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

Tan, Lit Yeen; Walker, Samantha; Lonergan, Tina; Lima, Nicole E.; Todd, Alison V.; Mokany, Elisa

doi:10.1371/journal.pone.0170087

Cited by 18 publications

(18 citation statements)

References 27 publications

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“…The ResistancePlus GC (beta) assay uses PlexPrime V R primers for improved amplification specificity of single base targets, coupled with a PlexZyme V R real-time PCR for robust multiplexing. 6 The assay reports on the presence of several genes associated with N. gonorrhoeae detection (porA and opa) as well as the S91F gyrA mutation. That test has recently been CE-marked.…”

Section: Methodsmentioning

confidence: 99%

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Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens

Allan‐Blitz

Ellis

Wee³

et al. 2019

Journal of Antimicrobial Chemotherapy

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Background The emergence of drug-resistant Neisseria gonorrhoeae has prompted the development of rapid molecular assays designed to determine antimicrobial susceptibility. One common assay uses high-resolution melt analysis to target codon 91 of the gyrase A gene (gyrA) to predict N. gonorrhoeae susceptibility to ciprofloxacin. Methods We extracted DNA from remnant clinical specimens that had previously tested positive for N. gonorrhoeae using the Aptima Combo 2 for CT/NG assay (Hologic, San Diego, CA, USA). We selected DNA extracts from specimens with indeterminate, WT and mutant gyrA genotype results from a previous study using high-resolution melt analysis to detect the gyrA codon 91 mutation. We re-tested those specimens using the recently CE-marked ResistancePlus GC (beta) assay (SpeeDx, Sydney, Australia). Results Of 86 specimens with indeterminate gyrA genotypes on high-resolution melt analysis, the ResistancePlus GC (beta) assay (SpeeDx) identified 30 (35%) WT, 22 (26%) mutant and 34 (40%) indeterminate gyrA genotypes. Conclusions The ResistancePlus GC (beta) assay showed improved N. gonorrhoeae gyrA genotype determination compared with a prior gyrA genotypic high-resolution melt assay.

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Section: Methodsmentioning

confidence: 99%

“…To compare our laboratory-developed assay, which utilizes PCR and high-resolution melt gyrA analysis, with a new gyrA genotyping assay that combines the detection of N. gonorrhoeae and the detection of ciprofloxacin susceptibility using a novel PCR technology, 6 we re-tested specimens with known gyrA genotypes.…”

Section: Introductionmentioning

confidence: 99%

Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens

Allan‐Blitz

Ellis

Wee³

et al. 2019

Journal of Antimicrobial Chemotherapy

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“…Recently, the multiplex qPCR assay developed by SpeeDx has shown to be sensitive and specific for the detection of M. genitalium and macrolide resistance-associated mutations [22][23][24]. Because of the promising results of the Mg Macrolide R qPCR, it would be of interest to compare the assays for ease of use and performance in a routine setting.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, a new multiplex qPCR assay has been developed by SpeeDx that allows simultaneous detection of M. genitalium and mutations commonly associated with macrolide resistance (including A2058G, A2059G, A2058T, A2058C, and A2059C) [22,23]. This multiplex qPCR has shown to be sensitive and specific for the detection of M. genitalium and macrolide resistance-associated mutations [24].…”

Section: Introductionmentioning

confidence: 99%

Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations

Braam

Marm

Severs

et al. 2018

Eur J Clin Microbiol Infect Dis

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Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistanceassociated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg Macrolide R qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other microorganisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg Macrolide R qPCR. The Mg Macrolide R qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg Macrolide R qPCR, indicating specificity. The Mg Macrolide R qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.

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“…The multiplex assay was developed using PlexPCR technology which employs novel PlexPrimers (Tan et al . ) coupled with PlexZyme (formerly known as MNAzyme) (Mokany et al . ) detection in qPCR.…”

Section: Methodsmentioning

confidence: 99%

One-step simultaneous detection ofUreaplasma parvumand genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology

Payne

Furfaro

Tucker³

et al. 2017

Lett Appl Microbiol

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Significance and Impact of the Study: Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest. AbstractUreaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 9 10 À5 ng DNA (SV3) and 0.4 9 10

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

Cited by 18 publications

References 27 publications

Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens

Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens

Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations

One-step simultaneous detection ofUreaplasma parvumand genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology

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