Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections

We developed a real-time PCR to detect Mycoplasma pneumoniae with a primer set designed for the 16S rRNA gene. Clinical samples (n = 937) were collected from children with community-acquired pneumonia between April 2002 and March 2004 at 12 Japanese medical institutions. Sensitivity of real-time PCR was calculated as 10 colony-forming units per reaction tube using a pMP01 plasmid carrying a 225-bp target DNA fragment of the 16S rRNA gene in M. pneumoniae M129, a standard strain. Results, obtained within 2 h, were compared with those of conventional culture and serologic methods. Of all cases tested, 151 (16.4%) and 129 (13.8%) were positive for M. pneumoniae by real-time PCR and by culture, respectively. Among the 151 cases, almost all of those tested serologically by passive agglutination showed a rise in M. pneumoniae antibody titre between acute and convalescent sera. We conclude that this real-time PCR can identify M. pneumoniae rapidly and fulfills the need for rapid identification, high sensitivity, and high specificity.Key words: real-time PCR, Mycoplasma pneumoniae identification, pediatrics, community-acquired pneumonia, Mycoplasma pneumoniae culture.

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Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4214-4219.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4214-4219 ◽

Cited By ~ 15

Author(s):

Abu Sadeque Md Selim ◽

Piyanuch Boonkumklao ◽

Teruo Sone ◽

Apinya Assavanig ◽

Masaru Wada ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Pure Culture ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pcr Assay ◽

Chicken Feces

ABSTRACT A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345T and Lactobacillus fermentum JCM 1173T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 103 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 104 cells/g feces on day 4 and 105 cells/g feces on days 11 to 18. However, cell populations of 106 to 107 cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 1011 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

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A Real-time PCR assay for the rapid determination of 16S rRNA genotype and discovery of 16S rRNA gene heterogeneity in individual isolates of Vibrio vulnificus

Journal of Microbiological Methods ◽

10.1016/j.mimet.2006.02.010 ◽

2006 ◽

Author(s):

M VICKERY ◽

W NILSSON ◽

M STROM ◽

J NORDSTROM ◽

A DEPAOLA

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Vibrio Vulnificus ◽

Rapid Determination ◽

Rrna Gene ◽

Pcr Assay

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Chlamydiales and hemotropic mycoplasma in captive and free-living bats

10.21203/rs.2.22015/v1 ◽

2020 ◽

Author(s):

Janine Fritschi ◽

Hanna Marti ◽

Helena M.B. Seth-Smith ◽

Sébastien Aeby ◽

Gilbert Greub ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Rrna Gene ◽

Free Living ◽

Hemotropic Mycoplasma ◽

Total Prevalence ◽

Pcr Targeting ◽

The 16S Rrna Gene

Abstract Background: Bats are hosts for a variety of microorganisms, however, little is known about the presence of Chlamydiales and hemotropic mycoplasma. This study investigated 475 free-living and captive bats from Switzerland, Germany and Costa Rica for the occurrence of Chlamydiales and hemotropic mycoplasma.Results: Screening for Chlamydiales was performed using a Chlamydiaceae-specific real-time PCR targeting the 23S rRNA gene and a pan-Chlamydiales PCR targeting the 16S rRNA gene resulting in a total prevalence of 31.4%. For sequencing, a PCR with the specifically designed inner primers panFseq and panRseq was performed, and criteria published by Pillonel et al. were used to classify the 19 obtained sequences, resulting in the formation of two groups. Groups one and two shared sequence identities to Chlamydiaceae and to Chlamydia-like organisms, including Rhabdochlamydiaceae and unclassified Chlamydiales from environmental samples, respectively.Analysis for the presence of hemotropic mycoplasma was performed using a universal SYBR Green hemoplasma screening real-time PCR targeting the 16S rRNA gene, real-time PCRs specific for M. haemofelis-like and 'Candidatus M. haemominutum'-like organisms and two conventional PCRs targeting an 871-bp and 1030-bp region of the 16S rRNA gene resulting in a total prevalence of 0.7%. Sequencing and phylogenetic analysis of the 871-bp and 1030-bp region of the 16S rRNA gene were used to classify positive specimens and infer their phylogenetic relationships. Three sequences with identities to other unidentified mycoplasma found in vampire bats and Chilean bats were obtained.Conclusions: Bats can harbor Chlamydiales and hemotropic mycoplasma and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than thought before. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat species or countries and free-living as well as captive bats can be colonized. In conclusion, bats represent another potential host or vector for novel, previously unidentified, Chlamydiales and hemotropic mycoplasma.

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How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis

International Journal of Molecular Sciences ◽

10.3390/ijms19103256 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3256 ◽

Cited By ~ 9

Author(s):

Leonardo Terranova ◽

Martina Oriano ◽

Antonio Teri ◽

Luca Ruggiero ◽

Camilla Tafuro ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Coefficient Of Variation ◽

Extraction Yield ◽

Rrna Gene ◽

Microbiota Analysis ◽

The 16S Rrna Gene

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

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