Microbial DNA qPCR Arrays

リアルタイムPCR によるアプリケーション特異的な微生物の同定およびプロファイリング

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適切なターゲット特異的アッセイおよびパネルを探すか、またはターゲットをカスタムデザインし、お客様のご興味のある生物学的ターゲット評価にお使いください。

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は分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

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特徴

微生物種、毒性因子の遺伝子、または抗生物質耐性遺伝子の同定
さまざまなサンプルに対応する操作手順
コントロールアッセイを含むことにより信頼できる結果

製品詳細

Microbial DNA qPCR Arrays は、様々なサンプルから関連した微生物種、病原性因子の遺伝子、または抗生物質耐性遺伝子の同定およびプロファイリングするためのアレイです。Microbial DNA qPCR Arrays は、様々なサンプルから関連した微生物種、病原性因子の遺伝子、または抗生物質耐性遺伝子の同定およびプロファイリングするためのアッセイパネルです。種の同定用アッセイは、バクテリアの16S rRNA遺伝子および真菌のrRNA遺伝子をターゲットとし、各アレイには、ホストDNA、バクテリアDNAの含有、PCR反応を確認するためのコントロールが含まれています。また、このキットには、Microbial qPCR Mastermixが含まれています。本アレイの簡便な操作手順は、リアルタイムPCRを使用するいかなる実験室においても、ルーチンで信頼できる微生物遺伝子の同定またはプロファイリングを可能にします。

パフォーマンス

直線性のあるダイナミックレンジ

10～10⁶コピーのDNAテンプレートと、Microbial DNA qPCR Assaysを用いた増幅の結果は直線性を示しました（図）。

定量下限（Lower limit of quantification；LLOQ）

LLOQとは標準曲線の直線状にのる下限濃度です（図）。全てのMicrobial DNA qPCR Assaysのうち、93％が100コピー以下のLLOQを示しました（図）。微生物同定用のアッセイでは92％、毒性因子遺伝子のアッセイは95％、抗生物質耐性遺伝子のアッセイの97％が100コピー以下の定量下限を示しました（図、、および）。

特異性

それぞれのMicrobial DNA qPCR Assayはターゲットとなる種および遺伝子を一つだけ検出するように厳密に検証されています（図）。その他のターゲットも検出するアッセイについては、in silicoで予測された検出対象ターゲットがspecifications sheetに記載されています。

この特異性は、糞便、唾液および歯垢のような複雑な生物種を持つサンプルにおいても維持され（図）、様々なシークエンス法でも検証されています（図）。

再現性

Microbial DNA qPCR Assaysは同一個体内比較および個体間比較を検証しており、どちらも高い再現性を示します（図）。

図参照

Linearity and sensitivity of Microbial DNA qPCR Arrays.

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.

Specificity of the Antibiotic Resistance Genes Microbial DNA qPCR Array is verified by pyrosequencing.

Microbial DNA qPCR Arrays generate reliably reproducible results.

原理

Microbial DNA qPCR Assaysは種を同定するために、バクテリアの16S rRNA遺伝子、真菌のrRNA遺伝子配列、病原性因子の遺伝子および抗生物質耐性遺伝子をプライマーおよび加水分解プローブを用いたPCR増幅で検出するようにデザインされています。

Microbial DNA qPCR Arraysは96または384ウェルプレートで関連性の高いアッセイ（例：呼吸器感染に関連する微生物種や、生物防御研究に関する病原体など）で構成されています。各プレートには、確実な結果を得るため、真菌、バクテリアおよび宿主ゲノムDNAの存在コントロール、PCR反応の確認コントロールが含まれております。

操作手順

Microbial DNA qPCR Arrayの操作は簡便で、さらに一般のラボにあるリアルタイムPCR装置にそのまま使用できます。DNAはサンプル種に適応するQIAampキットを用いて抽出し、適切なMicrobial qPCR Mastermixと混合します。反応ミックスをアレイプレートに分注し、リアルタイムPCRによりそれぞれのアッセイから生のC_T値が得られます。サンプル中の微生物種または遺伝子の同定およびプロファイリングを行なうことが出来ます。

アプリケーション

Microbial DNA qPCR Arraysは様々なサンプル中の微生物種または遺伝子の同定およびプロファイリングに最適です。たとえば、Vaginal Flora Microbial DNA qPCR Arrayは、細菌性膣炎の基本的な原因の解明に使用でき（図および）、Antibiotic Resistance Genes Microbial DNA qPCR Arrayは、腸内または汚泥サンプル中のこれらの遺伝子の同定に有効です（図および）。

図参照

The Vaginal Flora Microbial DNA qPCR Array provides accurate profiling for cervical swab samples.

Vaginal samples positive for Gardnerella vaginalis also show changes in commensal and bacterial vaginosis-related microbes compared to healthy samples.

The Microbial DNA qPCR Array screens gut microbiota for the presence of antibiotic resistance genes.

The Antibiotic Resistance Genes Microbial qPCR Array identified antibiotic resistance genes in sewage samples.

裏付けデータと数値

Linearity and sensitivity of Microbial DNA qPCR Arrays.

Linearity and sensitivity for each Microbial DNA qPCR Array was determined using synthetic templates over a 6 log serial dilution ranging from 1 copy to 1 million copies. The following are representative results for all the qPCR assays. [A] shows the real-time amplification curves of the KPC antibiotic resistance gene qPCR assay. In [B], a standard curve was prepared that shows that the primer efficiency equals 103% (calculated from slope = –3.3236) and the correlation coefficient is 0.9983, indicating optimum performance for the KPC qPCR assay. All Microbial DNA qPCR Assays have primer efficiencies between 80–120% and correlation coefficients (R)>0.995.

リソース

Download Safety Data Sheets for QIAGEN product components.

The work presented here thus offers an original assessment of the dynamics at play in the tree phyllosphere.

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

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