Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms.

Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.     

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.     

Kinetics quality assessment for relative quantification by real-time PCR

For proper relative quantification by real-time PCR, compared samples should have similar PCR efficiencies. To test this prerequisite, we developed two quality tests: (i) adjustment of a test for kinetic outlier detection (KOD) to relative quantification; and (ii) comparison of the efficiency variance of test samples with the efficiency variance of samples with highly reproducible quantification. The tests were applied on relative quantification of two genes in 30 sets of 5 replicate samples (same treatment, different animals). Ten low-quality sets and 28 outliers were identified. The low-quality sets showed higher coefficient of variation (cv)% of DNA quantities in replicate experiments than high-quality sets (63% versus 26%; P = 0.001) and contained a higher proportion of outlying quantities (35% versus 5.9%; P = 0.001) when individual samples were detected by adjusted KOD. Outlier detection with adjusted KOD reduced the false detection of outliers by 2/3 compared with the previous, nonadjusted version of KOD (20% versus 5.9%; P = 0.001). We conclude that the presented tests can be used to assign technical reasons to outlying observations.     

大鼠血管组织血管紧张素原基因表达的实时PCR定量分析

目的:探讨大鼠血管组织血管紧张素原(angiotensinogen,AGT)低丰度mRNA表达的实时PCR定量分析方法,并将其用于检测模拟失重大鼠基底和股动脉血管组织AGT mRNA的表达.方法:提取8周模拟失重(SUS)与对照组(CON)大鼠血管组织的总RNA,进行反转录后,对目的基因AGT与内参照基因GAPDH的mRNA进行实时PCR分析.应用TaqMan-MGB探针,测出上述mRNA实时PCR反应的放大效率(E)及阈循环数Ct,再依据一定数学模型由E与Ct得出经GAPDH归一化的AGT mRNA表达变化.结果:与CON相比,SUS大鼠基底动脉组织AGT mRNA表达增加240%,而股动脉组织则降低66%.结论:本工作为定量检测大鼠血管组织低丰度mRNA表达提示了一种特异、灵敏、精确、重复性好的简便方法.     

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.     

3 次连续重复提取DNA 能较好反映土壤微生物丰度

【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。     

Transcription dynamics of the functional tfdA gene during MCPA herbicide degradation by Cupriavidus necator AEO106 (pRO101) in agricultural soil

A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an alpha-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 10(5) cells g(-1) soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg(-1)). Mineralization rates were estimated by quantification of 14CO2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg(-1), the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg(-1) amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg(-1) scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.     

金黄色葡萄球菌基因表达的DNA扣除法

[目的]金黄色葡萄球菌作为一种分布广泛的致病微生物和研究革兰氏阳性菌遗传背景的模式菌株,利用real-time RT PCR对相关毒素及调控基因进行表达定量分析,在生物、医学、食品检测等领域具有较大研究价值.[方法]对制备好的反转录(RT,含有cDNA和DNA)和非反转录(RTˉ,仅含DNA)样品进行Real-time PCR检测,根据经典(1 E)ˉ△△Ct相对定量算法并结合PCR效率公式建立一种基因表达相对定量分析的DNA扣除法,将得到的Ct值转换为各样品含量,从RT样品中扣除RTˉ样品的量,无需DNaseⅠ酶解处理就可以去除DNA的影响,RTˉ样品的检测结果还可同时作为稳定的DNA内参.[结果]采用以上方法分析金黄色葡萄球菌肠毒素A基因(sea)、16S rRNA和RNA Ⅲ的表达情况,在含有葡萄糖的NB培养基中sea的相对转录水平随着葡萄糖浓度的增大而升高,RNAⅢ的相对转录水平随葡萄糖浓度的变化而产生小幅度的波动,16S rRNA在菌体生长初期时的表达量较为稳定;与绝对定量法比较,结果差异较小(均小于15%),且差异不显著(p＞0.05).[结论]这种基于DNA扣除法的Real-time RT PCR相对定量方法可以有效的对金黄色葡萄球菌的基因表达进行分析.