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Microbial DNA qPCR Assays

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

Features

  • Experimentally verified assays with high sensitivity and specificity
  • Detect microbial species, virulence genes or antibiotic resistance genes
  • Simple qPCR workflow
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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
SEK 6,960.00
For plates
Assay
Custom Assay
  CONFIGURE AT GeneGlobe
Microbial DNA qPCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

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,  Limit of detection versus lower limit of quantification). Across all Microbial DNA qPCR Assays, 93% have an LLOQ of <100 gene copies (see figure,  The LLOQ for all Microbial DNA qPCR Assays reveals high sensitivity). 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,  The LLOQ for microbial identification Microbial DNA qPCR Assays reveals high sensitivity The LLOQ for virulence factor genes Microbial DNA qPCR Assays reveals high sensitivity, and  The LLOQ for antibiotic resistance genes Microbial DNA qPCR Assays reveals high sensitivity).

Specificity
Each Microbial DNA qPCR Assay is stringently tested to ensure that it detects only one target species or gene (see figure,  Microbial DNA qPCR Assays are highly specific). 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,  Microbial DNA qPCR Assays display high sensitivity even in complex metagenomic samples).
See figures

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

Resources

Safety Data Sheets (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.
Brochures & Guides (2)
Microbial product configurator guide
For application-specific microbial identification and profiling by real-time PCR
Kit Handbooks (1)
For real-time PCR-based profiling/detection of microbial species, antibiotic resistance genes or virulence factor genes

FAQ

What species are detected by the Pan Bacteria 1 and Pan Bacteria 3 Assays?

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
What sequences are used to design the Microbial DNA qPCR Assays?
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
What are the storage conditions for the Microbial DNA qPCR products?
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
What is the difference between LLOQ and LOD?
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
What is the difference between Positive PCR Control (PPC) and Microbial DNA Positive Control?
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
Is the Microbial qPCR mastermix used in the Microbial DNA assay and in the Microbial DNA arrays free of genomic bacterial DNA?

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
Can I multiplex the Microbial DNA qPCR Assays?
No, multiplexing is not an option because all of the Microbial DNA qPCR Assay 5′ hydrolysis probes are labeled with FAM.
FAQ ID — 3408
What sample types can be tested on the arrays/assays?
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
Can I measure antibiotic resistance gene expression?
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
What is the expected amplicon size of the Microbial DNA qPCR Assays?
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
What is LLOQ?
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
Can I measure virulence factor gene expression?
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
Can I use the Microbial DNA-Free Water and Microbial qPCR Mastermix if they have been opened more than 3 times?
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
How can I calculate the number of bacterial cells that are present in a sample using the Microbial DNA qPCR Assays?
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
Are the Microbial DNA qPCR Assays wet-lab verified?
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
Are the assays species-specific?
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
Which Microbial qPCR Mastermix should I use?
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
What are the minimum sample requirements for Microbial DNA qPCR kits?
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
Which probe labels are available for the Microbial DNA qPCR Assays?
All probes for Microbial DNA qPCR Assays are labeled with FAM.
FAQ ID — 3407
What is the sensitivity for the Microbial DNA qPCR kits?
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