Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification ...Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was established in this study. Based on the sequence of MCMV coat protein coding gene, 3 sets of primers were designed and specificity test showed that the second set of primers was specific to MCMV, Similar sensitivities were observed on RT-LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescence under daylight allows naked easy detection of the amplification of MCMV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCMV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter. The method is rapid, specific, sensitive without the need for complicated equipment, and is suitable for rapid field detection of MCMV.展开更多
Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (...Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.展开更多
Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting ma...Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting maize,which caused devastating damage in the African region recently.MLND is induced by co-infection of maize chlorotic mottle virus and one of several cereal-infecting viruses in the Potyviridae family,with the symptoms ranging from chlorotic mottle to plant death at different infection stages.Integrated pest management for MLND needs strengthening detection,focusing on prevention and effective control.Early detection system of MLND has been successfully established by serological methods,nucleic acid-based methods,next-generation sequencing,etc.The practices,such as using certified seeds,sanitary measures,crop rotation,tolerant or resistant varieties etc.,have been considered as the effective,economical and eco-friendly way to prevent and control MLND.展开更多
Imported maize seeds were planted in a quarantine greenhouse and two seedlings with chlorotic mottle symptoms were observed.The ELISA results showed that the two seedlings reacted positively with antibody against Maiz...Imported maize seeds were planted in a quarantine greenhouse and two seedlings with chlorotic mottle symptoms were observed.The ELISA results showed that the two seedlings reacted positively with antibody against Maize chlorotic mottle virus(MCMV).The positive samples were further identified by RT-PCR method and the 711 bp size target bands were amplified specifically.The similarity range of nucleotide sequence of both RT-PCR product and six MCMV coat protein genes was 97%-99%.The amino acid sequence homology was 97.88%-99.89%.Based on the above results,the virus was identified as MCMV.It was the first time that MCMV was intercepted from imported maize seeds.展开更多
文摘Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was established in this study. Based on the sequence of MCMV coat protein coding gene, 3 sets of primers were designed and specificity test showed that the second set of primers was specific to MCMV, Similar sensitivities were observed on RT-LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescence under daylight allows naked easy detection of the amplification of MCMV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCMV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter. The method is rapid, specific, sensitive without the need for complicated equipment, and is suitable for rapid field detection of MCMV.
基金supported by the National Natural Science Foundation of China (No. 31272015)the Ministry of Education of China (No. 313052)the Zhejiang Provincial Natural Science Foundation of China (No. Z3090039)
文摘Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.
基金the National Natural Science Foundation of China(31930089)the Food and Agriculture Organization of the United Nations,the International Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAASTIP)(CAASZDRW202108)the Central Public-interest Scientific Institution Basal Research Fund,China(Y2022GH05).
文摘Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting maize,which caused devastating damage in the African region recently.MLND is induced by co-infection of maize chlorotic mottle virus and one of several cereal-infecting viruses in the Potyviridae family,with the symptoms ranging from chlorotic mottle to plant death at different infection stages.Integrated pest management for MLND needs strengthening detection,focusing on prevention and effective control.Early detection system of MLND has been successfully established by serological methods,nucleic acid-based methods,next-generation sequencing,etc.The practices,such as using certified seeds,sanitary measures,crop rotation,tolerant or resistant varieties etc.,have been considered as the effective,economical and eco-friendly way to prevent and control MLND.
文摘Imported maize seeds were planted in a quarantine greenhouse and two seedlings with chlorotic mottle symptoms were observed.The ELISA results showed that the two seedlings reacted positively with antibody against Maize chlorotic mottle virus(MCMV).The positive samples were further identified by RT-PCR method and the 711 bp size target bands were amplified specifically.The similarity range of nucleotide sequence of both RT-PCR product and six MCMV coat protein genes was 97%-99%.The amino acid sequence homology was 97.88%-99.89%.Based on the above results,the virus was identified as MCMV.It was the first time that MCMV was intercepted from imported maize seeds.
