蔬菜病毒不同检测技术灵敏度的比较研究

doi:10.3969/j.issn.1004-1524.20240150

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (5): 1072-1081.DOI: 10.3969/j.issn.1004-1524.20240150

蔬菜病毒不同检测技术灵敏度的比较研究

陈伟¹^,²(), 姚洁³^,⁴, 冯明峰², 李帅², 蒋西子², 蒋磊²^,⁵^,⁶^,^*(), 江彤²^,⁵^,⁶^,^*()

1.安徽辉隆集团农业发展有限责任公司,安徽合肥 230094
2.安徽农业大学植物保护学院,安徽合肥 230036
3.亳州职业技术学院药学院,安徽亳州 236800
4.安徽中药材种植联合研究中心,安徽亳州 236800
5.作物有害生物综合治理安徽省重点实验室,安徽合肥 230036
6.植物病虫害生物学与绿色防控安徽普通高校重点实验室,安徽合肥 230036

收稿日期:2024-02-19 出版日期:2025-05-25 发布日期:2025-06-11
作者简介:陈伟(1981—),女,安徽五河人,硕士研究生,主要从事植物病毒学研究。E-mail: 54324282@qq.com
通讯作者: *江彤,E-mail: jiangtong4650@sina.com;
蒋磊,E-mail: jianglei062x@ahau.edu.cn
基金资助:
安徽省高等学校科研计划项目(2022AH050920);安徽中药材种植联合研究中心科研项目(yjzx2023013);国家自然科学基金(32472518)

Comparative study on the sensitivity of different detection techniques for major viruses in vegetables

CHEN Wei¹^,²(), YAO Jie³^,⁴, FENG Mingfeng², LI Shuai², JIANG Xizi², JIANG Lei²^,⁵^,⁶^,^*(), JIANG Tong²^,⁵^,⁶^,^*()

1. Anhui Huilong Group Agricultural Development Co.,Ltd., Hefei 230094, China
2. School of Plant Protection, Anhui Agricultural University, Hefei 230036, China
3. School of Pharmacy, Bozhou Vocational and Technical College, Bozhou 236800, Anhui, China
4. Joint Research Center for Chinese Herbal Medicine of Anhui, Bozhou 236800, Anhui, China
5. Anhui Province Key Laboratory of Integrated Pest Management on Crops, Hefei 230036, China
6. Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, Hefei 230036, China

Received:2024-02-19 Online:2025-05-25 Published:2025-06-11

摘要/Abstract

摘要： 为了明确蔬菜病毒种类,探索快速、灵敏、低成本、高通量的病毒检测方法。将10种蔬菜病毒,烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)、蚕豆萎蔫病毒(Broad bean wilt virus,BBWV)、芜菁花叶病毒(Turnip mosaic virus,TuMV)、小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、番茄褪绿病毒(Tomato chlorosis virus,ToCV)、马铃薯Y病毒(Potato virus Y,PVY)和番茄环纹斑点病毒(Tomato zonate spot virus,TZSV)的cp基因质粒DNA顺次固定在同一张硝酸纤维素膜(NC膜)上,根据10种蔬菜病毒的cp基因序列制备地高辛(DIG)标记的特异性探针,利用dot-Southern blot方法一次性检测10种病毒。再采用dot-Southern blot、dot-Northern blot、PCR和ELISA方法检测TMV,比较不同检测方法的灵敏度。结果发现,dot-Southern blot方法可在同一张NC膜上一次性检出10种病毒的cp基因质粒DNA。dot-Southern blot检测TMV cp基因质粒DNA和cp基因cDNA的最低浓度分别为5×10^-2 ng·μL^-1和5×10^-1 ng·μL^-1,dot-Northern blot检测感染TMV蔬菜样本总RNA的最低浓度为5×10^-2 ng·μL^-1,PCR检测TMV cp基因质粒DNA的最低浓度为5×10^-3 ng·μL^-1,RT-PCR检测TMV cDNA的最低浓度为5×10^-2 ng·μL^-1,dot-ELISA和TAS-ELISA检测感染TMV蔬菜病汁液的最低浓度为9.8 ng·μL^-1和78 ng·μL^-1。说明dot-Southern blot能够一次性检出多种病毒,可用于蔬菜病毒复合侵染检测。dot-Southern blot、dot-Northern blot、RT-PCR和PCR等核酸检测方法的灵敏度相对较高,而ELISA检测方法的灵敏度相对较低。

关键词: 蔬菜病毒, 检测方法, 灵敏度, dot-Southern blot

Abstract:

To clarify the species of vegetable viruses, this study was designed to explore rapid, sensitive, low-cost and high-throughput virus detection methods. The plasmid DNA of cp genes of ten vegetable viruses containing Tobacco mosaic virus (TMV), Cucumber mosaic virus(CMV), Cucumber green mottle mosaic virus (CGMMV), Tomato mosaic virus (ToMV), Broad bean wilt virus(BBWV), Turnip mosaic virus(TuMV), Zucchini yellow mosaic virus(ZYMV), Tomato chlorosis virus(ToCV), Potato virus Y(PVY) and Tomato zonate spot virus(TZSV) were fixed on a same nitrocellulose membrane(NC membrane) in order. The DIG-labeled specific probes based on the sequences of the cp genes of these ten vegetable viruses were separately prepared, and these ten viruses could be simultaneously detected by dot-Southern blot. Thereafter, TMV was detected by dot-Southern blot, dot-Northern blot, PCR, and ELISA methods to compare the sensitivities of the diverse methods. The results revealed that the plasmid DNA of cp genes of ten vegetable viruses could be detected on the same NC membrane by dot-Southern simultaneously. The minimum detectable concentration (MDC) of plasmid DNA and the cDNA of TMV-cp gene by dot-Southern was 5 ×10^-2 ng·μL^-1 and 5×10^-1 ng·μL^-1 respectively. The MDC of the total RNA extracted from TMV-infected vegetable by dot-Northern blot was 5×10^-2 ng·μL^-1. The MDC of TMV plasmid DNA by PCR and TMV cDNA RT-PCR approaches was 5×10^-3 ng·μL^-1 and 5×10^-2 ng·μL^-1 respectively. The MDC of extracted diseased saps from TMV-infected vegetable samples by dot-ELISA and TAS-ELISA assays was 9.8 ng·μL^-1 and 78 ng·μL^-1, respectively. The results indicated that the various viruses could be detected by dot-Southern blot in this experiment at a time, which should be adaptable to detect the mixed infection of diverse vegetable viruses. The nucleic acid detection methods such as dot-Southern blot, dot-Northern blot, RT-PCR and PCR have higher sensitivity, but the sensitivity of the ELISA detection method is relatively low.

Key words: vegetable virus, detection method, sensitivity, dot-Southern blot

中图分类号:

S436.3

陈伟, 姚洁, 冯明峰, 李帅, 蒋西子, 蒋磊, 江彤. 蔬菜病毒不同检测技术灵敏度的比较研究[J]. 浙江农业学报, 2025, 37(5): 1072-1081.

CHEN Wei, YAO Jie, FENG Mingfeng, LI Shuai, JIANG Xizi, JIANG Lei, JIANG Tong. Comparative study on the sensitivity of different detection techniques for major viruses in vegetables[J]. Acta Agriculturae Zhejiangensis, 2025, 37(5): 1072-1081.

图/表 12

表1 检测10种蔬菜病毒cp基因特异性探针的PCR扩增引物

Table 1 List of primers used to detect the specific probe PCR fragments of cp genes of ten vegetable-infecting viruses

基因名称 Primer name	正向引物序列 Forward primer sequence (5'-3')	反向引物序列 Reverse primer sequence (5'-3')	序列长度 Fragment size/bp
TMV-CP	ACAGTATCACTACTCCATCTCAG	GTGTTTGAAACTGATTTCCTAAG	108
TuMV-CP	CAATGGTTTAATGGTCTGGTGC	GCGTGGTCAATGAGCGGTTTG	120
TSWV-N	TCAAGCAAGTTCTGCGAGTTTTG	AGCTCCAGCAATCCTAATGC	120
ZYMV-CP	GTGAACTGGCACGCTACCTAC	TTCTTTGTAGCCCCAGCGTCTG	118
TZSV-N	TCAAGCAAGTTCTGCGAGTTTTG	CAATTGAAGCAGATGGAGACTG	111
BBWV-CP	GGAGTATACCAAGCGCTTGATAG	TCTGAATGTAAGCTCTCCTTCC	126
PVY-CP	CAACCTCGACTTTTCGGGTTG	CTACATCACATGTTCTTGACTCC	126
CMV-CP	ATGGACAAATCTGAATCAACCAG	GCGAAAGCTGTTGCGACAG	121
CGMMV-CP-F	ATGGCTTACAATCCGATCACAC	ATCTCTTCCCGCTTGAGTCTG	129
ToCV-CP	ATGGAGAACGATGCTGTTACAA	GGAATGAGAGATGTCGAATCG	134

图1 10种蔬菜病毒cp基因质粒DNA检测排列示意图

Fig.1 Detection arrangement diagram of plasmid DNA of cp genes from ten vegetable-infecting viruses

图2 蔬菜病毒dot-Southern blot和dot-Northern blot检测流程图白色圆圈“○”代表点样位置,每点加2 μL样品,黑色圆圈“●”代表荧光扫描结果。

Fig.2 Schematic representation of vegetable-infecting viruses detected by dot-Southern blot and dot-Northern blot assays The white circle “○” represents the sample position, 2 μL sample is added to each spot. The black spot “●” represents the fluorescence scanning results.

图3 蔬菜病毒cp基因特异性探针的dot-Southern blot检测 a~j,分别利用TMV、CMV、ToMV、BBWV、TuMV、CGMMV、ZYMV、ToCV、PVY和TZSV cp基因特异性探针的检测结果;k,10种蔬菜病毒cp基因混合探针的检测结果。

Fig.3 Analysis of the specific probe of cp genes for vegetable-infecting viruses by dot-Southern blot a-j, Detection results of TMV, CMV, ToMV, BBWV, TuMV, CGMMV, ZYMV, ToCV, PVY and TZSV-specific cp gene probes, respectively; k, Detection results of mixed probe for cp gene of 10 vegetable viruses.

图4 dot-Southern blot检测梯度稀释的TMV质粒DNA 1~7, 5, 5×10-1, 5×10-2, 5×10-3, 5×10-4, 5×10-5, 5×10-6 ng·μL-1.

Fig.4 Analysis of the gradient diluted TMV plasmid DNA by dot-Southern blot

图5 dot-Northern blot检测梯度稀释的TMV病样总RNA 1~7, 5×102, 5×101, 5, 5×10-1, 5×10-2, 5×10-3, 5×10-4 ng·μL-1.

Fig.5 Analysis of the gradient dilutions of total RNA from TMV-infected samples by dot-Northern blot

图6 dot-Southern blot检测梯度稀释的TMV cDNA 1~7, 5×102, 5×101, 5, 5×10-1, 5×10-2, 5×10-3, 5×10-4 ng·μL-1.

Fig.6 Analysis of the gradient diluted TMV cDNA by dot-Southern blot

图7 PCR检测梯度稀释的TMV cp基因质粒DNA M,DNA marker; 1~9,5×102、5×101 、5、5×10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6 ng·μL-1; 10,阳性对照;11,阴性对照。

Fig.7 Analysis of the gradient diluted TMV plasmid DNA by PCR assay M, DNA marker; 1-9, 5×102, 5×101, 5, 5×10-1, 5×10-2, 5×10-3, 5×10-4, 5×10-5, 5×10-6 ng·μL-1; 10, Positive control; 11, Negative control.

图8 RT-PCR检测梯度稀释的TMV cDNA M,DNA marker; 1~11,5×102、5×101 、5、5×10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8 ng·μL-1; 12,阳性对照; 13,阴性对照。

Fig.8 Analysis the gradient diluted TMV cDNA detected by RT-PCR assay M, DNA marker; 1-11,5×102, 5×101, 5, 5×10-1, 5×10-2, 5×10-3, 5×10-4, 5×10-5, 5×10-6, 5×10-7, 5×10-8 ng·μL-1; 12,Positive control; 13, Negative control.

图9 dot-ELISA检测梯度稀释的TMV病汁液 1~6,病汁液分别稀释320倍、640倍、1 280倍、2 560倍、5 120倍和10 240倍; 7,健康蔬菜汁液; 8,提取缓冲液。

Fig.9 The gradient dilutions of diseased saps from the TMV-infected plant samples by dot-ELISA assay 1-6, The disease juice was diluted 320, 640, 1 280, 2 560, 5 120 and 10 240 times, respectively; 7, Healthy vegetable saps; 8, Extract buffer.

图10 TAS-ELISA检测梯度稀释的TMV病汁液 D405为2孔平均值。样品D405和阴性对照D405的比值大于2.1,样品被认为是阳性;HL,健康样本;CK,阴性对照。

Fig.10 Analysis of the gradient dilutions of diseased saps from the TMV-infected plant samples by TAS-ELISA assay D405 is the average value of two holes. D405 of samples/D405 of negative control>2.1, the samples are regard as positive; HL, Healthy sample; CK, Negative control.

表2 6种方法检测TMV的灵敏度比较

Table 2 Comparison of sensitivity of six detection methods for TMV

检测对象 Detection samples	检测方法 Detection methods	最低检测浓度 Minimum concentrations/ (ng·μL^-1)	灵敏度 Sensitivity
cp基因	dot-Southern blot	5×10^-2
质粒DNA	PCR	5×10^-3	+
cp gene plasmid DNA
病样总RNA	dot-Northern blot	5×10^-2
Diseased sample total RNA
病样cDNA	dot-Southern blot	5×10^-1	+++
Diseased sample cDNA	RT-PCR	5×10^-2
病汁液	dot-ELISA	9.8	++
Diseased sap	TAS-ELISA	78	+

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蔬菜病毒不同检测技术灵敏度的比较研究

Comparative study on the sensitivity of different detection techniques for major viruses in vegetables

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