Rattus species identification by cytb gene

Samples identified as non- Rattus norvegicus using the RFLP-method were subjected to further PCR and sequencing of the mitochondrial cytochrome b (cytb) gene to discriminate between closely related Rattus species. The PCR and sequencing of cytb gene were performed using primers mcytbHb (5’-GAATGGGAGAATGAAGTGGAATGCG-3’) and mcytb1 (5’-CCATCGTTGTAATTCAACTATAG-3’)^{8 (link)}. Each PCR reaction contained 1.5 µl of a prepared primer mix (with final concentrations of each primer being 2.5 µM), 1.5 µl extracted DNA from kidneys, 15 µl 2 × SYBR green master mix (Roche, Switzerland) and water to a final volume of 30 µl. The PCR reactions were performed and analysed using LightCycler 2.0 (Roche, Switzerland), with an initial holding temperature of 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s and 72 °C for 1 min, and 1 cycle of 10 min at 72 °C. Obtained PCR amplicons were purified using QIAquick PCR Purification Kit (QIAGEN, Germany) and sequenced using both the PCR amplification primers. The resulting DNA sequencing chromatogram were assembled using SeqMan Pro (DNASTAR Lasergene, USA). The sequences derived was aligned against the cytb gene sequence entries in GenBank using the online BLAST search engine of the National Center for Biotechnology Information (NCBI) to detect sequences with high similarity for species identification.