A diagnostic polymerase chain reaction forMycoplasma iowaeusing primers located in the intergenic spacer region and the 23S rRNA gene

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.

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Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

Nova Biotechnologica et Chimica ◽

10.1515/nbec-2016-0007 ◽

2016 ◽

Vol 15 (1) ◽

pp. 65-76

Author(s):

Zuzana Šramková ◽

Barbora Vidová ◽

Andrej Godány

Keyword(s):

Staphylococcus Aureus ◽

Polymerase Chain Reaction ◽

Food Poisoning ◽

Detection Sensitivity ◽

23S Rrna ◽

Rrna Gene ◽

Chain Reaction ◽

23S Rrna Gene ◽

Polymerase Chain ◽

Toxic Shock Syndrome Toxin

Abstract Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s) and staphylococcal enterotoxin-like proteins (SEl-s). Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED), new SE-s (SEH, SEI), SEl-s (SEK, SEL) and tsst-1 gene (toxic shock syndrome toxin). Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

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Point-of-care molecular diagnosis of Mycoplasma pneumoniae including macrolide sensitivity using quenching probe polymerase chain reaction

PLoS ONE ◽

10.1371/journal.pone.0258694 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258694

Author(s):

Nobuhisa Ishiguro ◽

Rikako Sato ◽

Toshihiko Mori ◽

Hiroshi Tanaka ◽

Mitsuo Narita ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Point Mutation ◽

Mycoplasma Pneumoniae ◽

Point Of Care ◽

23S Rrna ◽

Rrna Gene ◽

Chain Reaction ◽

23S Rrna Gene ◽

Polymerase Chain ◽

Domain V

Objectives Macrolides are generally considered to be the drugs of choice for treatment of patients with Mycoplasma pneumoniae infection. However, macrolide-resistant M. pneumoniae has been emerging since about 2000. The Smart Gene® system (MIZUHO MEDY Co., Ltd., Tosu, Japan) is a novel fully automated system for detection of pathogens using the method of quantitative polymerase chain reaction (qPCR) with QProbe (QProbe PCR). The entire procedure is completed within 50 min and the size of the instrument is small (15 x 34 x 30 cm). The purpose of this study was to evaluate the usefulness of the Smart Gene® system for detection of M. pneumoniae and detection of a point mutation at domain V of the 23S rRNA gene of M. pneumoniae. Materials Pharyngeal swab samples were collected from 154 patients who were suspected of having respiratory tract infections associated with M. pneumoniae. Results Compared with the results of qPCR, the sensitivity and specificity of the Smart Gene® system were 98.7% (78/79) and 100.0% (75/75), respectively. A point mutation at domain V of the 23S rRNA gene was detected from 7 (9.0%) of 78 M. pneumoniae-positive samples by the Smart Gene® system and these results were confirmed by direct sequencing. The minimum inhibitory concentrations of clarithromycin among the 5 isolates of M. pneumoniae with a point mutation at domain V of the 23S rRNA gene were >64 μg/ml and those among the 33 isolates without a mutation in the 23S rRNA gene were <0.0625 μg/ml. Conclusion The Smart Gene® system is a rapid and accurate assay for detection of the existence of M. pneumoniae and a point mutation at domain V of the 23S rRNA gene of M. pneumoniae at the same time. The Smart Gene® system is suitable for point-of-care testing in both hospital and outpatient settings.

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Genus- and species-specific detection ofListeria monocytogenesusing polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon

Canadian Journal of Microbiology ◽

10.1139/m96-147 ◽

1996 ◽

Vol 42 (11) ◽

pp. 1155-1162 ◽

Cited By ~ 27

Author(s):

Tom Graham ◽

Elizabeth J. Golsteyn-Thomas ◽

Victor P. J. Gannon ◽

James E. Thomas

Keyword(s):

Polymerase Chain Reaction ◽

Listeria Monocytogenes ◽

Sequence Data ◽

Intergenic Spacer ◽

23S Rrna ◽

Chain Reaction ◽

Nucleotide Sequence Data ◽

Intergenic Spacer Region ◽

Polymerase Chain ◽

Species Specific

In this study, the 16S/23S rRNA intergenic spacer (IGS) regions of six Listeria species were examined. DNA bands of 590 and 340 bp were observed following polymerase chain reaction (PCR) amplification of DNA from Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria ivanovii strains with generic rRNA IGS oligonucleotide primers. For strains of Listeria grayi subspp. grayi and murrayi, DNA band sizes of 550 and 340 bp were observed with this primer pair. DNA bands of these sizes were not observed for other Gram-negative or -positive bacteria in this PCR assay. Four RsaI digestion profiles were noted for the Listeria PCR products. Listeria monocytogenes strains had one profile; L. innocua strains had a second; L. seeligeri, L. welshimeri, and L. ivanovii strains had a third; and L. grayi strains had a fourth. The small and large 16S/23S rRNA IGSs of L. monocytogenes ATCC 15313 were identical in the first 58 5′ and the last 169 3′ nucleotides. However, the large rRNA IGS contained a central 267-bp region with tRNAIleand tRNAAlagenes. Large rRNA 16S/23S IGS nucleotide sequence data has not been previously reported. This data was used to develop novel Listeria genus-specific and L. monocytogenes species-specific PCR assays.Key words: polymerase chain reaction, 16S/23S rRNA intergenic spacer region, Listeria monocytogenes.

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Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region ofGeomyces destructans

Mycologia ◽

10.3852/12-242 ◽

2013 ◽

Vol 105 (2) ◽

pp. 253-259 ◽

Cited By ~ 80

Author(s):

Laura K. Muller ◽

Jeffrey M. Lorch ◽

Daniel L. Lindner ◽

Michael O’Connor ◽

Andrea Gargas ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Intergenic Spacer ◽

Polymerase Chain Reaction Test ◽

Chain Reaction ◽

Intergenic Spacer Region ◽

White Nose Syndrome ◽

Reaction Test ◽

Polymerase Chain ◽

Chain Reaction Test ◽

Taqman Polymerase Chain Reaction

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Differentiation of Moraxella Bovoculi sp. nov. from other Coccoid Moraxellae by the Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis of Amplified DNA

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870701900511 ◽

2007 ◽

Vol 19 (5) ◽

pp. 532-534 ◽

Cited By ~ 18

Author(s):

John A. Angelos ◽

Louise M. Ball

Keyword(s):

Polymerase Chain Reaction ◽

Dna Sequence ◽

Restriction Analysis ◽

Intergenic Spacer ◽

Restriction Enzyme Digestion ◽

Chain Reaction ◽

Gram Negative ◽

Intergenic Spacer Region ◽

Polymerase Chain ◽

Moraxella Bovoculi

Moraxella oris was historically the only coccoid Moraxella identified in cultures of ocular fluid from cattle with infectious bovine keratoconjunctivitis (IBK) and could be morphologically and biochemically differentiated from Moraxella bovis. Moraxella bovoculi sp. nov. is a recently characterized Moraxella isolated from ulcerated eyes of calves with IBK in northern California in 2002. Like Moraxella ovis, M. bovoculi sp. nov. is a gram-negative coccus/diplococcus. All 18 original isolates of M. bovoculi sp. nov. possessed phenylalanine deaminase (PADase) activity and could therefore be differentiated from M. ovis and M. bovis. During the characterization of 44 additional isolates of hemolytic gram-negative cocci that were cultured from ulcerated eyes of IBK-affected calves, 2 PADase-negative isolates were identified that could not be differentiated biochemically from M. ovis; however, the DNA sequence of the 16S-23S intergenic spacer region (ISR) of the isolates matched the 16S-23S ISR DNA sequence of M. bovoculi sp. nov. To facilitate the identification of PADase-negative moraxellae, a polymerase chain reaction (PCR) coupled with restriction enzyme digestion analysis of amplified DNA was developed. Amplification of the 16S-23S ISR followed by AfaI digestion of amplified DNA could differentiate M. bovoculi sp. nov. from M. ovis and other moraxellae. The DNA sequence analysis of the amplified 16S-23S ISR from the 42 PADase-positive isolates of hemolytic gram-negative cocci indicated that all were M. bovoculi sp. nov. and all possessed an AfaI site. A PCR coupled with restriction analysis of amplified DNA can aid in identifying M. bovoculi sp. nov.

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