Comparison of conventional, nested, and real-time quantitative PCR for diagnosis of scrub typhus

Abstract

Orientia tsutsugamushi is the causative agent of scrub typhus. For the diagnosis of scrub typhus, we investigated the performances of conventional PCR (C-PCR), nested PCR (N-PCR), and real-time quantitative PCR (Q-PCR) targeting the O. tsutsugamushi-specific 47-kDa gene. To compare the detection sensitivities of the three techniques, we used two template systems that used plasmid DNA (plasmid detection sensitivity), including a partial region of the 47-kDa gene, and genomic DNA (genomic detection sensitivity) from a buffy coat sample of a single patient. The plasmid detection sensitivities of C-PCR, N-PCR, and Q-PCR were 5 × 10(4) copies/μl, 5 copies/μl, and 50 copies/μl, respectively. The results of C-PCR, N-PCR, and Q-PCR performed with undiluted genomic DNA were negative, positive, and positive, respectively. The genomic detection sensitivities of N-PCR and Q-PCR were 64-fold and 16-fold (crossing point [Cp], 37.7; 426 copies/μl), respectively. For relative quantification of O. tsutsugamushi bacteria per volume of whole blood, we performed real-time DNA PCR analysis of the human GAPDH gene, along with the O. tsutsugamushi 47-kDa gene. At a 16-fold dilution, the copy number and genomic equivalent (GE) of GAPDH were 1.1 × 10(5) copies/μl (Cp, 22.64) and 5.5 × 10(4) GEs/μl, respectively. Therefore, the relative concentration of O. tsutsugamushi at a 16-fold dilution was 0.0078 organism/one white blood cell (WBC) and 117 organisms/μl of whole blood, because the WBC count of the patient was 1.5 × 10(4) cells/μl of whole blood. The sensitivities of C-PCR, N-PCR, and Q-PCR performed with blood samples taken from patients within 4 weeks of onset of fever were 7.3% (95% confidence interval [CI], 1.6 to 19.9), 85.4% (95% CI, 70.8 to 94.4), and 82.9% (95% CI, 67.9 to 92.8), respectively. All evaluated assays were 100% specific for O. tsutsugamushi. In conclusion, given its combined sensitivity, specificity, and speed, Q-PCR is the preferred assay for the diagnosis of scrub typhus.

Fig. 1.

(A) Locations of the selected primers and probes in the 47-kDa outer membrane protein gene (strain Kato, GenBank accession number L11697). External primers OtsuFR555 and OtsuRP771 were used to amplify a 238-bp segment of the O. tsutsugamushi-specific 47-kDa gene, and internal primers OtsuFP630 and OtsuRP747 were used to amplify a 118-bp segment. The probe OtsuPR665 was used for Q-PCR. (B) Locations of the selected primers in the 56-kDa outer membrane protein gene (strain Gilliam, GenBank accession number L31933). External primers p34 and p55 were used to amplify a 1,003-bp segment of the O. tsutsugamushi-specific 56-kDa gene, and internal primers p10 and p11 were used to amplify an internal 483-bp segment. The open reading frame of the 56-kDa gene is represented by a heavy line, and boxes I, II, III, and IV indicate the variable domains.

Fig. 2.

(A) Detection sensitivities of C-PCR and N-PCR. Lane 1, 100-bp ladder marker (Bioneer); lane 2, negative control (sterile distilled water); lane 3, positive-control strain Karp; lane 4, undiluted genomic DNA; lane 5 to lane 8, from 2-fold-diluted genomic DNA to 16-fold-diluted genomic DNA, respectively; lane 9, 100-bp ladder marker (Bioneer); lane 10, sterile D/W; lane 11, positive-control strain Karp; lane 12 and lane 13, from 32-fold-diluted genomic DNA and 64-fold-diluted genomic DNA, respectively; 47-kDa C-PCR (47-CPCR) and 47-kDa N-PCR (47-NPCR) target size, 118 bp; 56-kDa N-PCR (56-NPCR) target size, 483 bp. (B) Detection sensitivities of Q-PCR. Circle 1, undiluted genomic DNA (Cp, 33.30); circle 2, 2-fold-diluted genomic DNA (Cp, 34.01); circle 3, 4-fold-diluted genomic DNA (Cp, 35.21); circle 4, 8-fold diluted genomic DNA (Cp, 36.19); circle 5, 16-fold-diluted genomic DNA (Cp, 37.47); circle 6, D/W. The results of Q-PCR using 32-fold- and 64-fold-diluted genomic DNA were negative (Cp, 39.36 and >40.00, respectively) due to use of a cutoff value of >38 (data not shown).