Journal: Bio-protocol

Article Title: Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes

doi: 10.21769/BioProtoc.2504

Figure Lengend Snippet: . The 3’ and the 5’ target recognition arms bind to the multiplex PCR products of the β-lactamase genes. During the binding process, the padlock probes are circularized and subsequently ligated. The maximum distance of the padlock probe binding region to the 3’ or 5’-ends of the PCR product should not exceed 200 base pairs. Since the padlock probes gets concatenated with the PCR product upon the ligation reaction, longer distances cause an inhibition of subsequent RCAs. The C2CA sequence is needed for the circle-to-circle amplification and comprises an AluI restriction site for monomerization of the amplification products. The barcode sequence is derived from the 16S rRNA gene. This allowed us to halve the number of microarray probes because the C2CA products and 16S rRNA gene PCR products are detected on the same microarray probe but in different fluorescence channels.

Article Snippet: Pipettes ( e.g ., Sartorius, catalog numbers: 728020, 728050, 728060 and 728070) Microbiological incubator shaker ( e.g ., IKA, model: KS 4000 i control) Tabletop centrifuge for 1.5 ml tubes ( e.g ., Eppendorf, model: 5424) Roche MagNA Lyser Instrument (Basel, Switzerland) Thermomixer comfort (Eppendorf, Hamburg, Germany) Epoch Microplate spectrophotometer (Biotek, Winooski, VT, USA) Biosan DNA/RNA UV-cleaner box (Warren, MI, USA) (recommended, see Note 4) Thermal cycler ( e.g ., Thermo Fisher Scientific, Applied Biosystems, model: GeneAmp TM PCR System 2700) (Paisley, UK) Heraeus Megafuge 40R with a TX-750 rotor (Thermo Fisher Scientific, Thermo Scientific TM , model: Heraeus TM Megafuge TM 40R, catalog number: 75004518; TX-750 rotor: Thermo Fisher Scientific, Thermo Scientific TM , catalog number: 75003180) and inserts for plates (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: 75003617) and Falcon tubes (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: 75003608) Slide humidity incubation box ( e.g ., LabScientific, catalog number: HIC-3) GeneMachines Omnigrid 100 contact arrayer (Madison, WI, USA) International Microarray Pin Stealth 3 SMP3 (Telechem, catalog number: SMP3) Agilent SureScan DNA Microarray Scanner (Santa Clara, CA, USA) Autoclave Sartorius arium pro UV ultrapure water system (Sartorius, model: arium ® pro)

Techniques: Multiplex Assay, Binding Assay, Ligation, Inhibition, Sequencing, Amplification, Derivative Assay, Microarray, Fluorescence

Journal: Bio-protocol

Article Title: Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes

doi: 10.21769/BioProtoc.2504

Figure Lengend Snippet: For the species identification, the 16S rRNA gene is amplified using universal 16S primers (A1). Subsequently, the PCR products are labeled using a linear PCR and Cy5-modified cytosines (A2). In parallel, β-lactamase genes are pre-amplified in a 33-plex PCR (B1). Then, the padlock probes are hybridized (B2) and ligated (B3) using the β-lactamase PCR products as template and amplified in a first rolling circle amplification (RCA) (B4). The RCA products comprise a C2CA sequence and corresponding 16S rRNA barcode sequences. In a next reaction, the RCA products are monomerized (B5) using a restriction enzyme and circularized (B6) to serve as a new template for a second RCA (B7). These C2CA products are labelled in a linear PCR reaction using Atto532-modified cytosines (B8). Finally, the labelled amplification products from the 16S rRNA gene PCR and from the multiplex padlock assay are pooled together and hybridized to a microarray detecting the products at two different fluorescence wavelengths (C).

Article Snippet: Pipettes ( e.g ., Sartorius, catalog numbers: 728020, 728050, 728060 and 728070) Microbiological incubator shaker ( e.g ., IKA, model: KS 4000 i control) Tabletop centrifuge for 1.5 ml tubes ( e.g ., Eppendorf, model: 5424) Roche MagNA Lyser Instrument (Basel, Switzerland) Thermomixer comfort (Eppendorf, Hamburg, Germany) Epoch Microplate spectrophotometer (Biotek, Winooski, VT, USA) Biosan DNA/RNA UV-cleaner box (Warren, MI, USA) (recommended, see Note 4) Thermal cycler ( e.g ., Thermo Fisher Scientific, Applied Biosystems, model: GeneAmp TM PCR System 2700) (Paisley, UK) Heraeus Megafuge 40R with a TX-750 rotor (Thermo Fisher Scientific, Thermo Scientific TM , model: Heraeus TM Megafuge TM 40R, catalog number: 75004518; TX-750 rotor: Thermo Fisher Scientific, Thermo Scientific TM , catalog number: 75003180) and inserts for plates (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: 75003617) and Falcon tubes (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: 75003608) Slide humidity incubation box ( e.g ., LabScientific, catalog number: HIC-3) GeneMachines Omnigrid 100 contact arrayer (Madison, WI, USA) International Microarray Pin Stealth 3 SMP3 (Telechem, catalog number: SMP3) Agilent SureScan DNA Microarray Scanner (Santa Clara, CA, USA) Autoclave Sartorius arium pro UV ultrapure water system (Sartorius, model: arium ® pro)

Techniques: Amplification, Labeling, Modification, Sequencing, Multiplex Assay, Microarray, Fluorescence