PCR直接检测龈下菌斑主要可疑牙周致病菌

目的：应用PCR方法直接检测龈下菌斑主要可疑牙周致病菌与牙周病活动部位的关系,探讨其方法的可行性并探讨其主要可疑牙周致病菌的分布规律。方法：应用聚合酶链反应(polymerase chain reaction,PCR)直接检测龈下菌斑主要可疑致病菌16s RNA保守区域片段。40名受试者包括牙周病患者20人,每人同口取一个牙周病活动部位,一个相对健康或牙周病静止对照部位;成人健康者20人,每人各取一个标本。结果：龈下菌斑5种可疑牙周致病菌在牙周病活动部位的检出率牙龈卟啉菌为86％,福赛类杆菌为95％,螺旋体为86％,中间普氏菌和黑色普氏菌分别为95％和33％,均显著高于同口部位对照组和健康对照组。结论：PCR直接检测菌斑牙龈卟啉单胞菌、中间普氏菌、福赛类杆菌、齿密螺旋体及黑色普氏菌匀与牙周炎活动部位相关。     

Quantitative detection of periodontopathogens by real-time PCR

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.     

Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia, and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponema denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.     

Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers

doi: 10.1111/j.1741‐2358.2011.00506.x Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers Objective: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. Materials and methods: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher’s exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. Results: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue‐coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. Conclusion: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.     

Detection of dental plaque and its potential pathogenicity using quantitative light‐induced fluorescence

Quantitative light‐induced fluorescence (QLF) technology can detect some dental plaque as red fluorescence. This in vivo study aimed to identify the microbial characteristics of red fluorescent (RF) dental plaque using 16S rRNA gene sequencing and evaluate the correlations between RF plaque and the clinical symptoms of dental diseases. Paired supragingival plaque samples collected from each 10 subjects and consisted of RF and non‐RF dental plaques as observed by QLF technology using a 405 nm blue light source for excitation. The characteristics of the bacterial communities in the RF and non‐RF plaque samples were compared by sequencing analysis. An increase in microbial diversity was observed in RF plaque compared with the non‐RF plaque. There were significant differences in the community compositions between the 2 types of dental plaque. Periodontopathic bacteria were significantly more abundant in the RF plaque than non‐RF plaque. The fluorescence intensity of RF plaque was significantly related to the proportion of the periodontopathic bacterial community and the presence of gingival inflammation. In conclusion, the plaque red fluorescence is associated with changes in the microbial composition and enrichment of periodontopathic pathogens, which suggests that RF plaque detected by QLF technology could be used as a risk indicator for gingival inflammation.      

A rapid method for extraction and purification of DNA from dental plaque.

A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Comparison of culture methods and multiplex PCR for the detection of periodontopathogenic bacteria in biofilm associated with severe forms of periodontitis

Conventional culture methods and Multiplex PCR, both of which we have been used for a long time in our clinical microbiology laboratory, were compared for their ability to detect a selected panel of periodontopathic bacteria: Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. Tests were performed in a single subgingival sample taken from a periodontal diseased site with a probing depth equal to or greater than 6mm. The results were compared site-by-site, taking into account the quality and the presence or absence of pathogens. 529 samples of subgingival plaque were analysed and the prevalence of the six species monitored varied in relation to the species itself and the method of detection. The most represented species is F. nucleatum, with a percentage of positive variability between 44.9% PCR and 46.5% culture test. Generally, the lowest prevalence was determined by culture test, with the exception of E. corrodens and F. nucleatum, which, unlike other bacteria, have been seen in higher percentages in culture with respect to PCR. For both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans, C. rectus, E. corrodens, and P. gingivalis. It becomes weak for F. nucleatum and P. intermedia. Both culture and PCR techniques introduced many methodological problems when applied in oral microbiology, but the ideal technique for accurate detection of pathogens in subgingival plaque samples has yet to be developed.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.     

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Quantitation of Bacteroides forsythus in subgingival plaque comparison of immunoassay and quantitative polymerase chain reaction

Our objective was to compare three methods (enzyme-linked immunosorbent assay [ELISA], endpoint and quantitative polymerase chain reaction [E-PCR and Q-PCR]) for detection and quantitation of Bacteroides forsythus in 56 plaque samples from seven subjects with progressive periodontal disease. Samples collected in buffer were pelleted and resuspended in 500 microl of water. Fifty microl aliquots were removed for an ELISA performed on bacteria or plaque immobilized on 96-well plates and probed with B. forsythus specific antibody. An occurrence of 3.7+/-0.6 x 10(4) or more bacteria were detected by ELISA in pure culture; 26 of 54 plaque samples were positive, two samples could not be analyzed. Samples for PCR were autoclaved for 10 min prior to use. The detection level of E-PCR using primers specific for B. forsythus 16S rRNA was 200 cells and 42 out of 56 samples were positive based on ethidium bromide stained agarose gels. Q-PCR using the same primers combined with a nested fluorescent oligonucleotide probe detected 10+/-0.32 bacteria in pure culture; 43 of 56 plaque samples were positive. The ELISA and Q-PCR obtained identical results with 36 of the 54 samples assayed; there were one false positive and 17 false negative ELISA results using Q-PCR as standard. The positive proportions of plaque samples were almost the same for E-PCR and Q-PCR. We conclude that the PCR methods are more appropriate for a multicenter study because of greater sensitivity and convenience of sample transportation from clinics to a central laboratory.     

Possible translocation of periodontal pathogens into the lymph nodes draining the oral cavity

Numerous publications have reported the presence of periodontopathogenic bacteria in peripheral and central vascular lesions. However, it is unclear how this bacterial translocation occurs. The objective of this study was to investigate whether periodontopathic bacteria are translocated to lymph nodes proximal to the oral cavity. Obtaining lymph node samples is not ethically feasible unless they are excised as part of the surgical management of patients with cancer. This study analyzed formalin-fixed and paraffin-embedded lymph nodes, histologically negative for cancer cell invasion, that were excised from 66 patients with histories of head and neck cancer. Real-time PCR was performed to amplify the 16S ribosomal DNA fragments from Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Prevotella intermedia. The relationship between bacterial detection and cancer severity, gender, and the use of anti-cancer therapy was examined by Fisher??s exact test. P. gingivalis, T. forsythia, and P. intermedia were present in 17%, 8%, and 8% of the samples of submandibular and submental lymph nodes, respectively. There were no significant relationships between bacterial detection and the cancer disease status, patient gender or use of anticancer therapy. According to these data, it appears that the translocation of periodontopathic bacteria may occur via lymphatic drainage, irrespective of the cancer disease status, gender or anticancer therapy.     

Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR

AIMS: To detect quantitatively the total bacteria and Streptococcus mutans in dental plaque by real-time PCR with prbac, Sm and GTF-B primers, and to compare their presence with the prevalence of dental caries in Japanese preschool children. METHODS AND RESULTS: Human dental plaque samples were collected from the labial surfaces of the upper primary central incisors of 107 children. The dental status was recorded as dft by WHO caries diagnostic criteria. Positive dt and dft scores by the Sm or GTF-B primer were significantly higher than negative scores (P < 0.01). The proportions of Strep. mutans to the total bacteria from sound, and sound and/or filled upper primary incisors were significantly lower than those from decayed or filled, and decayed incisors, respectively (P < 0.01). CONCLUSIONS: The ratios of Strep. mutans to total bacteria in plaque detected by real-time PCR with Sm and GTF-B primers were closely associated with the prevalence of dental caries in Japanese preschool children. SIGNIFICANCE AND IMPACT OF THE STUDY: These assays may be useful for the assessment of an individual's risk of dental caries.     

Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries

The oral bacterial flora in the saliva from two patients with periodontitis and from a periodontally healthy subject were compared using a sequence analysis of 16S rDNA libraries without cultivation. 16S rDNAs were amplified from salivary DNA by PCR and cloned. Randomly selected clones were partially sequenced. On the basis of sequence similarities, the clones were classified into several clusters corresponding to the major phylum of the domain Bacteria. The major phylum in the libraries was the low G+C Gram-positive bacteria. There was no clonal sequence affiliated with periodontopathic bacteria in the salivary sample from the healthy subject, while a number of periodontal pathogens such as Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis and Treponema socranskii were detected in the salivary samples from the patients with periodontitis. In addition, a number of previously uncharacterized and uncultured microorganisms were recognized. These organisms may have some role in periodontal disease. This study reveals some potential for a molecular-biological technique to analyze the oral microflora associated with periodontal disease, including previously uncharacterized and uncultured microorganisms, without cultivation.     

Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

AIMS: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. METHODS AND RESULTS: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively. CONCLUSIONS: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.     

Identification of Treponema denticola in subgingival samples by PCR technology and its correlation with clinical diagnosis

Treponema denticola has been associated with gingivitis and chronic periodontitis. The aim of this study was to identify Treponema denticola in subgingival samples using PCR technology and to correlate it with clinical diagnosis of subjects. The study was carried out on seventy patients (20-84 years of age; mean age, 45.06 +/- 12.58) of which 22 individuals with no detectable gingivitis or periodontitis, 4 subjects with chronic gingivitis and 44 subjects with chronic periodontitis. Subgingival plaque samples were collected from five sites in each patient. DNA was extracted from the samples using QIAamp DNA Mini Kit (QIAGEN). Treponema denticola and other four periodontopathogens were found using multiplex polymerase chain reaction followed by a reverse hybridization. The relationship between clinical diagnoses and detection of Treponema denticola was determined with Fisher exact test. The results showed significant differences between diagnostic groups regarding subject proportion. Treponema denticola was detected in 2 out of 22 subjects with no detectable gingivitis or periodontitis, 2 out of 4 subjects with chronic gingivitis, and 40 out of 44 subjects with chronic periodontitis. Our findings suggest that Treponema denticola is closely connected to the initiation and progression of periodontal disease.     

Phylogenetic diversity and dietary association of rumen Treponema revealed using group-specific 16S rRNA gene-based analysis

Treponema spp. are a commonly detected bacterial group in the rumen that are involved in the degradation of soluble fibers. In this study, a ruminal Treponema group-specific PCR primer targeting the 16S rRNA gene was designed and used to assess the phylogenetic diversity and diet association of this group in sheep rumen. Total DNA was extracted from rumen digesta of three sheep fed a diet based on alfalfa/orchardgrass hay or concentrate. The real-time PCR quantification indicated that the relative abundance of the Treponema group in the total rumen bacteria was as high as 1.05%, while the known species Treponema bryantii accounted for only 0.02%. Fingerprints of the Treponema community determined by 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE) analysis tended to differ among the diets. Principal component analysis of the DGGE profiles distinguished those Treponema associated with either the hay or the concentrate diets. Analysis of a Treponema 16S rRNA gene clone library showed phylogenetically distinct operational taxonomic units for a specific dietary condition, and significant (P=0.001) differences in community composition were observed among clone libraries constructed from each dietary regimen. The majority of clones (75.4%) had <97% sequence similarity with known Treponema. These results suggest the predominance of uncultured Treponema that appear to have distinct members related to the digestion of either hay or concentrate diet.     

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.