A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area

AIMS: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes. METHODS AND RESULTS: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. CONCLUSIONS: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. Significance AND IMPACT OF THE STUDY: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.     

Quantitative microbial faecal source tracking with sampling guided by hydrological catchment dynamics

The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design.     

Improved detection of <Emphasis Type="Italic">Rhodococcus coprophilus</Emphasis> with a new quantitative PCR assay

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.     

Rapid identification and differentiation of agricultural faecal contamination sources using multiplex PCR

Aims:  To develop a quick, easy-to-use, robust and sensitive multiplex PCR assay to detect common sources of agricultural faecal contamination using a combination of bacterial and eukaryote-specific PCR targets.
Method and Results:  A novel multiplex PCR method was developed that utilizes primers specific for a conserved region of the eukaryote cytochrome-B gene as well as a universal 16S rRNA and the E. coli -specific uidA gene. This multiplex PCR assay was capable of identifying faecal amendments from pig, sheep, cow and goat sources in 24/30 (80%) of amended water samples.
Conclusions:  The method was capable of accurately identifying common agricultural sources.
Significance and Impact of the study:  The procedure described here is simple, rapid (<5 h) and can be used as a first step in microbial source tracking studies, particularly where agricultural faecal contamination is suspected.     

Escherichia coli and enterococci are sensitive and reliable indicators for human,livestock and wildlife faecal pollution in alpine mountainous water resources

Aims: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. Methods and Results: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7–7·0 CFU g^?1) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0–8·4 CFU g^?1). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3–7·7 CFU g^?1. To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi‐parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment ( Reischer et al. 2008 ; Environ Microbiol 10:2598–2608). Conclusion: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. Significance and Impact of the Study: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.     

Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay

Aim: To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. Methods and Results: Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571‐bp product was detected in 76% of poultry‐associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 10⁷–10⁹ gene copies per gram in soiled litter, up to 10⁵ gene copies per gram in spread‐site soils, and 10⁷ gene copies per litre in field run‐off water. Results were corroborated by a blinded study conducted by a second laboratory. Conclusion: The poultry‐specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land‐applied poultry litter. Significance and Impact of the Study: This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.     

A high‐throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)‐based approach to identify sources of faecal contamination in water bodies

As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides‐Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human‐associated Bacteroides spp. and three clusters of cow‐, pig‐ and dog‐specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high‐throughput method that can identify sources of faecal contamination.     

Sourcing faecal pollution from onsite wastewater treatment systems in surface waters using antibiotic resistance analysis

AIMS: To identify the sources of faecal contamination in investigated surface waters and to determine the significance of onsite wastewater treatment systems (OWTS) as a major contributor to faecal contamination. METHODS AND RESULTS: Antibiotic resistance patterns (ARP) were established for a library of 717 known Escherichia coli source isolates obtained from human, domesticated animals, livestock and wild sources. Eight commonly used antibiotics, including amoxicillin, cephalothin, erythromycin, gentamicin, ofloxacin, chlortetracycline, tetracycline and moxalactam, at four different concentrations were used to obtain ARPs for E. coli isolates. Discriminant analysis (DA) was used to differentiate between the ARP of sources isolates. The developed ARP library was found to be adequate for discriminating human from nonhuman isolates, and was used to classify 256 enumerated E. coli isolates collected from monitored surface water locations. CONCLUSIONS: The resulting ARP DA indicated that a majority of the faecal contamination in more rural areas was nonhuman; however, the percentage of human isolates increased significantly in urbanized areas using OWTS for wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study signifies the feasibility of using ARP for source tracking faecal contamination in surface waters, and linking faecal contamination to OWTS. The information will enable regulatory authorities to implement appropriate management practices to reduce the contamination of water resources caused by high densities and failing OWTS.     

Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR

AIMS: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR. METHODS AND RESULTS: A linear range of quantification between 0.1-10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30-4500 to 1.9 x 10(6)-6.0 x 10(6) target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification. CONCLUSIONS: Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.     

Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.     

Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.     

F+ RNA coliphage typing for microbial source tracking in surface waters

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.     

The influence of rainfall on the incidence of microbial faecal indicators and the dominant sources of faecal pollution in a Florida river

AIMS: An assessment of microbial densities in an urbanized Florida watershed was performed during a period of changing rainfall patterns to investigate the role of climate coupled with urbanization in declining water quality. METHODS AND RESULTS: Concentrations of traditional and alternative faecal indicators were assessed by standard methods over 24 months. Sources of faecal contamination were determined by antibiotic resistance analysis (ARA) of faecal coliform (FC) bacteria. Composite indices of indicator organisms based on a suite of microbial measurements were used to quantify pollution impacts in the river. ARA results found that FC from wild animal sources dominated during the drought, and the relative frequency of FC from human sources increased after cumulative rainfall increased to near-normal levels. CONCLUSIONS: Changes observed in faecal indicator densities and in FC sources during changing rainfall patterns strongly suggest a role of precipitation on the sources and extent of microbial pollution in urbanized coastal watersheds. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial source tracking coupled with a composite index of microbial contamination resulted in a more complete picture of microbial pollution within the river, as opposed to the general practice of reliance on one indicator organism. Improved land use decisions in urban areas are necessary to insure maintenance of coastal environmental health under changing climate patterns and population density.     

Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.     

Investigation of human sewage pollution and pathogen analysis at Florida Gulf coast beaches

Aims: Water quality at two Florida beaches was compared using faecal indicator bacteria measurements, microbial source tracking (MST) methods for detecting human source pollution and the assessment of pathogen presence. These values were also compared before and after remediation of wastewater infrastructure at one beach. Methods and Results: Faecal coliforms, Escherichia coli and enterococci were enumerated in estuarine water and sediment samples. PCR assays for the human‐associated esp gene of Enterococcus faecium and human polyomaviruses (HPyVs) were used to detect human sewage. Culturable Salmonella and enteric viruses were also analysed. MST identified human sewage contamination at one beach, leading to repair of a sewer main and relocation of portable restrooms. Exceedances of Florida recreational water regulatory standards were significantly reduced after remediation (by 52% for faecal coliforms and 39% for enterococci), and the frequency of detection of MST markers decreased. Coxsackie virus B4 and HPyVs were codetected following a major sewage spill, but Salmonella was not detected during the study. Conclusions: These data indicate that infrastructure remediation significantly reduced pollution from human sewage at the impacted beach. Significance and Impact of the Study: A comprehensive microbial water quality study that can identify contamination sources through the use of MST markers and close collaboration with local/and state agencies can result in tangible actions to improve recreational water quality and safety.     

Development of Faecalibacterium 16S rRNA gene marker for identification of human faeces

Aims:  The focus of this study was to identify a bacterial 16S rRNA gene sequence, unique to microbiota in the human gut, for use in development of a dependable PCR assay to detect human faecal pollution in water.
Methods and Results:  Suppression subtractive hybridization (SSH) and bioinformatics were used to identify a genetic marker, within the 16S rRNA gene of Faecalibacterium , for the detection of human faeces. DNA sequencing analysis demonstrated that a majority (16) of 74 clones of the SSH library contained insertion sequences identified as Faecalibacterium 16S rRNA genes . Human faeces-specific sequences were derived and six PCR primer sets designed and tested against faecal DNA samples from human and nonhuman sources. One PCR primer set, HFB-F3 and HFB-R5, was exclusively associated with human faeces. These primers generated a human faeces-specific amplicon of 399 bp from 60·2% of human faecal samples and 100% of sewage samples.
Conclusions:  The subject Faecalibacterium marker is specific for sewage.
Significance and Impact of the Study:  This study represents the initial report of a Faecalibacterium marker for human faeces, which may prove useful for microbial source tracking.     

Microbial Source Tracking in Adjacent Karst Springs

Modern man-made environments, including urban, agricultural, and industrial environments, have complex ecological interactions among themselves and with the natural surroundings. Microbial source tracking (MST) offers advanced tools to resolve the host source of fecal contamination beyond indicator monitoring. This study was intended to assess karst spring susceptibilities to different fecal sources using MST quantitative PCR (qPCR) assays targeting human, bovine, and swine markers. It involved a dual-time monitoring frame: (i) monthly throughout the calendar year and (ii) daily during a rainfall event. Data integration was taken from both monthly and daily MST profile monitoring and improved identification of spring susceptibility to host fecal contamination; three springs located in close geographic proximity revealed different MST profiles. The Giach spring showed moderate fluctuations of MST marker quantities amid wet and dry samplings, while the Zuf spring had the highest rise of the GenBac3 marker during the wet event, which was mirrored in other markers as well. The revelation of human fecal contamination during the dry season not connected to incidents of raining leachates suggests a continuous and direct exposure to septic systems. Pigpens were identified in the watersheds of Zuf, Shefa, and Giach springs and on the border of the Gaaton spring watershed. Their impact was correlated with partial detection of the Pig-2-Bac marker in Gaaton spring, which was lower than detection levels in all three of the other springs. Ruminant and swine markers were detected intermittently, and their contamination potential during the wet samplings was exposed. These results emphasized the importance of sampling design to utilize the MST approach to delineate subtleties of fecal contamination in the environment.     

Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

Aims:  To develop a strain-specific rapid assay for identification and quantification of Lactobacillus rhamnosus GG in human faecal samples.
Methods and Results:  A unique random amplified polymorphic DNA (RAPD) band of the L. rhamnosus GG strain was isolated and sequenced. Strain-specific polymerase chain reaction (PCR) primers and probes were designed based on the sequence. Quantification was performed by the real-time PCR using a fluorescent resonance energy transfer (FRET) system. The specificity of the assay was tested with DNA isolated from a set of known strains and human faecal samples. The analytical sensitivity of the method for L. rhamnosus GG was about 10 CFU per assay, which corresponds to 10⁵ CFU g⁻¹ of wet faeces.
Conclusions:  Quantitative real-time PCR is a suitable method for strain-specific identification of L. rhamnosus GG in human faecal samples.
Significance and Impact of the Study:  Lactobacillus rhamnosus GG is one of the most studied probiotic strains in clinical trials but still lacks a DNA-based identification method. This study describes a real-time PCR method for strain-specific identification and quantification of L. rhamnosus GG in human faecal samples.     

Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.     

粪便中肠球菌SYBR GreenI荧光定量PCR检测方法的建立

目的利用SYBR GreenI荧光定量PCR方法,建立肠球菌实时荧光PCR检测方法,并初步应用于粪便中肠球菌的检测。方法根据GenBank发表的肠球菌23S rRNA基因序列的保守区域设计合成特异性的引物;利用构建的质粒标准品绘制两种标准曲线,构建基因拷贝数、细菌数为分析指标的定量分析模型并初步应用于粪便标本的检测分析。结果所建立的SYBR GreenI荧光定量PCR方法检测灵敏度可达7个拷贝数/reaction。粪便样本根据实时荧光定量PCR方法所得的理论数值与培养菌值之间差异无显著性(P>0.05)。非炎性腹泻标本中菌数与健康成人标本中菌数差异无显著性(P>0.05)。灵敏度曲线所得的数值大于菌数标准曲线,可能由于DNA提取过程中存在部分的损失。检测粪便标本结果显示SYBR GreenI荧光定量PCR方法较平板计数法敏感、快捷、简便。结论本研究建立了一种灵敏、特异、简便易行的肠球菌定量检测方法。