Determination of mutations of the 23S rRNA gene ofHelicobacter pyloriby allele specific primer-polymerase chain reaction method

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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Detection and identification of azithromycin resistance mutations on Treponema pallidum 23S rRNA gene by nested multiplex polymerase chain reaction

Medical Journal of Indonesia ◽

10.13181/mji.v26i2.1543 ◽

2017 ◽

Vol 26 (2) ◽

pp. 90-6 ◽

Cited By ~ 1

Author(s):

Desy A. Gultom ◽

Yeva Rosana ◽

Ida Efendi ◽

Wresti Indriatmi ◽

Andi Yasmon

Keyword(s):

Multiplex Polymerase Chain Reaction ◽

Treponema Pallidum ◽

23S Rrna ◽

Rrna Gene ◽

Chain Reaction ◽

Detection And Identification ◽

23S Rrna Gene ◽

Polymerase Chain ◽

Primer Sets ◽

Blood Specimens

Background: Azithromycin-resistant strains of Treponema pallidum is associated with the mutation of 23S rRNA gene of T. pallidum. Although these strains are now prevalent in many countries, there is no laboratory test kit to detect and identify these mutations. Thus, in this study we developed a nested multiplex polymerase chain reaction (PCR) to detect and identify A2058G and A2059G mutations in 23S rRNA gene.Methods: Three primer sets were designed for nested PCR reactions. To obtain maximum PCR reaction, all parameters were optimized. The specificity of the primer sets was evaluated towards some microorganisms. A sensitivity test was conducted to get detection limit of deoxyribonucleic acid (DNA). Forty-five whole blood specimens were tested by PCR, and positive results were confirmed by the DNA sequencing.Results: The assay could detect at least 4,400 DNA copy number and showed no cross reaction with other microorganisms used in the specificity test. A total 13 of 45 whole blood specimens were PCR positive for T. pallidum, and no single mutations (either A2058G or A2059G) were detected. Two positive specimens were confirmed by the DNA sequencing and showed no mutation.Conclusion: Nested multiplex PCR developed in this study showed a specific and sensitive test for the detection and identification of A2058G and/or A2059G mutations of 23S rRNA T. pallidum gene.

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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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