scholarly journals Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Xue Li ◽  
Liqian Guo ◽  
Mengmeng Guo ◽  
Duo Qi ◽  
Xueping Zhou ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Large Scale ◽  
Sensitivity Analyses ◽  
Detection Methods ◽  
Tomato Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plant Samples ◽  
Tissue Print ◽  
Dot Elisa

AbstractIn recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato

Plant Disease ◽  
2021 ◽  
Vol 105 (4) ◽  
pp. 832-839
Author(s):  
Wanqin He ◽  
Deqing Huang ◽  
Jiayu Wu ◽  
Xue Li ◽  
Yajuan Qian ◽  
...  
Keyword(s):  
Sweet Potato ◽  
Cell Lines ◽  
Immunosorbent Assay ◽  
Root Rot ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dickeya Dadantii ◽  
Crude Extracts ◽  
Tissue Print ◽  
Hybridoma Cell Lines ◽  
Dot Elisa

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

Infection of red spruce, black spruce, and balsam fir seedlings with tomato mosaic virus

1992 ◽  
Vol 22 (7) ◽  
pp. 919-924 ◽  
Author(s):  
Volker Jacobi ◽  
John D. Castello
Keyword(s):  
Mosaic Virus ◽  
Immunoelectron Microscopy ◽  
Immunosorbent Assay ◽  
Black Spruce ◽  
Red Spruce ◽  
Balsam Fir ◽  
Tomato Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Spread ◽  
Root Extracts

Red spruce (Picearubens Sarg.), black spruce (Piceamariana (Mill.) B.S.P.), and balsam fir (Abiesbalsamea (L.) Mill.) seedlings were root inoculated with tomato mosaic virus, potted, and maintained in a cold frame. The virus was detected by enzyme-linked immunosorbent assay in root extracts of several inoculated seedlings of each species 6–12 months postinoculation, and in root extracts of approximately 35–40% of both inoculated and noninoculated seedlings of each species 12–18 months postinoculation. Virus spread apparently occurred from the roots of infected to noninfected seedlings within the cold frame. The presence of virus in root extracts was confirmed by immunoelectron microscopy. The virus was not detected in the needles of any seedling at any time by either enzyme-linked immunosorbent assay or immunoelectron microscopy. A transitory needle chlorosis was observed in approximately 14% of the inoculated black spruce and 30% of the inoculated red spruce seedlings, but tomato mosaic virus was not detected in all plants with symptoms.

scholarly journals Susceptibility of Vegetatively Propagated Petunia to Tobamovirus Infection and Its Possible Control

HortScience ◽  
1999 ◽  
Vol 34 (2) ◽  
pp. 292-293 ◽  
Author(s):  
J. Cohen ◽  
Noga Sikron ◽  
S. Shuval ◽  
A. Gera
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Phosphate Buffer ◽  
Sodium Phosphate ◽  
Tomato Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Inoculation ◽  
Sodium Phosphate Buffer ◽  
Local Lesions ◽  
Systemic Symptoms

In this study, 18 Petunia ×hybrida Hort. Volm.-Andr. cultivars were mechanically inoculated with the tobamoviruses tobacco mosaic (TMV) or tomato mosaic virus (ToMV) (20 μg·L-1 in 0.05 m sodium phosphate buffer). One and 2 weeks post-inoculation (PI), inoculated and noninoculated upper leaves were harvested and assayed for TMV infection using enzyme-linked immunosorbent assay (ELISA). Local lesions developed on inoculated leaves of 16 cultivars 3-5 days PI. A total of 11 and 16 of the cultivars developed systemic symptoms characteristic of tobamovirus infection 2 weeks after inoculation with TMV and ToMV, respectively. All cultivars were positive in ELISA tests. Large amounts of virus were recovered from the upper, noninoculated leaves of all cultivars, including symptomless plants. Up to 95% infection by TMV occurred when a sterilized knife was passed through an infected shoot of petunia prior to its being used to remove cuttings from healthy petunia plants. Heat sterilization of knives and/or treatment with 2.8 g·L-1 sodium troclosene was very effective in controlling TMV transmission.

scholarly journals Genome Sequence of a Tomato-Infecting Tomato Mosaic Virus Isolate from Zimbabwe

2018 ◽  
Vol 6 (11) ◽  
Author(s):  
Charles Karavina ◽  
Jacques D. Ibaba ◽  
Augustine Gubba
Keyword(s):  
Next Generation Sequencing ◽  
Mosaic Virus ◽  
Genome Sequence ◽  
Immunosorbent Assay ◽  
Virus Isolate ◽  
De Novo ◽  
Lateral Flow ◽  
Tomato Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Generation Sequencing

ABSTRACT A tomato-infecting tomato mosaic virus (ToMV) isolate was detected in Zimbabwe using lateral flow kits and double-antibody sandwich enzyme-linked immunosorbent assay. Next-generation sequencing and de novo assembly were subsequently performed to determine its genome sequence. The ToMV genome of the Zimbabwe isolate is the second to be reported in Africa.

scholarly journals Several viral diseases of Lycopersicon esculentum circulating in Ukraine

2014 ◽  
Vol 68 (3) ◽  
pp. 96-98
Author(s):  
Al Dalain Emad ◽  
A. Bysov ◽  
O. Shevchenko ◽  
T. Shevchenko ◽  
V. Polischuk
Keyword(s):  
Lycopersicon Esculentum ◽  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Plant Viruses ◽  
Tomato Mosaic Virus ◽  
Viral Diseases ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tomato Plants ◽  
Lycopersicon Esculentum Mill

This paper describes detection of some typical plant viruses infecting Lycopersicon esculentum Mill. plants in Ukraine. Diagnostics using enzyme-linked immunosorbent assay (ELISA) confirmed presence of antigens of viruses belonging to Tobamovirus (PMMoV, ToMV), Cucumovirus (CMV) and Tobravirus (TRV) genera in sap of tomato plants. When studying viral diseases of tomatoes, monoinfection was shown to be prevalent. Tomato mosaic virus (ToMV) was most common.

scholarly journals Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Saengsoon Charoenvilaisiri ◽  
Channarong Seepiban ◽  
Mallika Kumpoosiri ◽  
Sombat Rukpratanporn ◽  
Nuchnard Warin ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Mosaic Disease ◽  
Cassava Mosaic Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sri Lankan ◽  
Stem Cuttings ◽  
Field Samples ◽  
Cassava Stem ◽  
Cassava Mosaic Virus

Abstract Background Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). Methods Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. Results A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. Conclusions Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Mechanization of the enzyme-linked immunosorbent assay (ELISA) for large scale screening of sera

1977 ◽  
Vol 16 (4) ◽  
pp. 351-359 ◽  
Author(s):  
E.J. Ruitenberg ◽  
J.A. van Amstel ◽  
B.J.M. Brosi ◽  
P.A. Steerenberg
Keyword(s):  
Immunosorbent Assay ◽  
Large Scale ◽  
Enzyme Linked Immunosorbent Assay ◽  
Large Scale Screening

scholarly journals Avaliação da reação de genótipos de alface (Lactuca sativa L.) ao Lettuce mosaic virus (LMV)

Bragantia ◽  
2007 ◽  
Vol 66 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Rosa Maria Chung ◽  
Joaquim Adelino de Azevedo Filho ◽  
Addolorata Colariccio
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Lactuca Sativa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lettuce Mosaic Virus ◽  
Lactuca Sativa L

O trabalho teve como meta avaliar a reação de 18 linhagens superiores do programa de melhoramento de alface (Lactuca sativa L.) do IAC e de seis cultivares comerciais, ao Lettuce mosaic virus (LMV). Em condições de campo, na região de Atibaia (SP), foram observados sintomas de mosaico, nanismo e necrose em plantas das cultivares Rider, 'Karla H25' e Hortência. O vírus presente nos isolados foi identificado por meio de inoculação mecânica em plantas indicadoras e diferenciadoras e de testes sorológicos de Plate Trapped Antigen-Enzyme linked-immunosorbent assay (PTA-ELISA). Nas amostras avaliadas, identificou-se a espécie LMV pelo PTA-ELISA e do patotipo IV pela reação nas hospedeiras diferenciais. Para a avaliação do comportamento dos genótipos de alface, foi empregado o LMV isolado 'Karla H25'. Foram submetidos à inoculação 24 genótipos de alface empregando-se, como controle positivo, a alface 'White Boston' por sua suscetibilidade ao LMV. O delineamento experimental foi inteiramente ao acaso e analisado pelo teste do qui-quadrado. Detectaram-se genótipos com comportamento de suscetibilidade e de tolerância. Nos genótipos 3 e 4, foram observadas plantas com comportamento de tolerância ao LMV isolado 'Karla H25', enquanto nos demais genótipos, constataram-se plantas com comportamento suscetível. O plantio de cultivares tolerantes pode ser uma alternativa aos prejuízos causados pela infecção pelo LMV com conseqüente diminuição do uso de produtos químicos para o controle dos afídeos vetores.

scholarly journals First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia

Plant Disease ◽  
2021 ◽  
Author(s):  
Ahmed Sabra ◽  
Mohammed Ali Al Saleh ◽  
I. M. Alshahwan ◽  
Mahmoud A. Amer
Keyword(s):  
Saudi Arabia ◽  
Mosaic Virus ◽  
Solanum Lycopersicum ◽  
Tomato Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Pcr Products ◽  
Dark Green ◽  
Leaf Symptoms

Tomato (Solanum lycopersicum L.) is the most economically important member of family Solanaceae and cultivated worldwide and one of the most important crops in Saudi Arabia. The aim of this study is screening of the most common viruses in Riyadh region and identified the presence of tomato brown rugose fruit virus (ToBRFV) in Saudi Arabia. In January 2021, unusual fruit and leaf symptoms were observed in several greenhouses cultivating tomatoes commercially in Riyadh Region, Saudi Arabia. Fruit symptoms showed irregular brown spots, deformation, and yellowing spots which render the fruits non-marketable, while the leaf symptoms included mottling, mosaic with dark green wrinkled and narrowing. These plants presented the symptoms similar to those described in other studies (Salem et al., 2015, Luria et al., 2017). A total 45 Symptomatic leaf samples were collected and tested serologically against suspected important tomato viruses including: tomato chlorosis virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato chlorotic spot virus, tomato aspermy virus, tomato bushy stunt virus, tomato black ring virus, tomato ringspot virus, tomato mosaic virus, pepino mosaic virus and ToBRFV using Enzyme linked immunosorbent assay (ELISA) test (LOEWE®, Biochemica, Germany), according to the manufacturers' instructions. The obtained results showed that 84.4% (38/45) of symptomatic tomato samples were infected with at least one of the detected viruses. The obtained results showed that 55.5% (25/45) of symptomatic tomato samples were found positive to ToBRFV, three out of 25 samples (12%) were singly infected, however 22 out of 45 (48.8%) had mixed infection between ToBRFV and with at least one of tested viruses. A sample with a single infection of ToBRFV was mechanically inoculated into different host range including: Chenopodium amaranticolor, C. quinoa, C. album, C. glaucum, Nicotiana glutinosa, N. benthamiana, N. tabacum, N. occidentalis, Gomphrena globosa, Datura stramonium, Solanum lycopersicum, S. nigrum, petunia hybrida and symptoms were observed weekly and the systemic presence of the ToBRFV was confirmed by RT-PCR and partial nucleotide sequence. A Total RNA was extracted from DAS-ELISA positive samples using Thermo Scientific GeneJET Plant RNA Purification Mini Kit. Reverse transcription-Polymerase chain reaction (RT-PCR) was carried out using specific primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 bp of Open Reading Frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp). (Luria et al. 2017). RT-PCR products were analyzed using 1.5 % agarose gel electrophoresis. RT-PCR products were sequenced in both directions by Macrogen Inc. Seoul, South Korea. Partial nucleotide sequences obtained from selected samples were submitted to GenBank and assigned the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 98.99 % to 99.50 % among them and 98.87-99.87 % identity with ToBRFV isolates from Palestine (MK881101 and MN013187), Turkey (MK888980, MT118666, MN065184, and MT107885), United Kingdom (MN182533), Egypt (MN882030 and MN882031), Jordan (KT383474), USA (MT002973), Mexico (MK273183 and MK273190), Canada (MN549395) and Netherlands (MN882017, MN882018, MN882042, MN882023, MN882024, and MN882045). To our knowledge, this is the first report of occurrence of ToBRFV infecting tomato in Saudi Arabia which suggests its likely introduction by commercial seeds from countries reported this virus and spread in greenhouses through mechanical means. The author(s) declare no conflict of interest. Keywords: Tomato brown rugose fruit virus, tomato, ELISA, RT-PCR, Saudi Arabia References: Luria N, et al., 2017. PLoS ONE 12(1): 1-19. Salem N, et al., 2015. Archives of Virology 161(2): 503-506. Fig. 1. Symptoms caused by ToBRFV showing irregular brown spots, deformation, yellowing spots on fruits (A, B, C) and bubbling and mottling, mosaic with dark green wrinkled and narrowing on leaf (D).

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