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. 2020 Jan 30:11:4.
doi: 10.3389/fmicb.2020.00004. eCollection 2020.

Development of the Droplet Digital PCR to Detect the Teliospores of Tilletia controversa Kühn in the Soil With Greatly Enhanced Sensitivity

Jianjian Liu  1   2 , Chao Li  1 , Ghulam Muhae-Ud-Din  1 , Taiguo Liu  1 , Wanquan Chen  1 , Jianmin Zhang  2 , Li Gao  1
Affiliations

Development of the Droplet Digital PCR to Detect the Teliospores of Tilletia controversa Kühn in the Soil With Greatly Enhanced Sensitivity

Jianjian Liu et al. Front Microbiol. .

Abstract

Background and aims: The dwarf bunt disease of wheat is caused by Tilletia controversa Kühn. This pathogen is primarily involved in the stunted growth of wheat and affects seed quality. Many countries in the world have therefore imposed quarantine bans to prevent the spread of T. controversa. Morphological observations are the main method of detecting teliospores in soil. However, this is a lengthy and laborious process; this method is thus unable to quickly meet the demand for detection of teliospores in the soil.

Methods: We compared PCR, real-time PCR and droplet digital PCR (ddPCR) for the qualitative and quantitative measurement of the teliospores of T. controversa in soil.

Results: We suggest the use of ddPCR for detection of the soil samples, which was demonstrated to have the most sensitive detection at 2.1 copies/μL. In contract, SYBR Green I real-time PCR could detect 7.97 copies/μL of T. controversa in soil, and this sensitivity was 100 times more sensitive than that of simple PCR.

Conclusion: This study was the first report using ddPCR techniques to detect T. controversa teliospores in soil with greatly enhanced sensitivity.

Keywords: Tilletia controversa Kühn; detection; droplet digital PCR; dwarf bunt; soil born disease.

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Figures

FIGURE 1
FIGURE 1
Amplification of the target DNA from soil samples with specific primers. Line 1–10, T. controversa soil samples; line 11, Tilletia laevis soil samples; line 12, ddH2O; M, DL2000 Marker (100, 250, 500, 750, 1,000, 2,000 bp); black arrows show the target band of 372 bp.
FIGURE 2
FIGURE 2
Electrophoresis tests of the plasmid DNA standard. Line 1, T. controversa teliospores DNA; line 2–10, the ten-fold serially dilutions of plasmid DNA standard (CN = 7.97 × 108–7.97 × 100); line 11, T. laevis DNA; line 12, ddH2O; line M, DL2000 Marker (100, 250, 500, 750, 1,000, 2,000 bp); black arrow shows the target bands of 372 bp.
FIGURE 3
FIGURE 3
Establishment of standard curve by SYBR Green I Real-Time PCR. (A) Real-time amplified plot, red lines 1–9, ten-fold serially dilutions of plasmid DNA standard (CN = 7.97 × 108–7.97); line 10, negative control ddH2O. (B) Melt curve of SYBR Green I (peak temperature at 88.26°C). (C) Standard curve.
FIGURE 4
FIGURE 4
Detection of soil samples by SYBR Green I Real-Time PCR. Real-time amplified plot, yellow line with black arrow represents negative control T. laevis, and the blue lines represent the amplified curves of T. controversa soil samples. The red line represents threshold value.
FIGURE 5
FIGURE 5
Distribution diagram of droplets of soil samples by droplet digital PCR (ddPCR) detection. 1–10, T. controversa soil samples; 11–12, T. laevis soil samples; 13, ddH2O control; blue bots are positive droplets, and black bots are negative droplets.
FIGURE 6
FIGURE 6
Statistic analysis of soil samples by ddPCR detection. (A) Positive copy number analysis, 1–10, T. controversa soil samples; 11–12, T. laevis soil samples; 13, ddH2O control. (B) Number analysis of droplets, 1–10, T. controversa soil samples; 11–12, T. laevis soil samples; 13, ddH2O control; red pillars are positive droplets, and blue pillars are total droplets (positive+negative).
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