Quantitative Microbial Assays (qMA™)

Product Description

Branchpoint Biosciences’ pre-mixed qMA™ assays consist of two PCR primers and a 5’-hydrolysis probe that enable the rapid quantification of specific bacterial species within complex microbiome samples. Each assay is designed using a proprietary algorithm and up-to-date genomic databases before undergoing a rigorous experimental validation process to verify target specificity and sensitivity to ensure accurate quantification.

Assay Contents

For general lab use

Assays are 10x concentrated, ready-to-use pre-mixed reaction components for probe-based real-time quantitative PCR. 

Assays include: species-specific forward primer, reverse primer, and fluorogenic probe resuspended in stabilizing buffer (10 mM Tris-HCl pH 8.0/0.1 mM EDTA). 

All assays are optimized for annealing and extension at 60°C.

Storage and Stability

Guaranteed for 12 months in a consistent temperature freezer at -20°C protected from light. For convenience, pre-mixed assays can be stored at 4°C for up to 3 months.

Avoid excessive freeze-thaw cycles.

Protect from direct light.

Instrument Compatibility

Pre-mixed assays are compatible with all commercially available real-time qPCR systems (ROX-independent and ROX-dependent). System and analysis settings may need to be adjusted accordingly if using assays including ROX-labeled probes.

Multiplex Assays

Combining multiple qMA™ into a single reaction can save time and money. Visit our qMA™ list to see which species-specific assays have validated multiplex reactions. (Request a new multiplex set-up here [https://branchpointbiosciences.com/contact/]).

Be sure to consult your instrument-specific instructions to ensure capacity to collect data from fluorescent channels associated with fluorophores.

Reaction Mix Preparation and Thermal Cycling Protocol

  1. Thaw Branchpoint Assays and other frozen reaction components. Mix thoroughly, then centrifuge briefly. Store on ice protected from light.
  2. Prepare (on ice or at room temperature) enough reaction mix for all qPCR reactions by adding the required components, excluding DNA template, according to recommendations in Table 1. Include 2µl of each assay if multiplexing.

 

Table 1: Recommended Reaction Setup

 

*Master Mix not supplied. Branchpoint assays are compatible with most commercially-available probe-based qPCR Master Mixes. PrimeTime™ Gene Expression Master Mix (IDT) or iTaq Universal Probes Supermix (Bio-Rad) are recommended.

 

 

 

  1. Mix reaction components thoroughly, then distribute equal aliquots into each qPCR reaction tube or well. Practice good pipetting technique to avoid contamination and ensure assay precision and accuracy.
  2. Add DNA templates (and nuclease free water if needed) to qPCR tubes or wells containing the reaction mixtures.
  3. Seal tubes or wells, then spin to remove any air bubbles and collect reaction mixtures at the bottom of the vessel.
  4. Load the tubes or plate onto the real-time PCR instrument with the recommended cycling (Table 2) and data collection settings (Table 3) and start.

 

Table 2: Thermal Cycling Protocol      

Table 3: Fluorescence Detection

 

  1. Perform data analysis according to instrument-specific instructions upon completion.