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Emerging Microbes & Infections Volume 7, 2018 - Issue 1
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Original Articles

Detection of circulating Mycobacterium tuberculosis-specific DNA by droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis

Neng SongState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Yang TanState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Lingyun ZhangState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Wei LuoState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Qing GuanState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Ming-zhe YanWuhan Medical Treatment Center430071WuhanChina
,
Ruiqi ZuoState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Weixiang LiuState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChina
,
Feng-ling LuoState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChinaCorrespondence[email protected]
&
Xiao-Lian ZhangState Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of ImmunologyWuhan University School of Basic Medical Sciences430071WuhanChinaCorrespondence[email protected]
 show all
Pages 1-9 | Received 06 Oct 2017, Accepted 05 Mar 2018, Published online: 24 Apr 2018

Figures & data

Fig. 1 Standard curves and detection range of recombinant plasmids using qPCR and ddPCR.

a Optimum concentrations of CFP-10, Rv1768, and ESAT6 probes. Each sample was analyzed in three repetitive wells in one reaction, and each experiment was repeated three times. b Using qPCR, the CFP10 linear regression formula was y = −3.4274x + 35.604 (r2 = 0.9964), the Rv1768 linear regression formula was y = −3.3267x + 36.15 (r2 = 0.9961), and the ESAT-6 linear-regression formula was y = −2.7705x + 32.262 between the plasmid copy numbers and Ct values. c The linearity range of ddPCR for quantifying pET28a-CFP10 and pET28a-Rv1768 plasmid DNA. The formulae were y = 0.9277x + 0.1352 (r2 = 0.9931) for CFP10 and y = 0.6733x + 0.0325 (r2 = 0.998) for Rv1768.

Fig. 1 Standard curves and detection range of recombinant plasmids using qPCR and ddPCR.a Optimum concentrations of CFP-10, Rv1768, and ESAT6 probes. Each sample was analyzed in three repetitive wells in one reaction, and each experiment was repeated three times. b Using qPCR, the CFP10 linear regression formula was y = −3.4274x + 35.604 (r2 = 0.9964), the Rv1768 linear regression formula was y = −3.3267x + 36.15 (r2 = 0.9961), and the ESAT-6 linear-regression formula was y = −2.7705x + 32.262 between the plasmid copy numbers and Ct values. c The linearity range of ddPCR for quantifying pET28a-CFP10 and pET28a-Rv1768 plasmid DNA. The formulae were y = 0.9277x + 0.1352 (r2 = 0.9931) for CFP10 and y = 0.6733x + 0.0325 (r2 = 0.998) for Rv1768.
Fig. 2 Comparison of qPCR and ddPCR analysis of CFP10 and Rv1768 gene copy numbers in whole-blood DNA of the BCG-immunized and M. tb-infected RM models.

Each sample measured three times and each point represents the mean result of replicates. Each line (red or black) represents one monkey. ANOVA was used to compare the experimental data from qPCR and ddPCR. Differences were considered statistically significant for p < 0.05. a-c: for CFP10; d-f: for Rv1768. a-f have been provided in the figure legend.

Fig. 2 Comparison of qPCR and ddPCR analysis of CFP10 and Rv1768 gene copy numbers in whole-blood DNA of the BCG-immunized and M. tb-infected RM models.Each sample measured three times and each point represents the mean result of replicates. Each line (red or black) represents one monkey. ANOVA was used to compare the experimental data from qPCR and ddPCR. Differences were considered statistically significant for p < 0.05. a-c: for CFP10; d-f: for Rv1768. a-f have been provided in the figure legend.

Fig. 3 Representative data of the original microdrop pictures of the CFP10 ddPCR results forM. tb-infected RMs

Fig. 3 Representative data of the original microdrop pictures of the CFP10 ddPCR results forM. tb-infected RMs

Clinical characteristics of study participants

Comparison between ddPCR and qPCR for TB diagnosis in the clinical blood samples

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