Microbial DNA qPCR Assays

For real-time PCR-based microbial detection; includes one assay with Microbial qPCR Mastermix for 100 reactions

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Microbial DNA qPCR Assay

Cat. No. / ID: 330025

One 100 µl tube Microbial DNA qPCR Assay, one 1.35 ml tube Microbial qPCR Mastermix

Microbial DNA qPCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Features

Experimentally verified assays with high sensitivity and specificity
Detect microbial species, virulence genes or antibiotic resistance genes
Simple qPCR workflow

Product Details

Microbial DNA qPCR Assays are a mix of two PCR primers (10 µM each) and one 5′-hydrolysis probe (5 µM) that enables quantitative real-time PCR for 100 reactions. Microbial DNA qPCR Assays are designed using a proprietary and experimentally verified algorithm, providing uniform PCR efficiency and amplification conditions. Each Microbial DNA qPCR Assay undergoes rigorous experimental verification to ensure high PCR efficiency, and this high efficiency is guaranteed when the assays are used with Microbial qPCR Mastermixes and Microbial DNA-Free Water (sold separately).
What is the difference between microbial identification and profiling? Identification is determining the microbe’s presence or absence in your sample which requires you to run a No Template Control during your analysis. Profiling is determining the microbe’s relative expression in two or more experimental conditions and you will need to run a reference sample and a normalizer (provided by QIAGEN).

Performance

Lower limit of quantification (LLOQ)

The LLOQ is the lowest concentration of template that still falls into the linear range of the standard curve (see figure, ). Across all Microbial DNA qPCR Assays, 93% have an LLOQ of <100 gene copies (see figure, ). 92% of microbial identification assays meet this LLOQ, as do 95% of virulence factor gene assays and 97% of antibiotic resistance gene assays (see figures, , , and ).

Specificity

Each Microbial DNA qPCR Assay is stringently tested to ensure that it detects only one target species or gene (see figure, ). For assays that do detect more than one target, a list of detected targets and in silico predictions is included on the product sheet.
This specificity is maintained even when samples have high species complexity, such as in stool, sputum, and plaque (see figure, ).

See figures

Limit of detection versus lower limit of quantification.

The lower limit of quantification (LLOQ) for all Microbial DNA qPCR Assays reveals high sensitivity.

The lower limit of quantification (LLOQ) for microbial identification Microbial DNA qPCR Assays reveals high sensitivity.

The lower limit of quantification (LLOQ) for virulence factor gene detection Microbial DNA qPCR Assays reveals high sensitivity.

The lower limit of quantification (LLOQ) for antibiotic resistance gene detection Microbial DNA qPCR Assays reveals high sensitivity.

Microbial DNA qPCR Assays are highly specific.

Microbial DNA qPCR Assays display high sensitivity even in complex metagenomic samples.

Principle

Microbial DNA qPCR Assays are designed to detect bacterial 16S rRNA gene and fungal ribosomal rRNA gene sequences for species identification, as well as detecting virulence factor genes and antibiotic resistance genes using PCR amplification primers and hydrolysis-probe detection.

Procedure

The procedure for Microbial DNA qPCR Assays is simple, and can be performed in any laboratory with a real-time PCR instrument. DNA is isolated from the sample using the appropriate QIAamp kit, and then PCR reactions are set up using the appropriate Supplemental Microbial qPCR Mastermix for the PCR instrument. Four separate PCR reactions should be prepared for each sample, including a Positive PCR Control, a No Template Control, and the Microbial DNA Positive Control, as well as the Microbial DNA qPCR Assay. Real-time PCR is performed and data is analyzed using web-based data analysis software or Excel templates.

Applications

Microbial DNA qPCR Assays are highly suited for the detection of bacterial or fungal species, or microbial genes for antibiotic resistance or virulence factors, from a variety of samples including stool, sputum, vaginal swab, sewage, and others.

Supporting data and figures

Limit of detection versus lower limit of quantification.

This chart demonstrates the difference between the limit of detection (LOD) and the lower limit of quantification (LLOQ). The LOD is defined as the lowest concentration at which 95% of the positive samples are detected, whereas the LLOQ is the lowest concentration that falls within the linear range of a standard curve. LOD depends upon the precision of the assay, and requires at least 40 replicates for determination of a positive sample. For the Microbial DNA qPCR Assays, LLOQ is sufficient to determine assay sensitivity.

Resources

Microbial product configurator guide

For application-specific microbial identification and profiling by real-time PCR

For real-time PCR-based profiling/detection of microbial species, antibiotic resistance genes or virulence factor genes

FAQ

Pan Bacteria 1 and Pan Bacteria 3 are designed to detect the broadest possible collection of bacteria involved in human biology. We began by designing assays against evolutionarily conserved regions of the 16S rRNA gene. Bioinformatically these two assays are predicted to test positive for thousands of species, based off of perfect matches and near-perfect matches of the primers and probes with the target sequences. In order to confirm this on the bench, we assayed genomic DNA from 119 species. In the case of both assays, nearly all of the species showed amplification. Positive tests were recorded for species within these 7 phyla: Actinobacteria, Bacteroidetes, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, and Tenericutes. Additionally, Pan Bacteria 3 detected species within the phylum Spirochaetes.

For a finer breakdown of what our bench testing covered:

Pan Bacteria 1

Phylum	Class	Order	Family
Actinobacteria	Actinobacteria	Actinomycetales	Alcaligenaceae
Bacteroidetes	Alphaproteobacteria	Bacillales	Bacillaceae
Euryarchaeota	Bacilli	Bacteroidales	Bacteroidaceae
Firmicutes	Bacteroidia	Bifidobacteriales	Bartonellaceae
Fusobacteria	Betaproteobacteria	Burkholderiales	Bifidobacteriaceae
Proteobacteria	Clostridia	Campylobacterales	Brucellaceae
Tenericutes	Deltaproteobacteria	Caulobacterales	Burkholderiaceae
	Epsilonproteobacteria	Clostridiales	Campylobacteraceae
	Flavobacteria	Coriobacteriales	Caulobacteraceae
	Fusobacteriia	Desulfovibrionales	Clostridiaceae
	Gammaproteobacteria	Enterobacteriales	Coriobacteriaceae
	Methanobacteria	Flavobacteriales	Corynebacteriaceae
	Mollicutes	Fusobacteriales	Desulfovibrionaceae
		Lactobacillales	Enterobacteriaceae
		Legionellales	Enterococcaceae
		Methanobacteriales	Flavobacteriaceae
		Mycoplasmatales	Fusobacteriaceae
		Neisseriales	Helicobacteraceae
		Pasteurellales	Lactobacillaceae
		Pseudomonadales	Legionellaceae
		Rhizobiales	Listeriaceae
		Vibrionales	Methanobacteriaceae
		Xanthomonadales	Micrococcaceae
			Moraxellaceae
			Mycobacteriaceae
			Neisseriaceae
			Pasteurellaceae
			Peptostreptococcaceae
			Porphyromonadaceae
			Prevotellaceae
			Pseudomonadaceae
			Staphylococcaceae
			Streptococcaceae
			Veillonellaceae
			Vibrionaceae
			Xanthomonadaceae

Pan Bacteria 3

Phylum	Class	Order	Family
Actinobacteria	Actinobacteria	Actinomycetales	Alcaligenaceae
Bacteroidetes	Alphaproteobacteria	Bacillales	Bacillaceae
Euryarchaeota	Bacilli	Bacteroidales	Bacteroidaceae
Firmicutes	Bacteroidia	Bifidobacteriales	Bartonellaceae
Fusobacteria	Betaproteobacteria	Burkholderiales	Bifidobacteriaceae
Proteobacteria	Clostridia	Campylobacterales	Brucellaceae
Spirochaetes	Deltaproteobacteria	Caulobacterales	Burkholderiaceae
Tenericutes	Epsilonproteobacteria	Clostridiales	Campylobacteraceae
	Flavobacteria	Coriobacteriales	Caulobacteraceae
	Fusobacteriia	Desulfovibrionales	Clostridiaceae
	Gammaproteobacteria	Enterobacteriales	Clostridiales
	Methanobacteria	Flavobacteriales	Coriobacteriaceae
	Mollicutes	Fusobacteriales	Corynebacteriaceae
	Spirochaetes	Lactobacillales	Desulfovibrionaceae
		Legionellales	Enterobacteriaceae
		Methanobacteriales	Enterococcaceae
		Mycoplasmatales	Flavobacteriaceae
		Neisseriales	Fusobacteriaceae
		Pasteurellales	Helicobacteraceae
		Pseudomonadales	Lactobacillaceae
		Rhizobiales	Legionellaceae
		Spirochaetales	Listeriaceae
		Vibrionales	Methanobacteriaceae
		Xanthomonadales	Micrococcaceae
			Moraxellaceae
			Mycobacteriaceae
			Neisseriaceae
			Pasteurellaceae
			Peptostreptococcaceae
			Porphyromonadaceae
			Prevotellaceae
			Pseudomonadaceae
			Spirochaetaceae
			Staphylococcaceae
			Streptococcaceae
			Veillonellaceae
			Vibrionaceae
			Xanthomonadaceae

FAQ ID - 3427

The Microbial DNA qPCR Assays for bacterial species detection use the 16S rRNA gene as the template. Microbial DNA qPCR Assays for detection of fungal species use ribosomal (small subunit or larger subunit) genes as the template. Microbial DNA qPCR Assays for detection of microbial genes use various antibiotic resistance genes and virulence factor genes for the template.

FAQ ID — 3403

Store Microbial DNA qPCR Array plates at room temperature.
Store Microbial DNA qPCR Assays, Microbial DNA qPCR Assay Kits, and Microbial DNA qPCR Multi-Assay Kits at –20°C.
Store Microbial qPCR Mastermix at –20°C.
Store Microbial DNA Positive Control at –20°C.
Store Microbial DNA-Free Water at –20°C.

FAQ ID — 3394

Limit of detection (LOD) is defined as the lowest concentration at which 95% of positive samples are detected. Since LOD is not necessarily within the linear range of an assay, LOD can be lower than LLOQ.

FAQ ID — 3402

Positive PCR Control (PPC) is used to test for the presence of inhibitors in the sample or the efficiency of the polymerase chain reaction itself using a pre-dispensed artificial DNA sequence and the primer set that detects it. Microbial DNA Positive Control is a mixture of synthetic oligonucleotides that are targets for each primer/hydrolysis probe set. It is used to test that each Microbial DNA qPCR Assay is performing correctly.

FAQ ID — 3397

Yes, they are free of bacterial genomic DNA. Each Microbial DNA array contains a control for genomic DNA control, which gives a signal for gDNA contamination. A Ct below 35 indicates that there is a bacterial genomic DNA contamination.

FAQ ID - 3535

No, multiplexing is not an option because all of the Microbial DNA qPCR Assay 5′ hydrolysis probes are labeled with FAM.

FAQ ID — 3408

Microbial DNA qPCR Arrays and Assays / Multi-Assay Kits have been tested using genomic DNA from stool, sputum, tooth plaque, isolated bacterial colony, vaginal swab, and sewage samples, and can be used with a variety of other sample types.

FAQ ID — 3399

The Microbial DNA qPCR Arrays and Assay Kits do not allow measurement of antibiotic resistance gene expression because reverse transcriptase enzyme is not included. Microbial DNA qPCR Assays have not been tested on cDNA.

FAQ ID — 3404

The amplicon size of the Microbial DNA qPCR Assays varies from target to target. Each target is detected via an individual primer/probe set that determines the amplicon size.

FAQ ID — 3396

The lower limit of quantification (LLOQ) is the lower limit at which an assay can provide quantitative results. It is the lowest template concentration that is within the linear range of the assay.

FAQ ID — 3401

The Microbial DNA qPCR Arrays and Assay Kits do not allow measurement of virulence factor gene expression because reverse transcriptase enzyme is not included. Microbial DNA qPCR Assays have not been tested on cDNA.

FAQ ID — 3405

We recommend not using Microbial DNA-Free Water or Microbial qPCR Mastermix after opening more than 3 times due to increased chance of microbial contamination.

FAQ ID — 3398

Calculation of the number of bacterial cells present in a sample is determined by generating a standard curve using genomic DNA from a known amount of bacterial cells.

FAQ ID — 3411

Yes. The assays have been wet-lab verified to ensure optimal performance. Primer efficiency, correlation coefficient, sensitivity, and specificity have been determined for each assay.

FAQ ID — 3406

Most of the Microbial DNA qPCR Assays that detect bacterial species are species-specific. Assays that do detect more than one species will list which species are detected in the product specification sheet. These were determined by both experimental data and in silico prediction.

FAQ ID — 3400

The Microbial qPCR Mastermix (ROX) is suitable for use with the following real-time cyclers: all Applied Biosystems and Agilent (formerly Stratagene); BioRad Opticon, Opticon 2, and Chromo 4; Roche LightCycler 480; and Eppendorf Mastercycler ep realplex 2, 2S, 4, and 4S.

The Microbial qPCR Mastermix (Fluorescein) is suitable for use with the following real-time cyclers: BioRad iCycler, MyiQ, and iQ5.

FAQ ID — 3395


Array format	Metagenomic sample	Isolated colony
Array 96-well plate	500 ng	250 ng
Array 384-well plate	200 ng	100 ng
Single assay 96-well plate	5 ng	2.5 ng
Single assay 384-well plate	2 ng	1 ng

FAQ ID — 3393

All probes for Microbial DNA qPCR Assays are labeled with FAM.

FAQ ID — 3407

The sensitivity for Microbial DNA qPCR kits was determined by using the lower limit of quantification for each assay. Most of the assays had a lower limit of quantification less than 100 gene target copies. The sensitivity for each assay is listed in the product specification sheet.

FAQ ID — 3409