Qiaxcel screengel software

A set of 11 primer pairs were synthesized using the software Primer3 (Untergasser et al., 2012 (link)) and validated in 21 accessions of Piper representing 12 different species (Piper nigrum, Piper longum, Piper arboreum, Piper argyrophyllum, Piper attenuatum, Piper betel, Piper chaba, Piper hymenophyllum, Piper trichostachyon, Piper wallichi, Piper columbrinum, and Piper sarmentosum). Genomic DNA was isolated from the leaf using the cTAB DNA extraction method. The PCR reaction consisted of 1× PCR buffer, 2.5 mM of MgCl₂, 1 µM of primer, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 15 ng of template DNA in a total volume of 20 µl and cycled at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min followed by a final extension at 72°C for 10 min. The amplification products were resolved on a QIAxcel multicapillary system using QIAxcel High Resolution Kit 1200 (QIAGEN, No. 929002, New Delhi,Qiagen India Pvt. Ltd.) 50-800 bp v2.0 QX DNA size marker (QIAGEN, No. 929561, New Delhi,Qiagen India Pvt. Ltd.) and 15 bp/1,000 bp Qx alignment marker (QIAGEN No. 929521, New Delhi,Qiagen India Pvt. Ltd.) with the high-resolution run method OM700. The allelic sizes of each sample were calculated in the form of gel profiles and peaks using the QIAxcel ScreenGel software (QIAGEN, v1.5).

Crude bacterial template DNA for toxin profiling was prepared by resuspension of cells in a 5% (wt/vol) solution of Chelex-100 resin (Sigma-Aldrich, Castle Hill, NSW, Australia). All isolates were screened by PCR for the presence of toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB) genes (38 (link)). PCR ribotyping was performed as previously described (52 (link)). PCR ribotyping products were concentrated using a Qiagen MinElute PCR purification kit (Qiagen, Hilden, Germany). Visualisation of PCR products was performed with QIAxcel ScreenGel software (Qiagen, Hilden, Germany). Using the BioNumerics software package v.7.5 (Applied Maths, Sint-Martens-Latem, Belgium), the ribotyping patterns generated were compared to a reference library that consisted of over 16,000 C. difficile strains, including 54 internationally recognized RTs from the Anaerobe Reference Laboratory (ARL) (Cardiff, United Kingdom) and the European Centre for Disease Prevention and Control (ECDC) collection. Isolates that gave patterns that did not correspond to any internationally recognized RTs in our library but had previously been isolated by our laboratory were assigned an internal nomenclature prefixed with “QX.” RTs that were new to our library were assigned a new QX number.

The PCR products for α-thalassaemia were analysed by using agarose gel electrophoresis. PCR products of multiplex gap PCR were run on a 1.5% agarose gel electrophoresis using 1× Tris-Borate-EDTA (TBE) buffer. Whereas, PCR products for α-thalassaemia from multiplex ARMS PCR were run on a 1.2% agarose gel.
The PCR products from the β globin gene, except for reaction in MARMS-A, MARMS-B and MARMS-F, were separated using 2% agarose D1, Low EEO Pronadisa (Laboratorios CONDA, Madrid, Spain) in 1X TBE buffer (Biobasic, Markham, ON, Canada). DNA band was visualized under transilluminator (Vilber Lourmat, Sud Marne-la-Vallée, France). QIAxel Advanced System (QIAGEN GmBH, Hilden, Germany) was used to analyse MARMS-A, MARMS-B, and MARMS-F which used automated capillary electrophoresis to separate the products based on their sizes and visualised on the interfaced computer system using its dedicated QIAxcel ScreenGel Software (QIAGEN GmBH, Hilden, Germany).