Microbial DNA qPCR Assays

采用real-time PCR技术进行微生物检测

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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

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Assay

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Microbial DNA qPCR Assays 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

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

Features

经实验验证，具有高度灵敏性和特异性
可检测微生物物种、毒力因子基因、或抗生素耐药性基因
简单便利的qPCR工作流程

Product Details

Microbial DNA qPCR Assays含有两种PCR引物（每种10 µM）、以及一种5'水解探针（5 µM）用于定量real-time PCR。Microbial DNA qPCR Assays采用经实验验证的专有技术，其PCR效率高，扩增效果准确、稳定。所有的Microbial DNA qPCR Assay都经过严格的实验验证，PCR反应效率高。该产品搭配Microbial qPCR Mastermixes和Microbial DNA-Free Water使用，可确保PCR的高效率。

Performance

最低定量阈限（LLOQ）

LLOQ是指在标准曲线线性范围内的最低模板浓度（参见）。所有Microbial DNA qPCR Assays中的93%具有低至100个基因拷贝的LLOQ（参见）。该系列产品中的92%微生物检测产品能达到这一LLOQ，95%的毒力因子检测产品和97%的抗生素耐药性基因检测产品能达到这一LLOQ（参见、和）。

特异性

所有Microbial DNA qPCR Assay都经过严格检验，确保能够灵敏检测某一物种或基因（参见）。对于能够检测一种以上五种或基因的产品，产品说明页上附有电脑预测可检测靶标的列表。

即便样本中存在多种物种，该试剂盒仍有极高的检测特异性，此类样本包括粪便、痰和菌斑（参见）。

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在进行物种检测时，可检测细菌的16S rRNA基因和真菌核糖体rRNA基因序列；采用PCR扩增引物和水解探针，检测毒力因子基因和抗生素耐药性基因。

Procedure

Microbial DNA qPCR Assay的实验流程简单，能够在各种real-time PCR仪上使用。使用相应的QIAamp试剂盒从样本中分离DNA，然后使用与PCR仪相应的Supplemental Microbial qPCR Mastermix构建PCR反应体系。为每种样本构建4个独立的PCR反应体系，包括Positive PCR Control、No Template Control、Microbial DNA Positive Control以及Microbial DNA qPCR Assay。进行real-time PCR，然后使用在线数据分析软件或Excel模板文件进行数据分析。

Applications

Microbial DNA qPCR Assay高度适用于细菌或真菌物种检测，以及微生物的抗生素耐药性基因或毒力因子检测，适用样本包括粪便、痰、阴道拭子、污水等等。

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

Download Safety Data Sheets for QIAGEN product components.

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

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