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

doi:10.3969/j.issn.1004-1524.20240150

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (5): 1072-1081.DOI: 10.3969/j.issn.1004-1524.20240150

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S436.3

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.

Figures/Tables 12

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

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

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.

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.

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

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

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

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.

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.

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.

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.

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

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