scholarly journals Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

2005 ◽  
Vol 71 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Anne-Ga�lle Le Bourhis ◽  
Katiana Saunier ◽  
Jo�l Dor� ◽  
Jean-Philippe Carlier ◽  
Jean-Fran�ois Chamba ◽  
...  
Keyword(s):  
Temperature Gradient ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Clostridial Species ◽  
The 16S Rrna Gene

ABSTRACT A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

Temperature Gradient Gel Electrophoresis of the Amplified Product of a Small 16S rRNA Gene Fragment for the Identification of Listeria Species Isolated from Food

2000 ◽  
Vol 63 (5) ◽  
pp. 659-661 ◽  
Author(s):  
MARISA MANZANO ◽  
LUCA COCOLIN ◽  
CARLO CANTONI ◽  
GIUSEPPE COMI
Keyword(s):  
Polymerase Chain Reaction ◽  
Temperature Gradient ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gel Electrophoresis ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Gradient Gel Electrophoresis ◽  
Polymerase Chain ◽  
Temperature Gradient Gel Electrophoresis

The development of a rapid method for the identification of Listeria spp. is described. It is based on the polymerase chain reaction amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis. Forty-five strains of Listeria spp. (Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) were used for the optimization of the protocol. No differences were observed between the results of the identification of the strains tested using traditional methods and those obtained by polymerase chain reaction–temperature gradient gel electrophoresis analysis.

scholarly journals Genetic characterization of the microbiota of artisan fresh cheese from the Papaloapan region

2021 ◽  
Vol 6 (2) ◽  
pp. 61-85
Author(s):  
Miguel A. García-Muñoz ◽  
◽  
Nancy Cruz-Velazco ◽  
América Chávez-Martínez ◽  
Cirilo Nolasco-Hipólito ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gel Electrophoresis ◽  
Pathogenic Bacteria ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Fresh Cheese ◽  
The 16S Rrna Gene

The population of the Papaloapan region consume artisan fresh cheeses and no pathogen outbreaks have been reported recently. The microbiota is responsible to develop desirable characteristics of cheeses and undesirable characteristics due to the presence of certain pathogens microorganisms. Therefore, to identify the microorganisms of fresh cheeses is an important issue for the producers, consumers, and authorities. 11 Artisan fresh cheese samples from the Papaloapan region were collected in the summer and 11 samples in winter to characterize their microbiota. Traditional microbial techniques were used to identify the fungus and the amplification of the 16S rRNA gene and PCR-denaturing gradient gel electrophoresis (DGGE) The population of the Papaloapan region consume artisan fresh cheeses and no pathogen outbreaks have been reported recently. The microbiota is responsible to develop desirable characteristics of cheeses and undesirable characteristics due to the presence of certain pathogens microorganisms. Therefore, to identify the microorganisms of fresh cheeses is an important issue for the producers, consumers, and authorities. 11 Artisan fresh cheese samples from the Papaloapan region were collected in the summer and 11 samples in winter to characterize their microbiota. Traditional microbial techniques were used to identify the fungus and the amplification of the 16S rRNA gene and PCR-denaturing gradient gel electrophoresis (DGGE) The population of the Papaloapan region consume artisan fresh cheeses and no pathogen outbreaks have been reported recently. The microbiota is responsible to develop desirable characteristics of cheeses and undesirable characteristics due to the presence of certain pathogens microorganisms. Therefore, to identify the microorganisms of fresh cheeses is an important issue for the producers, consumers, and authorities. 11 Artisan fresh cheese samples from the Papaloapan region were collected in the summer and 11 samples in winter to characterize their microbiota. Traditional microbial techniques were used to identify the fungus and the amplification of the 16S rRNA gene and PCR-denaturing gradient gel electrophoresis (DGGE) method was used for bacteria identification. For all the samples, the presence of aerobic mesophiles, Streptococcus mesophiles and thermophiles, Lactobacillus mesophiles, Leuconostoc, total coliforms, Staphylococcus aureus, molds, and yeasts were identified. The complexity and variety of microorganisms in the summer and winter seasons samples were not significantly different. In conclusion, all samples of fresh artisan cheeses were under high microbial loads. Lactic Acid Bacteria (LAB) were in a typical load, as established by the quality and safety standards in the food industry. Conversely, pathogenic bacteria exceeded this limit. The microorganisms present in the fresh artisanal cheeses of the Papaloapan region were identified with precision, regarding the count and their diversity. A recommendation for the cheese manufacturers is to prepare starter cultures by selecting the appropriate microorganisms to produce the desirable characteristics such as aroma and flavor and reduce the risk of microbial infections by using pasteurized milk.

Detection of filamentous genus Gordonia in foam samples using genus-specific primers combined with PCR – denaturing gradient gel electrophoresis analysis

2007 ◽  
Vol 53 (6) ◽  
pp. 768-774 ◽  
Author(s):  
Fo-Ting Shen ◽  
Hsuan-Ru Huang ◽  
A.B. Arun ◽  
Hui-Ling Lu ◽  
Ta-Chen Lin ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Wastewater Treatment Plants ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis

A nested-PCR amplification combined with denaturing gradient gel electrophoresis (PCR–DGGE) approach was used to detect and identify Gordonia populations from wastewater treatment plant foam samples. The PCR-amplified region (position 722–1119) by specifically designed primers G699F and G1096R covered the hypervariable region of the Gordonia 16S rRNA gene sequence. This approach successfully distinguished Gordonia species to the interspecies level. The differential ability of PCR–DGGE analysis was effectively used to separate 12 Gordonia species belonging to different 16S rRNA gene-based phylogenetic lineages into 8 groups. Based on this method, the minimum limit of Gordonia detection was 5 × 104CFU·g–1in the seeded soil samples. The PCR–DGGE bands obtained were excised and identified by sequence analysis. Gordonia polyisoprenivorans , Gordonia amicalis , DGGE type II Gordonia species, and an uncertain Gordonia species dominated the activated sludge foam samples. Results of this study indicate that the detection and analyses of genus Gordonia within a complex microbial community could be accomplished using the PCR–DGGE approach to a larger extent, with certain limitations. Detection of diverse Gordonia populations in foam samples from wastewater treatment plants revealed the significant role of Gordonia in biological foaming during wastewater treatment. The nested-PCR amplification and DGGE can be used as a diagnostic tool for the early detection of foaming incidents in wastewater treatment plants using Gordonia as indicator organism.

Overestimation of Streptococcus mutans prevalence by nested PCR detection of the 16S rRNA gene

2006 ◽  
Vol 55 (1) ◽  
pp. 109-113 ◽  
Author(s):  
Ali Al-Ahmad ◽  
Thorsten Mathias Auschill ◽  
Gabriele Braun ◽  
Elmar Hellwig ◽  
Nicole Birgit Arweiler
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Streptococcus Mutans ◽  
Nested Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Direct Pcr ◽  
Specific Primers ◽  
The 16S Rrna Gene

This study was carried out in order to compare two PCR-based methods in the detection of Streptococcus mutans. The first PCR method was based on primers for the 16S rRNA gene and the second method was based on specific primers that targeted the glucosyltransferase gene (gtfB). Each PCR was performed with eight different streptococci from the viridans group, five other streptococci and 17 different non-streptococcal bacterial strains. Direct use of the S. mutans 16S rRNA gene-specific primers revealed that Streptococcus gordonii and Streptococcus infantis were also detected. After amplifying the 16S rRNA gene with universal primers and subsequently performing nested PCR, the S. mutans-specific nested primers based on the 16S rRNA gene detected all tested streptococci. There was no cross-reaction of the gtfB primers after direct PCR. Our results indicate that direct PCR and nested PCR based on 16S rRNA genes can reveal false-positive results for oral streptococci and lead to an overestimation of the prevalence of S. mutans with regards to its role as the most prevalent causative agent of dental caries.

scholarly journals Diversity and Seasonal Variability of β-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning

2003 ◽  
Vol 69 (8) ◽  
pp. 4463-4473 ◽  
Author(s):  
I. H. M. Brümmer ◽  
A. Felske ◽  
I. Wagner-Döbler
Keyword(s):  
Temperature Gradient ◽  
16S Rrna ◽  
Seasonal Variability ◽  
Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
High Temporal Resolution ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
River Biofilm

ABSTRACT The β-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for β-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described βI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga. RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the β-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.

scholarly journals First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1653-1653 ◽  
Author(s):  
M. Starović ◽  
S. Kojic ◽  
S. T. Kuzmanovic ◽  
S. D. Stojanovic ◽  
S. Pavlovic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Vaccinium Corymbosum ◽  
23S Rrna ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
First Report ◽  
Stolbur Phytoplasma ◽  
The 16S Rrna Gene

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20′10.9″ N, 20°38′39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5′ end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 μl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) J. Jović et al. Phytopathology 99:1053, 2009. (3) S. Pavlovic et al. J. Med. Plants Res. 6:906, 2012. (4) D. Valiunas et al. J. Plant Pathol. 86:135, 2004.

scholarly journals Analytical Sensitivity, Reproducibility of Results, and Clinical Performance of Five PCR Assays for Detecting Chlamydia pneumoniae DNA in Peripheral Blood Mononuclear Cells

2000 ◽  
Vol 38 (7) ◽  
pp. 2622-2627 ◽  
Author(s):  
J. B. Mahony ◽  
S. Chong ◽  
B. K. Coombes ◽  
M. Smieja ◽  
A. Petrich
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Chlamydia Pneumoniae ◽  
Mononuclear Cells ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Infected Cells ◽  
Pcr Assays ◽  
The 16S Rrna Gene

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P < 0.001), with thePstI fragment (P < 0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P < 0.001), and the other 3 assays detected no positive specimens (P < 0.001, compared with theompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detectC. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.

Sign in / Sign up

Export Citation Format

Share Document