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NextGenPCR™ 16S Full Gene Amplification Kit

NextGenPCR™ 16S Full Gene Amplification

Sequencing of the 16S ribosomal DNA (16S rDNA) gene is a well-established method to identify ­bacterial species in biological specimens. Taxonomic identification using 16S sequence is based on exploiting the unique combination of hyper conserved and variable regions (V1 – V9).

Ultrafast analysis of bacterial composition in a biological specimen

The NextGenPCR™ 16S Full Gene Amplification Kit is a complete PCR assay solution intended for ­ultrafast analysis of bacterial composition in a biological specimen. The kit includes all necessary materials (primer oligonucleotides, ready-to-use polymerase master mix and genomic bacterial DNA control) to amplify the full length 16S rDNA gene in ≤ 21 minutes total cycling time.

Performance of the NextGenPCR™ 16S Full Gene Amplification Kit was evaluated using DNA template material isolated from the Microbial Community Standard (ZymoBIOMICS) using a MagNA Pure 96 System (Roche). Using 10 ng gDNA template as input and the cycling program listed in Table 1, ­amplicons were generated on a NextGenPCR™ Thermal Cycler using either NextGenPCR™ Arctic Fox HF Chemistry-2× (Molecular Biology Systems) or a competitor’s polymerase mix, of which two were tested. Amplicons were barcoded using a Rapid Barcoding Kit (SQK-RBK114.96) and sequenced using a Flow Cell R9.4.1 on a MinION MK1B sequencer (Oxford Nanopore Technologies). Sequences were ­analyzed using a custom bioinformatics pipeline (MABA16S, available through GitHub).

In the application note from MBS, written by R.B. Struyk, we conclude that integration of ultrafast amplification of the 16S gene using NextGenPCR™ products and rapid sequencing provides a promising new avenue for bacterial identification in a biological sample within a single working day. The NextGenPCR™ 16S Full Gene Amplification Kit offers several pronounced advantages over existing bacterial identification methods such as Sanger sequencing and MALDI-TOF, including the capability to simultaneously detect multiple organisms in a sample and the ability to confidently determine negative results if no reads are obtained:

Highlights of the Application Note

  • Single-day laboratory workflow suited for standard microbiological laboratories

  • Ultrafast amplification of full length 16S rDNA sequences in ≤ 21 minutes

  • Complete PCR kit including all necessary chemistries for target sequence amplification

  • High fidelity sequence data generation for V1–V9 regions allowing species-level determination

  • Suitable for complex and difficult to amplify samples, i.e. polymicrobial specimens

 

Do you want to learn more about NextGenPCR and 16S Full Gene Amplification, please read the applicatione note: Application-Brief-16S-Full-Gene-Amplification.pdf

Or sent an email to: info@mbspcr.com

 

Authors:
Robin Struijk, PhD Director of Science
Maaike Mathijssen

26 March 2024