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Microbial DNA qPCR Arrays

real-time PCR方法的微生物鉴定或分析
  • 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
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。
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330261 BAID-1404Z varies
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330261 BAID-1901Z varies
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330261 BAID-1902Z varies
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Microbial DNA qPCR Arrays适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


0
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.
1
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.
2
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.
3
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.
4
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.
5
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.
6
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 CT 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 CT call (26.8) while the others gave a CT>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.
7
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 CT 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 CT<35 in stool, plaque, or sputum samples alone, then ΔCT was calculated (i.e., CTstool + pooled synthetic template targets – CTpooled synthetic template targets). This calculation was performed for all the assays. For each assay, the ΔCT<3, indicating that a complex metagenomic background does not affect the performance of each Microbial DNA qPCR Assay.
8
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.
9
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.
10
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 CT 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.
11
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 ΔΔCT 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.
12
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.
13
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 CT 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.
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Linearity and sensitivity of Microbial DNA qPCR Assays)。

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The LLOQ for microbial identification Microbial DNA qPCR Assays reveals high sensitivityThe LLOQ for virulence factor gene detection Microbial DNA qPCR Assays reveals high sensitivityThe LLOQ for antibiotic resistance gene detection 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);其特异性可通过测序验证(参见Specificity of the Antibiotic Resistance Genes Microbial DNA qPCR Array is confirmed by pyrosequencing)。


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CT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The Vaginal Flora Microbial DNA qPCR Array provides accurate profiling for cervical swab samplesVaginal samples positive for Gardnerella vaginalis also show changes in commensal and bacterial vaginosis-related microbes compared to healthy samples)。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The Microbial DNA qPCR Array screens gut microbiota for the presence of antibiotic resistance genesThe Antibiotic Resistance Genes Microbial DNA qPCR Array identified antibiotic resistance genes in sewage samples)。

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New version – For real-time PCR-based profiling/detection of microbial species, antibiotic resistance genes or virulence factor genes
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