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: ‘Fresenius’, no catalog number) Ultrapure water, hereafter simply referred to as water or H 2 O (Note 1) The universal bacterial 16S rRNA primers 45f++ (5’-GCYTAAYACATGCAAGTCGARCG-3’) and 783R (5’-TGGACTACCAGGGTATCTAATCCT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The fungal 18S rRNA (ITS region) primers ITS3 (5’-GCATCGATGAAGAACGCAGC-3’) and ITS4+ (5’-TCCT-CCGCTTATTGATATGCTTAAGT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The circle-to-circle amplification (C2CA) oligonucleotides C2CA- (5’-TACTCGAGGAGCTGCATACAC-3’) and C2CA + (5’-GTGTATGCAGCTCCTCGAGTA-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The Cy5-labelled hybridization control (complimentary to ‘Bsrev’) (5’-Cy5-AAGCTCACTGGCCGTCGTTTTAAA-3’) was ordered from Microsynth (Balgach, Switzerland) T4 DNA ligase (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EL0011) T4 polynucleotide kinase (10 U/µl) supplied with 10x reaction buffer A (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EK0031) ATP solution (100 mM) (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: R0441) Mastermix 16S Basic, DNA-free (Molzym, catalog number: S-040-0250) containing a 2.5x complete master mix, Moltaq 16S DNA polymerase and PCR-grade water (Note 2) dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: R0192) Cy5-dCTP (1 mM solution) (GE Healthcare, catalog number: PA55021) Vent R (exo-) DNA polymerase (New England Biolabs, catalog number: M0257S) supplied with 10x ThermoPol reaction buffer and 100 mM MgSO 4 ExpressHyb hybridization solution (Takara Bio, Clontech, catalog number: 636831) Ampligase thermostable DNA ligase (5 U/µl) and Ampligase 10x reaction buffer (Epicentre, catalog number: A32750) Bovine serum albumin (BSA), molecular biology grade (New England Biolabs, catalog number: B9000S) phi29 DNA polymerase supplied with 10x phi29 DNA polymerase reaction buffer (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EP0091) Alu I restriction enzyme (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: ER0011) Atto532-dCTP (MoBiTec, Göttingen, Germany) SDS solution 10% for molecular biology (AppliChem, catalog number: A0676) Tryptic soy broth, also referred to as CASO medium (Casein-peptone soymeal-peptone broth) (Merck, catalog number: 105459) PBS (10x), pH 7.2 (Thermo Fisher Scientific, Gibco TM , catalog number: 70013) Betaine monohydrate (Sigma-Aldrich, catalog number: B2754) UltraPure SSC, 20x (Thermo Fisher Scientific, Invitrogen TM , catalog number: 15557036) CASO medium (see Recipes) 1x phosphate-buffered saline (PBS) (see Recipes) 2x spotting buffer (6x SSC, 3 M betaine) (see Recipes)

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: Multiplex PCR program for 16S rRNA gene identification and for pre-amplification of β-lactamase-encoding gene information

Article Snippet: ‘Fresenius’, no catalog number) Ultrapure water, hereafter simply referred to as water or H 2 O (Note 1) The universal bacterial 16S rRNA primers 45f++ (5’-GCYTAAYACATGCAAGTCGARCG-3’) and 783R (5’-TGGACTACCAGGGTATCTAATCCT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The fungal 18S rRNA (ITS region) primers ITS3 (5’-GCATCGATGAAGAACGCAGC-3’) and ITS4+ (5’-TCCT-CCGCTTATTGATATGCTTAAGT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The circle-to-circle amplification (C2CA) oligonucleotides C2CA- (5’-TACTCGAGGAGCTGCATACAC-3’) and C2CA + (5’-GTGTATGCAGCTCCTCGAGTA-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The Cy5-labelled hybridization control (complimentary to ‘Bsrev’) (5’-Cy5-AAGCTCACTGGCCGTCGTTTTAAA-3’) was ordered from Microsynth (Balgach, Switzerland) T4 DNA ligase (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EL0011) T4 polynucleotide kinase (10 U/µl) supplied with 10x reaction buffer A (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EK0031) ATP solution (100 mM) (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: R0441) Mastermix 16S Basic, DNA-free (Molzym, catalog number: S-040-0250) containing a 2.5x complete master mix, Moltaq 16S DNA polymerase and PCR-grade water (Note 2) dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: R0192) Cy5-dCTP (1 mM solution) (GE Healthcare, catalog number: PA55021) Vent R (exo-) DNA polymerase (New England Biolabs, catalog number: M0257S) supplied with 10x ThermoPol reaction buffer and 100 mM MgSO 4 ExpressHyb hybridization solution (Takara Bio, Clontech, catalog number: 636831) Ampligase thermostable DNA ligase (5 U/µl) and Ampligase 10x reaction buffer (Epicentre, catalog number: A32750) Bovine serum albumin (BSA), molecular biology grade (New England Biolabs, catalog number: B9000S) phi29 DNA polymerase supplied with 10x phi29 DNA polymerase reaction buffer (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EP0091) Alu I restriction enzyme (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: ER0011) Atto532-dCTP (MoBiTec, Göttingen, Germany) SDS solution 10% for molecular biology (AppliChem, catalog number: A0676) Tryptic soy broth, also referred to as CASO medium (Casein-peptone soymeal-peptone broth) (Merck, catalog number: 105459) PBS (10x), pH 7.2 (Thermo Fisher Scientific, Gibco TM , catalog number: 70013) Betaine monohydrate (Sigma-Aldrich, catalog number: B2754) UltraPure SSC, 20x (Thermo Fisher Scientific, Invitrogen TM , catalog number: 15557036) CASO medium (see Recipes) 1x phosphate-buffered saline (PBS) (see Recipes) 2x spotting buffer (6x SSC, 3 M betaine) (see Recipes)

Techniques: Multiplex Assay

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: ‘Fresenius’, no catalog number) Ultrapure water, hereafter simply referred to as water or H 2 O (Note 1) The universal bacterial 16S rRNA primers 45f++ (5’-GCYTAAYACATGCAAGTCGARCG-3’) and 783R (5’-TGGACTACCAGGGTATCTAATCCT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The fungal 18S rRNA (ITS region) primers ITS3 (5’-GCATCGATGAAGAACGCAGC-3’) and ITS4+ (5’-TCCT-CCGCTTATTGATATGCTTAAGT-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The circle-to-circle amplification (C2CA) oligonucleotides C2CA- (5’-TACTCGAGGAGCTGCATACAC-3’) and C2CA + (5’-GTGTATGCAGCTCCTCGAGTA-3’) were ordered from Integrated DNA Technologies (Coralville, IA, USA) The Cy5-labelled hybridization control (complimentary to ‘Bsrev’) (5’-Cy5-AAGCTCACTGGCCGTCGTTTTAAA-3’) was ordered from Microsynth (Balgach, Switzerland) T4 DNA ligase (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EL0011) T4 polynucleotide kinase (10 U/µl) supplied with 10x reaction buffer A (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EK0031) ATP solution (100 mM) (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: R0441) Mastermix 16S Basic, DNA-free (Molzym, catalog number: S-040-0250) containing a 2.5x complete master mix, Moltaq 16S DNA polymerase and PCR-grade water (Note 2) dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: R0192) Cy5-dCTP (1 mM solution) (GE Healthcare, catalog number: PA55021) Vent R (exo-) DNA polymerase (New England Biolabs, catalog number: M0257S) supplied with 10x ThermoPol reaction buffer and 100 mM MgSO 4 ExpressHyb hybridization solution (Takara Bio, Clontech, catalog number: 636831) Ampligase thermostable DNA ligase (5 U/µl) and Ampligase 10x reaction buffer (Epicentre, catalog number: A32750) Bovine serum albumin (BSA), molecular biology grade (New England Biolabs, catalog number: B9000S) phi29 DNA polymerase supplied with 10x phi29 DNA polymerase reaction buffer (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: EP0091) Alu I restriction enzyme (Thermo Fisher Scientific, Thermo Scientific TM , catalog number: ER0011) Atto532-dCTP (MoBiTec, Göttingen, Germany) SDS solution 10% for molecular biology (AppliChem, catalog number: A0676) Tryptic soy broth, also referred to as CASO medium (Casein-peptone soymeal-peptone broth) (Merck, catalog number: 105459) PBS (10x), pH 7.2 (Thermo Fisher Scientific, Gibco TM , catalog number: 70013) Betaine monohydrate (Sigma-Aldrich, catalog number: B2754) UltraPure SSC, 20x (Thermo Fisher Scientific, Invitrogen TM , catalog number: 15557036) CASO medium (see Recipes) 1x phosphate-buffered saline (PBS) (see Recipes) 2x spotting buffer (6x SSC, 3 M betaine) (see Recipes)

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