Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus

ABSTRACT Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus.

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O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

Human Reproduction ◽

10.1093/humrep/deab125.021 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

E Panacheva ◽

D Pochernikov ◽

E Voroshilina

Keyword(s):

Cluster Analysis ◽

Real Time ◽

Real Time Pcr ◽

Detection Rate ◽

Bacterial Load ◽

Rrna Gene ◽

Rt Pcr ◽

Silhouette Index ◽

Enterococcus Spp ◽

Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

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Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches

Food Chemistry ◽

10.1016/j.foodchem.2017.11.068 ◽

2018 ◽

Vol 245 ◽

pp. 1034-1041 ◽

Cited By ~ 10

Author(s):

Telmo J.R. Fernandes ◽

Joana Costa ◽

M. Beatriz P.P. Oliveira ◽

Isabel Mafra

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Rrna Gene ◽

Allergenic Food ◽

Detection And Quantification

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Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

Canadian Journal of Microbiology ◽

10.1139/w08-157 ◽

2009 ◽

Vol 55 (5) ◽

pp. 611-616 ◽

Cited By ~ 32

Author(s):

Ingrid H. Franke-Whittle ◽

Marta Goberna ◽

Heribert Insam

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Biogas Production ◽

Pcr Primers ◽

Sybr Green ◽

Rrna Gene ◽

Pcr Assays ◽

Design And Testing ◽

Uncultured Archaea ◽

Detection And Quantification

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta , Methanosarcina , and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter , and a group of yet uncultured archaea from anaerobic habitats.

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Detection and Quantification of Milk Ingredients as Hidden Allergens in Meat Products by a Novel Specific Real-Time PCR Method

Biomolecules ◽

10.3390/biom9120804 ◽

2019 ◽

Vol 9 (12) ◽

pp. 804 ◽

Cited By ~ 4

Author(s):

Caterina Villa ◽

Joana Costa ◽

Isabel Mafra

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Meat Products ◽

Milk Proteins ◽

12S Rrna ◽

Processed Meat ◽

Rrna Gene ◽

Wide Range ◽

Detection And Quantification

Milk ingredients are often included in a wide range of meat products, such as cooked hams and sausages, to improve technological characteristics. However, milk proteins are also important food allergens. The aim of this study was the development of a highly sensitive and specific real-time PCR system targeting the 12S rRNA gene of Bos domesticus for the detection and quantification of milk as an allergenic ingredient in processed meat products. The method was able to achieve an absolute limit of detection (LOD) of 6 fg of milk DNA. Using a normalized approach (∆Ct method) for the detection of milk protein concentrate (MPC), it was possible to obtain sensitivities down to 0.01% (w/w) of MPC in model hams (raw and cooked) and autoclaved sausages, and 0.005% in raw sausage mixtures. The developed systems generally presented acceptable PCR performance parameters, being successfully validated with blind samples, applied to commercial samples, and further compared with an immunochemical assay. Trace amounts of milk material were quantified in two out of 13 samples, but the results mostly infer the excessive practice of the precautionary labeling.

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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Nocardioides panacihumi sp. nov., isolated from soil of a ginseng field

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64806-0 ◽

2007 ◽

Vol 57 (9) ◽

pp. 2143-2146 ◽

Cited By ~ 12

Author(s):

Dong-Shan An ◽

Wan-Taek Im ◽

Sung-Taik Lee ◽

Min-Ho Yoon

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Major Fatty Acid ◽

Rrna Gene ◽

Strictly Aerobic ◽

Biochemical Tests ◽

Phenotypic Differentiation ◽

The Family ◽

Established Species ◽

Type Strains

A novel bacterial strain designated Gsoil 616T was isolated from a soil sample of a ginseng field in Pocheon province (South Korea) and was characterized taxonomically by using a polyphasic approach. The isolate was Gram-positive, strictly aerobic, non-motile, non-spore-forming and rod- or coccoid-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Nocardioides in the family Nocardioidaceae but was clearly separated from established species of this genus. The 16S rRNA gene sequence similarities between strain Gsoil 616T and the type strains of Nocardioides species with validly published names ranged from 91.8 to 96.1 %. The G+C content of the genomic DNA was 73 mol%. Phenotypic and chemotaxonomic data [major menaquinone MK-8(H4) and major fatty acid iso-C16 : 0] supported the affiliation of strain Gsoil 616T to the genus Nocardioides. However, the results of physiological and biochemical tests allowed phenotypic differentiation of the isolate from other Nocardioides species. Therefore, strain Gsoil 616T represented a novel species within the genus Nocardioides, for which the name Nocardioides panacihumi sp. nov. is proposed. The type strain is Gsoil 616T (=KCTC 19187T =DSM 18660T).

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Real-time PCR for detection and quantification of Erwinia amylovora, the causal agent of fireblight

Plant Pathology ◽

10.1111/j.1365-3059.2004.01066.x ◽

2004 ◽

Vol 53 (5) ◽

pp. 602-610 ◽

Cited By ~ 40

Author(s):

H. Salm ◽

K. Geider

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Erwinia Amylovora ◽

Causal Agent ◽

Detection And Quantification

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PX-10 VALIDATION OF EASYARTUS CMV AUTOMATED REAL TIME PCR SYSTEM FOR DETECTION AND QUANTIFICATION OF CMV DNA.

Journal of Clinical Virology ◽

10.1016/s1386-6532(09)70232-x ◽

2009 ◽

Vol 46 ◽

pp. S49

Author(s):

C. Pope ◽

K. Capaldi ◽

P. Rice

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Detection And Quantification

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Kordia aestuariivivens sp. nov. and Olleya sediminilitoris sp. nov., isolated from a tidal flat

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004815 ◽

2021 ◽

Vol 71 (6) ◽

Author(s):

Sooyeon Park ◽

Jung-Sook Lee ◽

Wonyong Kim ◽

Jung-Hoon Yoon

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Tidal Flat ◽

Genomic Sequences ◽

Rrna Gene ◽

Phenotypic Properties ◽

Content Type ◽

Link Type ◽

The Family ◽

Type Strains

Two Gram-stain-negative and non-flagellated bacteria, YSTF-M3T and YSTF-M6T, were isolated from a tidal flat from Yellow Sea, Republic of Korea, and subjected to a polyphasic taxonomic study. Neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strains YSTF-M3T and YSTF-M6T belong to the genera Kordia and Olleya of the family Flavobacteriaceae , respectively. The 16S rRNA gene sequence similarities between strain YSTF-M3T and the type strains of Kordia species and between strain YSTF-M6T and the type strains of Olleya species were 94.1–98.4 and 97.3–98.3 %, respectively. The ANI and dDDH values between genomic sequences of strain YSTF-M3T and the type strains of five Kordia species and between those of strain YSTF-M6T and the type strains of three Olleya species were in ranges of 77.0–83.2 and 20.7–27.1 % and 79.4–81.5 and 22.3–23.9 %, respectively. The DNA G+C contents of strain YSTF-M3T and YSTF-M6T from genomic sequences were 34.1 and 31.1 %, respectively. Both strains contained MK-6 as predominant menaquinone and phosphatidylethanolamine as only major phospholipid identified. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strains YSTF-M3T and YSTF-M6T are separated from recognized species of the genera Kordia and Olleya , respectively. On the basis of the data presented, strains YSTF-M3T (=KACC 21639T=NBRC 114499T) and YSTF-M6T (=KACC 21640T=NBRC 114500T) are considered to represent novel species of the genera Kordia and Olleya , respectively, for which the names Kordia aestuariivivens sp. nov. and Olleya sediminilitoris sp. nov. are proposed.

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