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Qiagen/Custom Microbial DNA qPCR Arrays/330161

商品编号： 330161

品牌： Qiagen

市场价： ¥0.00

美元价： 0.00

产地：美国(厂家直采)

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产品分类：多肽合成

公司分类： peptide

联系Q Q： 3392242852

电话号码： 4000-520-616

电子邮箱： info@ebiomall.com

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商品介绍

qPCR Assays & Instruments
CNV qPCR Assays & Arrays3
Food Safety Testing26
Microbial DNA qPCR Assays & Panels8
miRNA qPCR Assay & Panels28
mRNA/IncRNA qPCR Assays & Panels31
Somatic Mutations qPCR Assays & Panels3

Custom Microbial DNA qPCR Arrays

For real-time PCR-based, customized microbial identification or profiling

Enables profiling of a customized panel of microbial genes or species
Contains highly sensitive and specific Microbial DNA qPCR Assays
A variety of plate layouts available to suit your research needs

The Custom Microbial DNA qPCR Array contains user-defined assays for detection of microbial species or microbial genes.

To order a Custom Microbial DNA qPCR Array, please use this excel file.

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Cat No./ID:330161

Custom Microbial DNA qPCR Array

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Array plates and master mix for detection of a user-defined set of microbial species or genes

Custom 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

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

Municipal biosolids generated by wastewater treatment plants are significant reservoirs for antibiotic resistance genes, since they originate from fecal microbiota. The end product from the treatment plants can either be disposed of in landfills or sold as fertilizer for agricultural use, where antibiotic-resistant bacteria may be reintroduced into the food supply. To determine the diversity of antibiotic resistance genes in municipal biosolids, genomic DNA from belt-filter presscake sewage samples was isolated and analyzed for the presence of antibiotic resistance genes using the Antibiotic Resistance Genes Microbial qPCR Array (cat.no. BAID-1901Z). Raw C_T values were exported into the data analysis software and identification criteria was followed. Figure [A] shows the results from the sewage sample. There were 14 antibiotic resistance genes from different resistance classifications that were present in the metagenomic sample. In addition, there were genes that gave an inconclusive result. To determine the presence/absence of the antibiotic resistance genes from the inconclusive results, the “Determination of Inconclusive Microbial DNA qPCR Array/Assay Results” protocol was followed for SHV, ACT-1 group, MIR and LAT [B]. The verification protocol determined that these genes were present in the sewage sample. This highlights the importance of increased surveillance of antibiotic resistance reservoirs to identify potential sources of antibiotic-resistant bacteria that may affect the food supply.

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

The vaginal microbiota is a key component influencing women’s urogenital health. To determine what changes in the vaginal microbiota occurs during bacterial vaginosis, the Vaginal Flora Microbial DNA qPCR Array (cat. no. BAID-1902Z), which detects up to 90 different microbial species, was used to test cervical swabs from healthy individuals and from patients with bacterial vaginosis. Genomic DNA from vaginal samples originating from three patients that tested negative for bacterial vaginosis, three patients that tested positive for Candida, three patients that tested positive for Garderella vaginalis, and one patient that tested positive for Trichomonas vaginalis by BD Affirm™ VPIII Microbial Identification Test were run on the Vaginal Flora Microbial DNA qPCR Array. Genomic DNA from ThinPrep samples were isolated using QIAGEN’s QIAamp MinElute Media Kit and 500 ng genomic DNA from each sample was analyzed. After the PCR run on a Roche LightCycler 480, raw C_T values were exported to the Microbial DNA qPCR data analysis software. Positive (+) / negative (blank) / inconclusive (+/-) results for each microbial species were determined using the identification criteria. The results from the Vaginal Flora Microbial DNA qPCR Array were in concordance with the BD Affirm VPIII Microbial Identification Test.

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.

Microbial DNA qPCR Assays display high sensitivity even in complex metagenomic samples.

To ensure that Microbial DNA qPCR Assays performed comparably in a complex sample, where there may be up to a thousand different microbial species, each assay was tested using stool, tooth plaque, and sputum samples. For each sample, synthetic template targets were spiked in and the C_T was compared to synthetic template alone. PCR was performed using several sample types, which included pooled synthetic template targets alone, stool, stool plus pooled synthetic template targets, plaque, plaque plus pooled synthetic template targets, sputum, and sputum plus pooled synthetic template targets. If the C_T<35 in stool, plaque, or sputum samples alone, then ΔC_T was calculated (i.e., C_T^{stool + pooled synthetic template targets}– C_T^{pooled synthetic template targets}). This calculation was performed for all the assays. For each assay, the ΔC_T<3, indicating that a complex metagenomic background does not affect the performance of each Microbial DNA qPCR Assay.

The lower limit of quantification (LLOQ) for microbial identification Microbial DNA qPCR Assays reveals high sensitivity.

This chart shows the distribution of LLOQ for microbial identification Microbial DNA qPCR Assays. 92% of all microbial identification assays have a LLOQ of <100 gene copies.

The lower limit of quantification (LLOQ) for antibiotic resistance gene detection Microbial DNA qPCR Assays reveals high sensitivity.

This chart shows the distribution of LLOQ for Microbial DNA qPCR Assays for antibiotic resistance gene detection. 95% of all antibiotic resistance gene assays have a LLOQ of <100 gene copies.

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

To verify the specificity of the Antibiotic Resistance Genes Microbial DNA qPCR Array (cat no. BAID-1901Z) results from Klebsiella pneumoniae isolates, pyrosequencing assays were designed to detect for the presence and sequences of SHV-156G, SHV-156D, SHV-238G240E, SHV-238S240K, SHV-238S240E, SHV-238G240K, ermB, mefA, tetA, tetB,CTX-M-1 Group, CTX-M-2 Group, AAC(6′)-lb-cr and aadA1. For each Klebsiella pneumoniae isolate, results from the Antibiotic Resistance Genes Microbial DNA qPCR Array were confirmed by pyrosequencing. Representative pyrograms for [A] SHV-156G, [B] SHV-238/240, [C] KPC and [D] CTX-M-1 group are shown. For SHV variants, the Antibiotic Resistance Gene Microbial DNA qPCR Array was able to reliably distinguish single nucleotide polymorphisms occurring at different sites.

Microbial DNA qPCR Assays are highly specific.

To determine the specificity of Microbial DNA qPCR Assays, each assay was tested against 119 genomic DNA samples from different bacteria and fungi. To facilitate testing, genomic DNA from different microbial species was pooled (10 different genomic DNA samples per pool) and each assay was tested against the different pools. None of the pools contained DNA from the same genus, to facilitate identification of cross-reacting species. Each pool contained the equivalent of 2000 genome copies for each microbial species. In addition, each assay was tested against human, mouse, and rat genomic DNA. A representative example for Streptococcus pyogenes is shown. The assay for Streptococcus pyogenes gave a C_T of 26.9 and 26.6 for the Staphylococcus/Streptococcus pool and complete pool [A]. Both pools contained genomic DNA for Streptococcus pyogenes. To determine which genomic DNA was detected by the Streptococcus pyogenes assay, each individual genomic DNA comprising the Staphylococcus/Streptococcus pool was tested separately [B]. Only Streptococcus pyogenes genomic DNA gave an acceptable C_T call (26.8) while the others gave a C_T>35. Most of the assays were specific as they did not detect unintended targets. For assays that detected other species, the list of detected targets along with in silico predictions are given in the specifications sheet.

The lower limit of quantification (LLOQ) for all Microbial DNA qPCR Assays reveals high sensitivity.

This chart shows the distribution of LLOQ for all Microbial DNA qPCR Assays. 93% of all Microbial DNA qPCR Assays have a LLOQ of <100 gene copies.

The lower limit of quantification (LLOQ) for virulence factor gene detection Microbial DNA qPCR Assays reveals high sensitivity.

This chart shows the distribution of LLOQ for Microbial DNA qPCR Assays for virulence factor gene detection. 97% of all virulence factor gene assays have a LLOQ of <100 gene copies.

Microbial DNA qPCR Arrays generate reliably reproducible results.

To determine the reproducibility of the Microbial DNA qPCR Array, both intra-individual and inter-individual variability was tested. In this experiment, 500 ng genomic DNA isolated from belt-filter presscake sewage sample was loaded onto the Antibiotic Resistance Genes Microbial DNA qPCR Array (cat. no.BAID-1901Z). To determine intra-individual variability, the same operator ran two different PCR arrays on different days with four technical repeats. To determine inter-individual variability, two different operators ran PCR arrays with four technical repeats. The results show low inter- and intra-individual variation of the qPCR array.

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

To compare any differences in the vaginal microbiome between healthy women and women with bacterial vaginosis, each sample that tested positive for Gardnerella vaginalis using the Vaginal Flora Microbial DNA qPCR Array was compared to samples from healthy women (n=3). Fold-change in microbial species abundance was calculated by the ΔΔC_T method using human genomic DNA to normalize. The results show that as the relative abundance of Gardnerella vaginalis increases, the abundance of the commensal species Lactobacillus crispatus decreases. Also, an increase in Gardnerella vaginalis was associated with an increase in other bacterial vaginosis-associated microbial species. This suggests that Lactobacillus crispatus protects the vagina from bacterial vaginosis-associated microbial species.

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

The human gut microbiota is known to act as a reservoir for antibiotic resistance genes, where they can be transferred horizontally to potential pathogenic bacteria. To detect the presence of antibiotic resistance genes from gut microbiota, stool samples from five healthy adults were collected and genomic DNA was isolated using QIAGEN’s QIAamp DNA Stool Mini Kit. 500 ng genomic DNA from each stool sample was analyzed for presence of antibiotic resistance genes using the Antibiotic Resistance Genes Microbial DNA qPCR Array (cat.no. BAID-1901Z). The Antibiotic Resistance Genes Microbial DNA qPCR Array contains assays for 83 antibiotic resistance genes, assays to identify methicillin-resistant Staphylococcus aureus, and control assays. ErmB, mefA, and tetA were found in all or most of the stool samples tested, showing that they may be highly prevalent in the gut. These antibiotic resistance genes have been reported to be isolated from bacterial strains originating from food, suggesting a possible source of origin. This highlights the importance of increased monitoring of antibiotic resistance reservoirs to identify potential sources of antibiotic-resistant bacteria.

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.

Performance

Linearity and dynamic range

Microbial DNA qPCR Assays display linear amplification across a dynamic range from 10 to 10⁶copies of DNAtemplate (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 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 factorgene detectionMicrobial DNA qPCR Assays reveals high sensitivity, andThe LLOQ for antibiotic resistance gene detectionMicrobial 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).

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 genesusing PCR amplification primers and hydrolysis-probe detection. Custom Microbial DNA qPCR Arrays are 96- or 384-well plates with assays defined by the customer, arranged according to your needs; for example, one can order a 96-well plate with 48 assays to be used with 2 samples, or with 12 assays to be tested with 8 samples. 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 C_T values for each assay.The complimentary dataanalysis software is then used to profile genes or species in the sample.

Applications

Custom Microbial DNA qPCR Arrays are highly suited for the rapid and accurate profiling of a custom panel of microbial species, virulence factor genes, or antibiotic resistance genes.For example, a microbial species identification panelassembled by our experts was used toexplore the underlying causes of bacterial vaginosis (see figures,The Vaginal Flora Microbial DNA qPCR Array provides accurate profiling for cervical swab samples andVaginal samples positive for Gardnerella vaginalis also show changes in commensal and bacterial vaginosis-related microbes compared to healthy samples), while a panel for antibiotic resistance genes identified 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).

Product Resources

Brochures & Guides (2)

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	Microbial Product Configurator Microbial product configurator guide	Show details
	Microbial DNA qPCR Arrays For application-specific microbial identification and profiling by real-time PCR	Show details

Analysis Software (1)

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	Microbial Custom PCR Array Universal Patch	Show details

Kit Handbooks (1)

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	Microbial DNA qPCR Handbook New version – For real-time PCR-based profiling/detection of microbial species, antibiotic resistance genes or virulence factor genes	Show details

Scientific Posters (1)

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	Detection and Surveillance of Antibiotic Resistance Genes From Food and Fertilizer Sources Using qPCR Technology	Show details

Technical Information (2)

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	Microbial DNA qPCR Assay/Assay Kits	Show details
	Antibiotic Resistance Gene Resource and Assay List	Show details

Safety Data Sheets (1)

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	MSDS Custom Microbial DNA qPCR Array

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品牌介绍

QIAGEN是一家专业化致力于生物分子样品制备解决方案的跨国经营企业，总部位于德国。1984年，QIAGEN在德国成立，1996年在美国纽约纳斯达克上市。QIAGEN拥有超过1000项专利和认可证明，在18个国家设立了分公司，代理商服务国超过40个，在全球有超过400000的用户。QIAGEN提供的产品超过500类，包括各种试剂，耗材和自动化纯化工作站。这些产品用于样品采集，稳定，核酸或蛋白的分离，纯化和检测中，不仅广泛的应用于科研领域的各个方面，在生物技术，制药，法医研究，食品安全检测，畜牧业和分子诊断领域也得到了广泛的应用。QIAGEN产品的卓越品质和在应用中的出色表现使得其成为样品处理中标准的代名词。

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发现与翻译研究人类识别与取证 Diagnostics & Clinical Research 仪器与自动化

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Custom Microbial DNA qPCR Arrays

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Product Details

Linearity and dynamic range

Lower limit of quantification (LLOQ)

Specificity

Reproducibility

Product Resources

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