Microbial DNA qPCR Arrays

real-time PCR方法的微生物鉴定或分析

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Cat. No. / ID: 330261

Array plate and master mix for detection of microbial species or genes

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Microbial DNA qPCR Arrays 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

可检测微生物物种、毒力因子基因、或抗生素耐药性基因
适用于多种类型样本，实验流程简单
含有内参，可确保获得可靠结果

Product Details

Microbial DNA qPCR Arrays含有多种引物、探针、试剂、对照等，用于鉴定和分析微生物物种、毒力因子基因、或抗生素耐药性基因，适用于多种类型样本。物种鉴定试剂可检测细菌的16S rRNA基因和真菌的核糖体RNA基因，每个芯片都含有宿主DNA对照、细菌DNA对照、和监测PCR反应是否成功的对照。该芯片试剂盒中还提供Microbial qPCR Mastermix。该产品规格便利、操作简单，可用于各种实验室常用real-time PCR仪，实现对微生物物种和样本中基因的可靠鉴定和分析。

Performance

线性扩增与动态范围

Microbial DNA qPCR Assays可对10至10⁶个拷贝的DNA模板进行符合现行规律的扩增（参见）。

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%微生物鉴定产品能达到这一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）；其特异性可通过测序验证（参见）。

可重复性

同一实验员和不同实验员使用Microbial DNA qPCR Assays进行多次检测，都具有可重复性（参见）。

See figures

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.

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。

Microbial DNA qPCR Arrays为96孔板或384孔板规格，含有相应的引物、探针、对照和试剂等；例如，与呼吸道感染相关的微生物物种、或生物反恐研究的微生物等。每个孔板都含有多种对照，用于检测是否存在真菌DNA、细菌DNA、宿主基因组DNA，以及监测PCR反应是否成功，确保获得可靠结果。

Procedure

Microbial DNA qPCR Assay的实验流程简单，能够在各种实验室real-time PCR仪上使用。使用适用于该类型样本的QIAamp试剂盒从样本中分离DNA，然后使用与PCR仪相应的Microbial qPCR Mastermix构建PCR反应体系。将混合液等分后加入孔板各孔，然后进行real-time PCR，获得每孔的原始C_T值。然后使用免费的数据分析软件，分析样本中的基因或微生物物种。

Applications

Microbial DNA qPCR Arrays用于鉴定和分析微生物物种或基因，适用于多种类型样本。例如，Vaginal Flora Microbial DNA qPCR Array可用于研究细菌性阴道炎的致病原因（参见和）。而Antibiotic Resistance Genes Microbial DNA qPCR Array可分析肠道或污水样本中的抗生素耐药性基因（参见和）。

See figures

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.

Supporting data and figures

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.

Resources

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

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

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

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