All species-specific probes were based on the sequence data from the lab data. Multiple-sequence alignments were carried out using BioEdit version 7.0 software. These species-specific probes were newly designed (Table 2 ). Probes were synthesized with a 5′-end amino C-12 modification (AuGCT, Beijing, China) and coupled to carboxylated beads (Bio-Rad Laboratories, Hercules, CA, United States) as described by the manufacture’s manual. Briefly, a total of 2.5 × 105 carboxylated beads were suspended in 8.5 μL of 0.1 M MES (pH 4.5) with 2 μL of 0.1 nmol/μL oligonucleotide probes. A 2.5 μL of 10 mg/mL freshly prepared EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] was added, and vortexed immediately, then incubated at room temperature in the dark for 30 min. After being washed by 0.02% Tween 20 and 0.1% SDS, pellets were centrifuged and resuspended in 20 μL of TE (pH 8.0), and stored at 4°C in the dark until used.
Carboxylated beads
Manufactured by Bio-Rad
Sourced in China
Carboxylated beads are a type of spherical particles with a carboxyl functional group on the surface. They are designed for a variety of applications in biotechnology and life science research.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using carboxylated beads
1
Probe Coupling to Carboxylated Beads
2
Serotype-specific Probe Design Protocol
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Serotype-specific probes were designed based on the wzx/wzy genes (S3 Table ). BioEdit software 7.0 version was used for multiple sequence alignments. The carboxylated beads (Bio-Rad, Hercules, CA) were coupled to specific probes with an amino C-12 modifiication at the 3’-end (AuGCT, China).
3
Oligonucleotide Probe Coupling for Bead-Based Detection
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All species-specific probes were designed based on the sequencing data obtained in
this study. Multiple-sequence alignments were performed using BioEdit version 7.0
software. These species-specific probes were newly designed (S4 Table ). The
probes were synthesized with a 5’-end amino C-12 modification (AuGCT, Beijing, China)
and coupled to carboxylated beads (Bio-Rad Laboratories, Hercules, CA) according to
the instructions in the manufacturer’s manual. Briefly, 2.5 x 105carboxylated beads were suspended in 8.5 μL of 0.1 M MES (pH 4.5) with 2 μL of 0.1
nmol/μL oligonucleotide probes. A 2.5 μL volume of 10 mg/mL freshly prepared EDC was
added, and the mixture was immediately vortexed. It was then incubated at room
temperature in the dark for 30 min. This step was repeated a second time. After the
beads were washed with 0.02% Tween 20 and 0.1% SDS, the pellets were centrifuged and
resuspend in 20 μL of TE (pH 8.0) and then stored at 4°C in the dark until used.
this study. Multiple-sequence alignments were performed using BioEdit version 7.0
software. These species-specific probes were newly designed (
probes were synthesized with a 5’-end amino C-12 modification (AuGCT, Beijing, China)
and coupled to carboxylated beads (Bio-Rad Laboratories, Hercules, CA) according to
the instructions in the manufacturer’s manual. Briefly, 2.5 x 105carboxylated beads were suspended in 8.5 μL of 0.1 M MES (pH 4.5) with 2 μL of 0.1
nmol/μL oligonucleotide probes. A 2.5 μL volume of 10 mg/mL freshly prepared EDC was
added, and the mixture was immediately vortexed. It was then incubated at room
temperature in the dark for 30 min. This step was repeated a second time. After the
beads were washed with 0.02% Tween 20 and 0.1% SDS, the pellets were centrifuged and
resuspend in 20 μL of TE (pH 8.0) and then stored at 4°C in the dark until used.
4
Serotype-Specific PCR Primer and Probe Design
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Primer Premier (v5.0, Premier Biosoft International, Palo Alto, CA, USA) was used to design serotype-specific PCR primers based on DNA sequences of the processing genes wzx/wzy and wzm/wzt that were obtained in this study. The forward primer was biotinylated at the 5’-end to allow binding to the reporter dye streptavidin-R-phycoerythrin for detection on a Bio-Plex platform. The primers generated PCR fragments of 151–217 bp (S2 Table ) that were then used to design serotype-specific probes based on the processing genes (S3 Table ) using multiple-sequence alignments with MUSCLE (v3.8.31). The final probes were 18–25 bp in length, and synthesized with a 5’-end amino C-12 modification (AuGCT, China) and coupled to carboxylated beads (Bio-Rad Laboratories, Hercules, CA).
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