title: Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.
abstract: This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.
authors.affiliation: Anses, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Technopôle Agricole et Vétérinaire, CS 40009, 54220 Malzéville, France.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Picard-Meyer
authors.affiliation: Anses, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Technopôle Agricole et Vétérinaire, CS 40009, 54220 Malzéville, France.
authors.forename: Carine
authors.initials: C
authors.lastname: Peytavin de Garam
authors.affiliation: Anses, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Technopôle Agricole et Vétérinaire, CS 40009, 54220 Malzéville, France.
authors.forename: Jean Luc
authors.initials: JL
authors.lastname: Schereffer
authors.affiliation: Anses, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Technopôle Agricole et Vétérinaire, CS 40009, 54220 Malzéville, France.
authors.forename: Clotilde
authors.initials: C
authors.lastname: Marchal
authors.affiliation: Anses, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Technopôle Agricole et Vétérinaire, CS 40009, 54220 Malzéville, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Robardet
authors.affiliation: Anses, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Technopôle Agricole et Vétérinaire, CS 40009, 54220 Malzéville, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Cliquet
mesh_terms: D000818:Animals; D002685:Chiroptera / Q000821:virology*; D018114:Lyssavirus / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry*; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D009930:Organic Chemicals; D011933:Reagent Kits, Diagnostic