Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sources of Orientia Genomic DNA
2.2. qPCR Condition for Single Copy 47 kDa Gene and Multiple Copy traD Gene
2.3. Comparison of Performance of Single-Copy Gene vs. Multiple-Copy Gene Using Genomic DNA
2.4. Demonstration of Specificity of traD Gene qPCR
2.5. Evaluation of traD qPCR Using DNA Extracted from Confirmed Scrub Typhus Positive and Negative Patient Blood
2.6. Statistical Analysis
3. Results
3.1. Multiple-Copy Gene qPCR Shows Lower Detection Limit in Comparison to Single-Copy Gene in DNA Extracted from Purified Organisms, Chigger-Mites and Chigger-Mite Infected Mouse Liver
3.2. traD Gene qPCR Is Specific in the Presence of Excessive Chigger-Mite or Mouse Liver DNA
3.3. traD Gene qPCR Is Sensitive and Specific in Clinically Confirmed Scrub Typhus Positive and Negative Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Orientia Isolate | Country of Origin | ΔCt * |
---|---|---|
AFC-27 | Thailand | 8.29 ± 0.72 |
18-030641 | Malaysia | 8.14 ± 0.34 |
18-032029 | Malaysia | 8.70 ± 1.24 |
AFC-12 | Thailand | 7.14 ± 1.40 |
Gilliam | Burma | 5.09 ± 0.32 |
MAK 119 | Taiwan | 4.60 ± 0.35 |
MAK 243 | Taiwan | 9.17 ± 1.03 |
Karp | New Guinea | 5.10 ± 0.26 |
AFPL12 | Thailand | 7.54 ± 0.49 |
TA763 | Thailand | 7.26 ± 1.19 |
AFC-1 | Thailand | 8.30 ± 0.34 |
Citrano | Australia | 7.68 ± 1.63 |
Garton | Australia | 11.74 ± 1.59 |
Boryong | South Korea | 5.40 ± 0.13 |
Kato | Japan | 7.41 ± 1.17 |
Sample 1 | Sample 2 | |
---|---|---|
Original DNA preparation (copy/μL in 200 μL NHS) | 0.4 | 10 |
Extracted DNA (copy/μL in 50 μL water) | 1.6 | 40 |
expected copy # in a qPCR reaction a | 3.2 (4.7, 5.9) | 80 (31.3 ± 15.4) |
% 47 kDa qPCR positive (range of Ct) b | 28.6% (33.7, 34.1) | 100% (30.3–33.3) |
% traD qPCR positive (range of Ct) | 100% (30.2–30.5) | 100% (25.2–26.1) |
ΔCt | 3.62 c | 5.66 ± 0.65 |
Sample | Based on Melting Curve | Ct | PCR/IFA Result of Acute Sample |
---|---|---|---|
NHB1 | Neg. | 36.5 | PCR(−) IFA(−) |
NHB2 | Neg. | Undetermined | PCR(−) IFA(−) |
NHB3 | Neg. | 36.5 | PCR(−) IFA(−) |
NHB4 | Neg. | 38.0 | PCR(−) IFA(−) |
NHB5 | Neg. | 38.5 | PCR(−) IFA(−) |
Other Disease 1 | Neg. | Undetermined | PCR(−) IFA(−) |
Other Disease 2 | Neg. | Undetermined | PCR(−) IFA(−) |
Other Disease 3 | Neg. | 38.4 | PCR(−) IFA(−) |
Other Disease 4 | Neg. | Undetermined | PCR(−) IFA(−) |
Other Disease 5 | Neg. | 36.8 | PCR(−) IFA(−) |
Other Disease 6 | Neg. | 35.5 | PCR(−) IFA(−) |
Scrub typhus 1 | Pos. | 32.3 | PCR(+) IFA(+) |
Scrub typhus 2 | Pos. | 25.8 | PCR(−) IFA(+) |
Scrub typhus 3 | Pos. | 31.9 | PCR(−) IFA(+) |
Scrub typhus 4 | Pos. | 32.4 | PCR(+) IFA(−) |
Scrub typhus 5 a | Pos. | 38.3 | PCR(+) IFA(−) |
Scrub typhus 6 | Pos. | 28.1 | PCR(+) IFA(+) |
Scrub typhus 7 | Pos. | 30.2 | PCR(−) IFA(+) |
Scrub typhus 8 | Pos. | 30.3 | PCR(+) IFA(+) |
Scrub typhus 9 | Pos. | 24.7 | PCR(+) IFA(−) |
Scrub typhus 10 b | Pos. | 22.4 | PCR(−) IFA(−) |
Patients are ST Positive or Negative Based on the Results from | |||
---|---|---|---|
IFA | PCR | Clinical Diagnosis | |
Sensitivity (%) | 100 | 100 | 100 |
Specificity (%) | 73.3 a | 73.3 a | 100 |
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Chao, C.-C.; Belinskaya, T.; Zhang, Z.; Jiang, L.; Ching, W.-M. Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA. Trop. Med. Infect. Dis. 2019, 4, 113. https://doi.org/10.3390/tropicalmed4030113
Chao C-C, Belinskaya T, Zhang Z, Jiang L, Ching W-M. Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA. Tropical Medicine and Infectious Disease. 2019; 4(3):113. https://doi.org/10.3390/tropicalmed4030113
Chicago/Turabian StyleChao, Chien-Chung, Tatyana Belinskaya, Zhiwen Zhang, Le Jiang, and Wei-Mei Ching. 2019. "Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA" Tropical Medicine and Infectious Disease 4, no. 3: 113. https://doi.org/10.3390/tropicalmed4030113