Table 1.
List of primer sequences.
Table 2.
List of the equations for calculating the performance of multiplex PCR.
Fig 1.
Optimization of singleplex and multiplex PCR.
Lane M; 100bp marker, lane 1; DNA mixture derived from Orientia (166 bp), Leptospira (243 bp), and Rickettsia (434 bp) was amplified using multiplex PCR. Lane 2–4; O. tsutsugamushi (Karp, Kato, and Gilliam strains), lane 5; negative control, lane 6–8; L. interrogans (serovar Bratislava, Pyrogenes, and Pomona), lane 9; L. biflexa serovar Patoc, lane 10; negative control, lane 11; R. typhi, and lane 12; negative control. Lane 2–12; DNA was amplified using singleplex PCR.
Fig 2.
Specificity of multiplex PCR assay.
(A) Multiplex PCR was tested with other pathogenic microorganisms. Lane M; 100bp marker, lane 1; Positive control (Orientia, Leptospira, and Rickettsia DNA mixture), lane 2; O. tsutsugamushi (166 bp), lane 3; L. interrogans (243 bp), lane 4; R. typhi (434 bp), lane 5; E. faecalis, lane 6; S. aureus, lane 7; Salmonella spp., lane 8; K. pneumoniae, lane 9; Dengue virus I, lane 10; Dengue virus II, lane 11; Dengue virus III, lane 12; Dengue virus IV, lane 13; P. falciparum, lane 14; P. vivax, lane 15; E. coli, and lane 16; negative control. (B) Multiplex PCR was tested in masking condition. Lane M; 100bp marker, lane 1; Positive control (Orientia, Leptospira, and Rickettsia DNA mixture), lane 2; DNA mixture of O. tsutsugamushi, S. aureus, and dengue virus, lane 3; DNA mixture of L. interrogans, S. aureus, and dengue virus, lane 4; DNA mixture of R. typhi, S. aureus, and dengue virus, lane 5; negative control.
Fig 3.
Sensitivity of multiplex PCR assay.
(A) Limit of detection of multiplex PCR with a serial dilution of Orientia, Leptospira, and Rickettia DNA mixture, and (B) limit of detection with three bacterial DNA-spiked blood sample in different concentration. In both figures: lane M; 100bp marker, lane 1; 5 ng/μl, lane 2; 0.5 ng/μl, lane 3; 50 pg/μl, lane 4; 5 pg/μl, lane 5; 0.5 pg/μl, lane 6; 50 fg/μl, lane 7; 5 fg/μl, and lane 8; negative control.
Fig 4.
Performance of the multiplex PCR.
(A-H) showed the multiplex PCR results when tested with blood specimens from Thai patients presented with acute undifferentiated febrile illness.
Table 3.
Comparison of multiplex PCR results and standard serological test results.