四种主要黄萎病致病菌微滴数字PCR检测方法的建立及应用

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

摘要/Abstract

摘要：

目的建立一种同时检测4种黄萎病致病菌的微滴式数字PCR（ddPCR）方法，为及时、准确定量监测该病原真菌的生长动态，进行早期诊断和风险评估奠定基础。方法通过比对4种黄萎病致病菌大丽轮枝菌（Verticillium dahliae, Vd）、长孢轮枝菌（V. longisporum, Vl）、非苜蓿轮枝菌（V. nonalfalfae, Vna）和黑白轮枝菌（V. albo-atrum, Vaa）的ITS（Internal transcribed spacer）序列（Vd，KY039312.1；Vl，KX058040.1；Vna，KT362917.1和Vaa，MH856937.1），选取保守区域设计引物和探针。结合微滴式数字PCR和实时荧光定量PCR（qPCR）筛选最佳引物，优化ddPCR最佳反应体系，并测定方法的特异性与灵敏度。结果建立方法的最佳引物/探针组为Ver5；最佳退火温度为58 ℃，引物浓度为500 nmol/L和探针浓度为250 nmol/L。特异性检测结果显示，该方法能够特异性识别4种黄萎病致病菌，对包括7种真菌和6种细菌在内的非靶标微生物无交叉扩增；对于Vd、Vl、Vna、Vaa的检测限分别为2.1×10^-6、1.6×10^-6、6.9×10^-4、3.6×10^-5 ng/μL。选取50个棉花和50份土壤样品展开检测分析，结果表明相较于qPCR，ddPCR方法的检出率呈现出显著优势，且检测灵敏度分别提高了46%和51%。结论建立的ddPCR方法检测4种黄萎病致病菌特异性强，灵敏度高，稳定可靠，为黄萎病的精准检测提供了重要的技术手段。该方法有利于海关检验检疫与植物病虫害监管等领域，提高病害防控的科学性与时效性。

关键词: 作物黄萎病, 轮枝菌, 数字PCR, 实时荧光定量, 精准检测

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

翁慧婷, 郭惠明, 程红梅, 李君, 张超, 刘海洋, 苏晓峰. 四种主要黄萎病致病菌微滴数字PCR检测方法的建立及应用[J]. 生物技术通报, 2025, 41(10): 186-195.

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.

图/表 11

图1 进化树分析及引物和探针在ITS序列中的位置A：轮枝菌属及其他真菌的进化树构建； B：引物和探针在ITS序列中的位置

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

表1 本研究所使用的引物和探针

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

图2 琼脂糖凝胶电泳分析M：DNA marker DL5 000；1-4：分别为Vd、Vl、Vna、Vaa基因组DNA

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

表2 不同引物和探针的Ct值

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

表3 引物/探针特异性分析

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.

图3 不同引物和探针的ddPCR检测结果A-D：分别为Ver2、Ver3、Ver5和Ver6进行ddPCR试验的引物组合展示。A1-D1：Vd液滴图；A2-D2：Vl液滴图；A3-D3：Vna液滴图，A4-D4：Vaa液滴图

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

图4 Vd（A）、Vl（B）、Vna（C）、Vaa（D）不同退火温度下的ddPCR结果10个ddPCR反应用黄色纵虚线划分，退火温度梯度为54-62 ℃。蓝色是阳性微滴，灰色是阴性微滴

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

图5 Vd（A）、Vl（B）、Vna（C）和Vaa（D）不同浓度引物和探针的ddPCR结果6个ddPCR反应用垂直黄色虚线划分，引物梯度范围为400-600 nmol/L，探针梯度范围为100-400 nmol/L。蓝色为阳性微滴，灰色为阴性微滴

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

图6 ddPCR的特异性检测1：Vd；2：Vl；3：Vna；4：Vaa；5：M. oryzae；6：B. maydis；7：E. turcicum；8：Foc；9：R. cerealis；10：M. incognita；11：F. pseudograminearum；12：U. virens；13：A. citrulli；14：Xoo；15：P. syringae；16：R. solanacearum；17：Xcc

Fig. 6 Specific detection of ddPCR

图7 ddPCR灵敏度检测结果以Vd（A）、Vl（B）、Vna（C）和Vaa（D）基因组DNA为模板。76个ddPCR反应用黄虚线垂直划分，菌液浓度分别为2.1 × 10-3 ng/μL-2.1 × 10-7 ng/μL、1.6 × 10-3 ng/μL-1.6 × 10-6 ng/μL、6.9 × 10-2 ng/μL-6.9 × 10-6 ng/μL和3.6 × 10-3 ng/μL-3.6 × 10-7 ng/μL。粉红色的线是阈值，蓝色部分为阳性液滴，灰色部分为阴性液滴

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

表4 qPCR和ddPCR检测50份棉花和土壤样本

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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