Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology

Tabrizi, Sepehr N.; Tan, Lit Yeen; Walker, Samantha; Twin, Jimmy; Poljak, M; Bradshaw, Catriona; Fairley, Christopher K.; Bissessor, Melanie; Mokany, Elisa; Todd, Alison V.; Garland, Suzanne M.

doi:10.1371/journal.pone.0156740

Cited by 70 publications

(56 citation statements)

References 26 publications

(29 reference statements)

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“…The ResistancePlus assay utilises a novel real-time PCR technology, PlexPCR, for the specific amplification of M. genitalium and concurrent detection of macrolide resistance-associated mutations in DNA extracted from clinical specimens 8. Master mix was prepared according to the manufacturer’s instructions, and reactions contained 5 µL template DNA.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance

et al. 2017

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The ResistancePlus assay generally performed well in comparison to methods currently employed at the reference laboratory. It detected a range of different mutations; however, a small number of specimens that were genotyped as macrolide resistant by Sanger sequencing were either not detected by the assay or were genotyped as susceptible. This could impact on treatment outcomes if assay results were used for patient management.

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Section: Methodsmentioning

confidence: 99%

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance

et al. 2017

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Section: Introductionmentioning

confidence: 99%

“…genitalium and the common mutations associated with macrolide resistance (including A2058G, A2059G, A2058T, A2058C, and A2059C) in one well (21). The previous evaluation assessed clinical performance in an infected population and also analytical performance (21). In this study, a prospective clinical evaluation of this assay was conducted in a routine clinical setting, thereby reflecting the prevalence of M.…”

Section: Introductionmentioning

confidence: 99%

Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

Tabrizi

Bradshaw

et al. 2017

J Clin Microbiol

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Mycoplasma genitalium is a significant pathogen for which first-line treatment is becoming less effective due to increased resistance to macrolides. As conventional culture and antimicrobial susceptibility testing is not feasible for routine detection of this pathogen, molecular markers such as detection of mutations in the 23S rRNA gene have been described to predict resistance. Recently, a novel multiplex quantitative PCR (qPCR) assay, ResistancePlus MG, has been described for the simultaneous detection of Mycoplasma genitalium and macrolide resistance. In the current study, the clinical performance of the assay was evaluated on 1,089 consecutive urine and anogenital swab samples in symptomatic and asymptomatic male and female patients. Overall, 6.0% were positive for M. genitalium, with 63.1% having macrolide resistance-associated mutations. Compared to the laboratory-validated qPCR method targeting the 16S rRNA gene and Sanger sequencing to determine 23S rRNA mutations, the sensitivity and specificity of M. genitalium detection were 98.5% and 100% and for detection of macrolide resistance mutations were 100.0% and 96.2%, respectively. This assay offers a considerable advantage in clinical settings for M. genitalium testing by making the results of macrolide resistance and mutation analyses simultaneously available, which is increasingly important with escalating macrolide resistance.

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“…This assay has since been validated on several hundred urine and urogenital samples with very high clinical sensitivity and specificity [28]. …”

Section: Discussionmentioning

confidence: 99%

Superior Multiplexing Capacity of PlexPrimers Enables Sensitive and Specific Detection of SNPs and Clustered Mutations in qPCR

Tan¹,

Walker²,

Lonergan³

et al. 2017

PLoS ONE

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BackgroundWhilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered.MethodPlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene.ResultsThe combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%.ConclusionPlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information.

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Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology

Cited by 70 publications

References 26 publications

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance

Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

Superior Multiplexing Capacity of PlexPrimers Enables Sensitive and Specific Detection of SNPs and Clustered Mutations in qPCR

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