PCR-Based Total Microbial Quantification

Overview

In the fields of clinical and veterinary diagnostics and food protection, real-time PCR (quantitative PCR, qPCR) is now an excellent tool for the identification, quantification, and typing of various microbial agents. qPCR calculates the number of DNA area copies identified by a specific PCR primer(s). Using this procedure, the 16S amplicon is precisely amplified and the total bacterial content in each sample is quantified to determine the total bacterial load.

Our Advantages:

Extensive experience in handling various samples.
Makes gene number discrimination across a larger dynamic spectrum than the end-point PCR.
High-resolution and high-reproducibility data.
Efficient and accurately quantitative approach for the study of gene expression.

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Introduction to our PCR-based microbial quantification platform

qPCR is a method for measuring the copy number of a region of DNA defined by a specific PCR primer. Using this method, we can specifically amplify 16S amplicons and quantify the total bacterial content in each sample to determine the total bacterial load. By providing an accurate measure of bacterial abundance in a sample, this technique can also be used as a method to determine the feasibility of microbiome studies. Prior to sequencing samples suspected of having low biomass, use this technique to determine if these samples will result in positive amplification.

CD Genomics provides not only the identification of the complete total of target DNA in the sample on the basis of a calibration curve made up of normal serially diluted samples of defined concentrations or copy numbers, but also semi-quantitative outcomes without criteria but with controls used as a guide material. Our qPCR-based microbial quantification service can offer: 1) an exact calculation of bacterial abundance in your cultures as a way to assess the viability of your microbiome research; 2) the identification of the quantification target prototype by tracking the PCR product amplification through a potential increase in the fluorescent signal correlated with the development of the product during each step in the PCR; 3) information in accordance with differences in abiotic or biotic factors and/or biological behaviors and process levels, the quantitative data produced can be used to correlate variability in gene abundances and/or levels of gene expression (in terms of transcript numbers).

PCR-Based Total Microbial Quantification Service Workflow

Bioinformatics Analysis

CD Genomics provides a tiered analysis and documentation kit that can be personalized according to the goals of the test.

Livak-Schmittgen method
Linear and Non-linear Method
Evaluation of essential PCR parameters
Novel five-parameter models

Sample Requirement

1. 1.8 < OD_260/280 < 2.0, no degradation or contamination.
2. Mixed microbial genomic DNA: total amount > 1 ug, concentration > 10 ng/ul.

Gram-positive bacteria are not easy to extract from a complex microbial community due to the relatively thick cell wall, and the use of conventional methods may result in the loss of strains. Please provide us with more than 10 ml of microbial solution (OD₆₀₀ > 1.5) or the microbial pellet.

Sampling kits: We provide a complete range of microbial sampling kits for clients, including microbial collection products, DNA/RNA isolation kits, and accessories for storage and mailing.

Deliverables: The experimental report includes information on experimental methods, primers, PCR conditions, gel electropherogram,etc.

* For Research Use Only. Not for use in diagnostic procedures or other clinical purposes.