Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves
Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the le...
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| Format: | Artículo |
| Language: | Inglés |
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Springer
2020
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| Online Access: | http://hdl.handle.net/20.500.11939/6440 https://link.springer.com/article/10.1007/s10658-019-01831-x#auth-4 |
| _version_ | 1855492084470906880 |
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| author | Pereira, W. V. Bertolini, Edson Cambra, Mariano Massola Junior, N. S. |
| author_browse | Bertolini, Edson Cambra, Mariano Massola Junior, N. S. Pereira, W. V. |
| author_facet | Pereira, W. V. Bertolini, Edson Cambra, Mariano Massola Junior, N. S. |
| author_sort | Pereira, W. V. |
| collection | ReDivia |
| description | Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD. |
| format | Artículo |
| id | ReDivia6440 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Springer |
| publisherStr | Springer |
| record_format | dspace |
| spelling | ReDivia64402025-04-25T14:47:06Z Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves Pereira, W. V. Bertolini, Edson Cambra, Mariano Massola Junior, N. S. Postbloom fruit drop Nested PCR qPCR H20 Plant diseases Citrus sinensis PCR Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD. 2020-05-14T10:56:46Z 2020-05-14T10:56:46Z 2019 article publishedVersion Pereira, W. V., Bertolini, E., Cambra, M., & Junior, N. M. (2019). Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves. European Journal of Plant Pathology, 155(4), 1047-1059. 1573-8469 http://hdl.handle.net/20.500.11939/6440 10.1007/s10658-019-01831-x https://link.springer.com/article/10.1007/s10658-019-01831-x#auth-4 en Atribución-NoComercial-SinDerivadas 3.0 España Atribución-NoComercial-SinDerivadas 3.0 España http://creativecommons.org/licenses/by-nc-nd/3.0/es/ Springer electronico |
| spellingShingle | Postbloom fruit drop Nested PCR qPCR H20 Plant diseases Citrus sinensis PCR Pereira, W. V. Bertolini, Edson Cambra, Mariano Massola Junior, N. S. Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves |
| title | Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves |
| title_full | Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves |
| title_fullStr | Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves |
| title_full_unstemmed | Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves |
| title_short | Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves |
| title_sort | multiplex real time pcr for detection and quantification of colletotrichum abscissum and c gloeosporioides on citrus leaves |
| topic | Postbloom fruit drop Nested PCR qPCR H20 Plant diseases Citrus sinensis PCR |
| url | http://hdl.handle.net/20.500.11939/6440 https://link.springer.com/article/10.1007/s10658-019-01831-x#auth-4 |
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