Microbial DNA qPCR Multi-Assay Kits

用于real-time PCR方法的微生物鉴定或分析

S_1084_5_GEN_V2
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Microbial DNA qPCR Multi-Assay Kit

Cat. No. / ID:  330043

One 20 µl tube for each Microbial DNA qPCR Assay, 1 tube Positive PCR Control (20 µl), 1 tube Microbial DNA Positive Control (50 µl), 1 or 2 tubes Microbial DNA-Free Water (1.35 ml each), 1 or 2 tubes Microbial qPCR Mastermix (1.35 ml each)
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Microbial DNA qPCR Multi-Assay Kits 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。
Configure at GeneGlobe
Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Features

  • 微生物检测分析的完整试剂盒,包含所需的引物、探针、对照和试剂
  • 包含经多个实验验证的Microbial DNA qPCR Assay
  • 采用5'水解探针进行高度灵敏的特异性检测 

Product Details

Microbial DNA qPCR Multi-Assay Kits是real-time PCR微生物检测的完整解决方案。Microbial DNA qPCR Multi-Assay Kits含有多个Microbial DNA qPCR Assay,后者含有两种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。该试剂盒还含有成功进行qPCR所需的所有试剂和对照,包括Microbial DNA Positive Control、Positive PCR Control、Microbial DNA-Free Water和Microbial qPCR Mastermix。

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 Limit of detection versus lower limit of quantification)。所有Microbial DNA qPCR Assays中的93%具有低至100个基因拷贝的LLOQ(参见 The LLOQ for all Microbial DNA qPCR Assays reveals high sensitivity)。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 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 The LLOQ for antibiotic resistance genes Microbial DNA qPCR Assays reveals high sensitivity)。

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 Microbial DNA qPCR Assays are highly specific)。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 Microbial DNA qPCR Assays display high sensitivity even in complex metagenomic samples)。
See figures

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 Multi-Assay Kit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Microbial qPCR Mastermix构建PCR反应体系。每种样本进行三个对照反应,包括Positive PCR Control、No Template Control和Microbial DNA Positive Control、Microbial DNA qPCR Assays。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 Multi-Assay Kits高度适用于检测多种样本中相关的细菌或真菌物种,或微生物的抗生素耐药性基因或毒力因子基因。

Supporting data and figures

Resources

安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
产品介绍与指南 (2)
Microbial product configurator guide
For application-specific microbial identification and profiling by real-time PCR
试剂盒操作手册 (1)
For real-time PCR-based profiling/detection of microbial species, antibiotic resistance genes or virulence factor genes
Safety Data Sheets (1)
Certificates of Analysis (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