Fig 1.
Principle of closed dumbbell-mediated isothermal amplification (CDA) method.
(A) Overall scheme for primers used in CDA method. (B) Partial sequence of the ompA gene of R. raoultii used for designing the primers of CDA method.
Table 1.
Screening of R-CDA basic primer sequences for the mapping genes of Rickettsia raoultii.
Table 2.
Primer sequences of RO-CDA and PCR targeting at map gene of Rickettsia raoultii.
Fig 2.
Amplifications of R. raoultii DNA by R-CDA monitored by real-time PCR carried out at 60°C for 60 min.
(A) Real-time R-CDA of different groups for R. raoultii DNA (each set 2 positive reactions and 2 negative reactions). (B) Melting curve analysis of R-CDA products by real-time PCR. NC, negative control reaction without the template.
Fig 3.
Amplifications of R. raoultii DNA by R-CDA and RO-CDA monitored by real-time PCR.
(A) Amplification plot of real-time R-CDA and RO-CDA for R. raoultii DNA. (B) Melting curve analysis of CDA and RO-CDA products. (C) Repeatability analysis of the RO-CDA monitored by real- time PCR at 60°C. (D) Repeatability analysis of melting curve analysis of RO-CDA products. PC, positive reaction with the template; NC, negative control reaction without the template.
Fig 4.
Sensitivity analysis of the R. raoultii RO-CDA method by real-time and visual approaches.
(A) RO-CDA amplification monitored by real-time PCR every 1 min at different concentrations of DNA. Reaction was performed at 60°C for 60 min. (B) Melting curve analysis of RO-CDA products at different concentrations of DNA. (C) Sensitivity analysis of R. raoultii detection by visually RO-CDA. The DNA concentrations were as follows: 106 copies/μL, 105 copies/μL, 104 copies/μL, 103 copies/μL, 102 copies/μL, 10 copies/μL, 1 copy/μL and negative control (nuclease-free water). NC, negative control reaction without the template.
Fig 5.
Real-time RO-CDA reactions were carried out at 60°C for 60 min. (A) Real-time RO-CDA for R. raoultii DNA positive controls (Rickettsia PC), different R. raoultii samples (HLBE 407 and 408), other rickettsial and non rickettsial samples, (Rickettsia sibirica, Anaplasma ovis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Shigella, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes and Streptococcus agalactiae) and no template control (NC). (B) Melting curve analysis of the RO-CDA products by real-time PCR. PC, positive reaction with the template; NC, negative control reaction without the template.
Fig 6.
Colorimetric RO-CDA assay using HNB.
Line 1, positive controls (106 copies of templates); Line 2 and Line 3, samples extracted from R. raoultii; Line 4, samples extracted from Rickettsia sibirica; Line 5, samples extracted from Anaplasma ovis; Line 6, samples extracted from Klebsiella pneumoniae. PC, positive reaction with the template. Due to the limited space of the manuscript, other samples are not displayed.
Table 3.
Determination of sensitivity and specificity of RO-CDA assay for Rickettsia raoultii.