Establishment and Application of Droplet Digital PCR Detection Methods for Four Major Verticillium Wilt Pathogens

doi:10.13560/j.cnki.biotech.bull.1985.2025-0421

Abstract

Abstract:

Objective A droplet digital PCR (ddPCR) method was established for the simultaneous detection of four Verticillium wilt pathogens, which may lay the foundation for timely and accurate quantitative monitoring of pathogen growth dynamics, early diagnosis, and risk assessment. Method Based on the alignment of the internal transcribed spacer (ITS) sequences of four Verticillium wilt pathogens-Verticillium dahliae (Vd, KY039312.1), V. longisporum (Vl, KX058040.1), V. nonalfalfae (Vna, KT362917.1), and V. albo-atrum (Vaa, MH856937.1)-conserved regions were selected for the design of primers and probes. Droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR) were used to screen for the optimal primers, optimize the ddPCR reaction system, and evaluate the specificity and sensitivity of the method. Result The optimal primer/probe set for the established method was Ver5; the optimal annealing temperature was 58 ℃, with primer and probe concentrations of 500 nmol/L and 250 nmol/L, respectively. Specificity testing showed that this method specifically identified the four Verticillium wilt pathogens without cross-amplification for non-target microbes, including 7 fungal and 6 bacterial species. The detection limits for Vd, Vl, Vna, and Vaa were 2.1×10^-⁶, 1.6×10^-⁶, 6.9×10^-⁴, and 3.6×10^-⁵ ng/μL, respectively. Detection analysis of 50 cotton and 50 soil samples demonstrated that, compared to qPCR, the ddPCR method showed a significant advantage in detection rate, with sensitivities improved by 46% and 51%, respectively. Conclusion The established ddPCR method for detecting the four Verticillium wilt pathogens demonstrates high specificity, excellent sensitivity, and robust reliability, providing an important technical tool for the accurate detection of Verticillium wilt. This method is advantageous for applications in customs inspection and quarantine, as well as in the monitoring and regulation of plant diseases and pests, thereby enhancing the scientific accuracy and timeliness of disease prevention and control.

Key words: crop Verticillium wilt, Verticillium, ddPCR, qPCR, precise detection

WENG Hui-ting, GUO Hui-ming, CHENG Hong-mei, LI Jun, ZHANG Chao, LIU Hai-yang, SU Xiao-feng. Establishment and Application of Droplet Digital PCR Detection Methods for Four Major Verticillium Wilt Pathogens[J]. Biotechnology Bulletin, 2025, 41(10): 186-195.

Figures/Tables 11

Fig. 1 Phylogenetic analysis and the position of primers and probes in ITS sequencesA: Construction of the phylogenetic tree for Verticillium and other fungi; B: position of primers and probes in ITS sequence

Table 1 Primers and probes used in this study

引物名称 Primer name	序列 Sequence （5′-3′）	长度 Length （bp）
Ver1	F：GCGATATGTAGTGTGAATTG	152
	R：GCCTACAGACGTAGATCC
	P：ATGCCTGTCCGAGCGTCGTTTCA
Ver2	F：CTCTTGGCTCTAGCATCG	189
	R：GCCTACAGACGTAGATCC
	P：ATGCCTGTCCGAGCGTCGTTTCA
Ver3	F：GCAGAATTCAGTGAATCATC	203
	R：GCGGGACTCCGATGCGAGC
	P：ATGCCTGTCCGAGCGTCGTTTCA
Ver4	F：CATCGATGAAGAACGCAGCG	97
	R：TACGCAAGGAAGGGCCACG
	P：ATGCCTGTCCGAGCGTCGTTTCA
Ver5	F：CATCGATGAAGAACGCAGCG	218
	R：TACGCAAGGAAGGGCCACG
	P：ATGCCTGTCCGAGCGTCGTTTCA
Ver6	F：CCGCCGGTACATCAGTCTCT	337
	R：ACTCCGATGCGAGCTGTAA
	P：ATGCCTGTCCGAGCGTCGTTTCA

Fig. 2 Analysis via agarose gel electrophoresisM: DNA marker DL5 000; 1-4: indicate the genomic DNA of Vd, Vl, Vna, and Vaa, respectively

Table 2 Ct values in different sets of primers and probes

菌株DNA Strain DNA	DNA浓度 DNA concentration （ng/μL）	Ver1	Ver2	Ver3	Ver4	Ver5	Ver6
Vd	18.2	19.67	17.70	18.28	18.63	15.02	16.24
Vl	19	15.65	13.53	14.42	14.53	11.19	11.93
Vna	22.2	17.96	16.04	16.70	16.99	13.48	14.25
Vaa	23.8	17.19	15.14	15.85	16.13	12.49	14.07
阴性对照 Negative control	0	Un.	37	37	Un.	37	37

Table 3 Specific analysis of primers/probe

植物病原体 Plant pathogen	C_t值 C_t value
植物病原体 Plant pathogen	Ver2	Ver3	Ver5	Ver6
稻瘟病菌 Magnaporthe oryzae	Un.	Un.	Un.	Un.
玉米小斑病菌 Bipolaris maydis	Un.	Un.	Un.	Un.
玉米大斑病菌 Exserohilum turcicum	Un.	Un.	Un.	Un.
尖孢镰刀菌粘团专化型 Fusarium fujikuroi f. sp. nirenbergiae	Un.	Un.	Un.	Un.
禾谷丝核菌 Rhizoctonia solani	Un.	Un.	Un.	Un.
南方根结线虫 Meloidogyne incognita	Un.	Un.	Un.	Un.
假禾谷镰孢菌 Fusarium pseudograminearum	Un.	Un.	Un.	Un.
水稻稻曲病菌 Ustilaginoidea virens	Un.	Un.	Un.	Un.
西瓜嗜酸菌 Acidovorax citrulli	Un.	Un.	Un.	Un.
水稻白叶枯病菌 Xanthomonas oryzae pv. oryzae	Un.	Un.	Un.	Un.
丁香假单胞菌 Pseudomonas syringae	Un.	Un.	Un.	Un.
青枯菌 Ralstonia solanacearum	Un.	Un.	Un.	Un.
野油菜黄单胞菌 Xanthomonas campestris pv. campestris	Un.	Un.	Un.	Un.

Fig. 3 ddPCR plots in different sets of primers and probesA-D: Primer combinations of ddPCR tests for Ver2, Ver3, Ver5 and Ver6, respectively. A1-D1: The raindrop map of Vd; A2-D2: the raindrop map of Vl; A3-D3: the raindrop pattern of Vna; A4-D4: the raindrop pattern of Vaa

Fig. 4 ddPCR results of Vd (A), Vl (B), Vna (C) and Vaa (D) at different annealing temperaturesThe 10 ddPCR reactions are divided by yellow dotted lines, and the annealing temperature gradient is 54-62 ℃. The blue is the positive drop, and gray is the negative drop

Fig. 5 ddPCR results with different concentration of primers and probe in Vd (A), Vl (B), Vna (C), and Vaa (D)Six ddPCR reactions are divided by vertical dotted yellow lines with a primer gradient ranged from 400-600 nmol/L, and a probe gradient ranged from100-400 nmol/L. The blue is the positive drop, and gray is the negative drop

Fig. 6 Specific detection of ddPCR

Fig. 7 Test results of ddPCR sensitivityThe Vd (A), Vl (B), Vna (C) and Vaa (D) genomic DNAs were used as template. Seventy-six ddPCR reactions are divided by vertical dotted yellow lines with bacteria solution concentration from 2.1 × 10-3 ng/μL to 2.1 × 10-7 ng/μL, 1.6 × 10-3 ng/μL to 1.6 × 10-6 ng/μL, 6.9 × 10-2 ng/μL to 6.9 × 10-6 ng/μL and 3.6 × 10-3 ng/μL to 3.6 × 10-7 ng/μL, respectively. The pink line is the threshold, blue part is positive droplet and gray part is negative droplet

Table 4 Detection of 50 cotton and soil samples using qPCR and ddPCR

样本 Sample	分析 Analysis	阳性/全部（阳性率%） Positive/total（% Positive）
植物 Plant	qPCR	15/50（30%）
植物 Plant	ddPCR	22/50（44%）
土壤 Soil	qPCR	27/50（54%）
土壤 Soil	ddPCR	41/50（82%）

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