DNA was isolated from the remaining body parts with a QIAamp® DNA Mini Kit 250 (Qiagen, Hilden, Germany). PCR amplification of a 658-basepairs (bp) fragment of the cytochrome c oxidase subunit I (coxI) gene was performed using the primers LCO-1490/CoxUniEr following the protocol of Kniha et al. [22 ].
PCR was performed with an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany). Bands were analysed with a Gel Doc™ XR + Imager (Bio-Rad Laboratories Inc., California, USA), cut out from the gel and purified with an Illustra™ GFX™ PCR DNA and Gel Purification Kit (GE Healthcare, Buckinghamshire, UK). Sequencing was performed with a Thermo Fisher Scientific SeqStudio (Thermo Fisher Scientific, Massachusetts, USA). Obtained sequences from both strands were aligned with ClustalX 2.1, edited with GeneDoc 2.7.0 and consensus sequences were blasted in the NCBI sequence database (GenBank) and compared to reference sequences.
MALDI-TOF protein profiling was done as previously described [23 (link), 24 (link)]. The protein extracts from thoraces of chosen specimens were mixed with a sinapinic acid matrix and mass spectra were acquired with an Ultraflex III MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany). The spectra were visualized by FlexAnalysis 3.4 software, processed by MALDI Biotyper 3.1 and compared with an in-house reference database.
PCR was performed with an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany). Bands were analysed with a Gel Doc™ XR + Imager (Bio-Rad Laboratories Inc., California, USA), cut out from the gel and purified with an Illustra™ GFX™ PCR DNA and Gel Purification Kit (GE Healthcare, Buckinghamshire, UK). Sequencing was performed with a Thermo Fisher Scientific SeqStudio (Thermo Fisher Scientific, Massachusetts, USA). Obtained sequences from both strands were aligned with ClustalX 2.1, edited with GeneDoc 2.7.0 and consensus sequences were blasted in the NCBI sequence database (GenBank) and compared to reference sequences.
MALDI-TOF protein profiling was done as previously described [23 (link), 24 (link)]. The protein extracts from thoraces of chosen specimens were mixed with a sinapinic acid matrix and mass spectra were acquired with an Ultraflex III MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany). The spectra were visualized by FlexAnalysis 3.4 software, processed by MALDI Biotyper 3.1 and compared with an in-house reference database.
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