNA was synthesized with use of SuperScript III Reverse Transcriptase (Invitrogen) following the manufacturer's protocol with gene-specific primers (S1 Table). PCR and RT-PCR were performed with use of a high fidelity Taq polymerase Pfu (Invitrogen). Amplicons of RT-PCR were directly sequenced by use of ABI PRISM 3730xl. To further verify the TS and RE ratio, RT-PCR amplicons were cloned, then sequenced using ABI PRISM 3730xl. The TS and RE ratio was inferred from the ratio of different sequences of clones.

We used direct and clone-sequencing methods to verify the real-time PCR results when detecting mixed infections of two hMPV genotypes. The amplified products were analysed by electrophoresis on a 2% agarose gel stained with ethidium bromide (EB), and the sizes of the amplified fragments were compared with those of standard molecular weight markers. To validate the amplification process and to exclude the presence of carryover contamination, positive and negative controls were included in each PCR. The amplified fragments were purified with a QIAquick PCR purification kit (Qiagen, Germany). The sequences were determined using an ABI Prism 3730 XL automated capillary DNA sequencer located at the company of Sangon (Shanghai, China).

Whole genomic DNA was extracted from the foot tissues using a NucleoSpin Tissue kit (MACHEREY-NAGEL, Germany), following the manufacturer’s standard protocol. Fragments of the cytochrome c oxidase subunit I (COI) mitochondrial gene was amplified and used to estimate molecular phylogeny. Primer sets used for polymerase chain reaction (PCR) and sequencing were either LCO1490 and HCO2198 (Folmer et al. 1994 (link)) or LoboF1 and LoboR1 (Lobo et al. 2013 (link)), depending on the specimen. The PCR amplification was conducted in a final volume of 20 µl containing 1 µl of each primer (10 mM), 10 ng of the extracted genomic DNA, 10 µl of EmeraldAmp PCR Master Mix (TAKARA BIO INC., Japan) and distilled water up to 20 μL total volume. Each PCR reaction was performed using a T100 thermal cycler (BIO-RAD, USA). The thermal cycling was started at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C (LCO1490 and HCO2198) or 46 °C (LoboF1 and LoboR1) for 60 s, extension at 72 °C for 90 s, then a final 72 °C for 5 min. All PCR products were purified by using a MicroSpin purification kit (Qiagen, USA) and then sent to BIONEER, Republic of Korea, for bi-directional sequencing on an automated sequencer (ABI Prism 3730XL). Nucleotide sequences were deposited in the GenBank database under GenBank submission numbers: OP491195 to OP491284 (Table 1).