Objectives Our goal was to develop a rapid diagnostic tool based on a multiplex Taqman real time qPCR or RT-qPCR and High Resolution Melting (HRM) platform to detect Staphylococcus and Streptococcus spp. determinants allowing an early identification and determination of virulence and/or antibiotic-resistance profiles towards drugs used in clinical practice and having a significant clinical impact in the patient outcome and treatment costs. Methods We set up a new single-multiplex Taqman real time qPCR and HRM platform, on Rotor-GeneQ, with Primers and Probes designed using Beacon Designer. On control strain staphylococcal DNA we identified: i) Staphylococcus aureus and coagulase-negative Staphylococci (CoNS) (variation in gap gene and presence of nuc gene); ii) Hospital- and Community-Acquired S.aureus (pvl); iii) Resistant genes responsible for methicillin (mecA), vancomycin (vanA), linezolid (cfr), macrolide and lincosamide resistance (ermA-ermC); iv) vancomycin (hVISA-VISA) and daptomycin reduced-susceptibility (hld transcription). Among Streptococci, Streptococcus pyogenes and Streptococcus pneumoniae were identified by spy1258 (TetR family transcriptional regulator) and ply (pneumolysin). On S.pyogenes, we detect the covR/S and speB virulence genes. We performed an S.pneumoniae serotyping (serotype 1,3,4, 5, 6A/B,7A/B, 8, 9V/A, 10A/B, 12A/B/F,14, 15, 18B/C, 19A, 19B/F, 20, 22A/F, 23F, 33A/F) by SYBR-GREEN real time qPCR. These targets were also tested on blood and nasal pharyngeal swabs. On resistant and susceptible staphylococcal strain DNA, HRM analysis was used to detect point mutations related to resistance versus: i) Linezolid (G2576T in 23SrRNA gene); ii) Rifampicin (C1441A and C1586T in rpoB); iii) Fluoroquinolones (C251T in gyrA and C239T in grlA). Results We evaluated the absolute copy number of all targets with a high sensitivity (detection limit 103-102 copies) and specificity (No cross-reaction with the DNA of other species). No inhibition was found with increasing concentrations (up to 103 copies) of other species DNA. All tests were repeated more than 10 times to optimize assays. All probes, both in Singleplex and Duplex assays, showed high-quality standard curve parameters in terms of R2 (coefficient of correlation), M (slope) and E (efficiency) of ≥0.99, -3.1/-3.6, 90-100%, respectively. All 10-fold DNA dilutions, ranging from 108 to 102 copy number, were successfully amplified producing CT values from 13 to 37 included in the theoretical CT range [13.6 (108 UG) – 36.7 (10 UG)]. Streptococcal identification probes did not show interference with biological matrix. HRM melting peaks and normalized graphs vs Wild Types allowed us to correctly distinguish the linezolid, rifampicin and fluoroquinolone resistant S.aureus vs the susceptible ones with a confidence threshold of 90%. Conclusion We describe a new combined real time qPCR and HRM platform for the identification of Staphylococci, Streptococci, their antibiotic resistance and major virulence genes, meeting the need to have rapid diagnostic methods for early patient treatment and ongoing pathogen-oriented therapy management.

A new combined Multiplex real time qPCR and HRM platform to rapidly detect Staphylococci, Streptococci, antibiotic-resistance and major virulence genes

Viviana Cafiso;SANTAGATI, Maria Carmela
2015

Abstract

Objectives Our goal was to develop a rapid diagnostic tool based on a multiplex Taqman real time qPCR or RT-qPCR and High Resolution Melting (HRM) platform to detect Staphylococcus and Streptococcus spp. determinants allowing an early identification and determination of virulence and/or antibiotic-resistance profiles towards drugs used in clinical practice and having a significant clinical impact in the patient outcome and treatment costs. Methods We set up a new single-multiplex Taqman real time qPCR and HRM platform, on Rotor-GeneQ, with Primers and Probes designed using Beacon Designer. On control strain staphylococcal DNA we identified: i) Staphylococcus aureus and coagulase-negative Staphylococci (CoNS) (variation in gap gene and presence of nuc gene); ii) Hospital- and Community-Acquired S.aureus (pvl); iii) Resistant genes responsible for methicillin (mecA), vancomycin (vanA), linezolid (cfr), macrolide and lincosamide resistance (ermA-ermC); iv) vancomycin (hVISA-VISA) and daptomycin reduced-susceptibility (hld transcription). Among Streptococci, Streptococcus pyogenes and Streptococcus pneumoniae were identified by spy1258 (TetR family transcriptional regulator) and ply (pneumolysin). On S.pyogenes, we detect the covR/S and speB virulence genes. We performed an S.pneumoniae serotyping (serotype 1,3,4, 5, 6A/B,7A/B, 8, 9V/A, 10A/B, 12A/B/F,14, 15, 18B/C, 19A, 19B/F, 20, 22A/F, 23F, 33A/F) by SYBR-GREEN real time qPCR. These targets were also tested on blood and nasal pharyngeal swabs. On resistant and susceptible staphylococcal strain DNA, HRM analysis was used to detect point mutations related to resistance versus: i) Linezolid (G2576T in 23SrRNA gene); ii) Rifampicin (C1441A and C1586T in rpoB); iii) Fluoroquinolones (C251T in gyrA and C239T in grlA). Results We evaluated the absolute copy number of all targets with a high sensitivity (detection limit 103-102 copies) and specificity (No cross-reaction with the DNA of other species). No inhibition was found with increasing concentrations (up to 103 copies) of other species DNA. All tests were repeated more than 10 times to optimize assays. All probes, both in Singleplex and Duplex assays, showed high-quality standard curve parameters in terms of R2 (coefficient of correlation), M (slope) and E (efficiency) of ≥0.99, -3.1/-3.6, 90-100%, respectively. All 10-fold DNA dilutions, ranging from 108 to 102 copy number, were successfully amplified producing CT values from 13 to 37 included in the theoretical CT range [13.6 (108 UG) – 36.7 (10 UG)]. Streptococcal identification probes did not show interference with biological matrix. HRM melting peaks and normalized graphs vs Wild Types allowed us to correctly distinguish the linezolid, rifampicin and fluoroquinolone resistant S.aureus vs the susceptible ones with a confidence threshold of 90%. Conclusion We describe a new combined real time qPCR and HRM platform for the identification of Staphylococci, Streptococci, their antibiotic resistance and major virulence genes, meeting the need to have rapid diagnostic methods for early patient treatment and ongoing pathogen-oriented therapy management.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11769/112787
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