Microbial DNA qPCR Arrays

For real-time PCR-based, application-specific microbial identification or profiling

Features

  • Profile microbial species, virulence genes, or antibiotic resistance genes
  • Simple procedure for a variety of sample types
  • Integrated controls ensure reliability of results
Microbial DNA qPCR Arrays

Cat. No. / ID: 330261

Array plate and master mix for detection of microbial species or genes
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Microbial DNA qPCR Arrays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Microbial DNA qPCR Arrays are collections of assays to identify and profile microbial species, virulence factor genes or antibiotic resistance genes from a variety of sample types. Species identification assays target bacterial 16S rRNA genes and fungal ribosomal RNA genes, and each array includes controls for host DNA, presence of bacterial DNA and success of the PCR reaction. Microbial qPCR Mastermix is also included in the array kit. The simplicity of the product format and operating procedure allow routine, reliable identification or profiling of microbial species and genes from samples in any research laboratory with access to a real-time PCR instrument.

What is the difference between microbial identification and profiling? Identification is determining the microbe’s presence or absence in your sample which requires running a No Template Control during your analysis. Profiling is determining the microbe’s relative expression in two or more experimental conditions for which you will need to run a reference sample and a normalizer (provided by QIAGEN).

Performance

Linearity and dynamic range
Microbial DNA qPCR Assays display linear amplification across a range from 10 to 106 copies of DNA template (see figure,  Linearity and sensitivity of Microbial DNA qPCR Assays).

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 gene detection Microbial DNA qPCR Assays reveals high sensitivity, and  The LLOQ for antibiotic resistance gene detection 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), and is verifiable by sequencing methods (see figure,  Specificity of the Antibiotic Resistance Genes Microbial DNA qPCR Array is confirmed by pyrosequencing). 

Reproducibility
Microbial DNA qPCR Assays are highly reproducible, both in intra- and inter-individual variability tests (see figure,  Microbial DNA qPCR Arrays generate reliably reproducible results).
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. 

Microbial DNA qPCR Arrays are 96- or 384-well plates comprising related assays; for example, microbial species associated with respiratory infections, or microbes that are studied for biodefense. Controls are integrated on each plate to test for the presence of fungal DNA, bacterial DNA, host genomic DNA, and success of the PCR reaction, ensuring confidence in the results.

Procedure

The Microbial DNA qPCR Array procedure is simple and can be carried out in any laboratory with a real-time PCR instrument. DNA is isolated using the QIAamp kit that is suited for the sample type, and then mixed with the appropriate Microbial qPCR Mastermix. The mixture is aliquotted across the array plate, and real-time PCR is performed to obtain the raw CT values for each assay. The complimentary data analysis software is then used to profile genes or species in the sample.

Applications

Microbial DNA qPCR Arrays are highly suited for identification or profiling of microbial species or genes in a variety of sample types. For example, the Vaginal Flora Microbial DNA qPCR Array can be used to explore the underlying causes of bacterial vaginosis (see figures,  The Vaginal Flora Microbial DNA qPCR Array provides accurate profiling for cervical swab samples and  Vaginal samples positive for Gardnerella vaginalis also show changes in commensal and bacterial vaginosis-related microbes compared to healthy samples), while the Antibiotic Resistance Genes Microbial DNA qPCR Array can identify these genes in samples from the gut or from sewage (see figures,  The Microbial DNA qPCR Array screens gut microbiota for the presence of antibiotic resistance genes and  The Antibiotic Resistance Genes Microbial DNA qPCR Array identified antibiotic resistance genes in sewage samples).
See figures

Supporting data and figures

Resources

Download Files (20)
Webinars (1)
The work presented here thus offers an original assessment of the dynamics at play in the tree phyllosphere.
Analysis Software (1)
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
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