Early diagnosis of a Mexican variant of Papaya meleira virus (PMeV-Mx) by RT-PCR.
Papaya meleira disease was identified in Brazil in the 1980s. The disease is caused by a double-stranded RNA virus known as Papaya meleira virus (PMeV), which has also been recently reported in Mexico. However, previously reported PMeV primers failed to diagnose the Mexican form of the disease. A genomic approach was used to identify sequences of the Mexican virus isolate, referred here to as PMeV-Mx, to develop a diagnostic method. A mini cDNA library was generated using total RNA from the latex of fruits; this RNA was also sequenced using the Illumina platform. Sequences corresponding to the previously reported 669-base pair sequence for PMeV from Brazil (PMeV-Br) were identified within the PMeV-Mx genome, exhibiting 79-92% identity with PMeV-Br. In addition, a new sequence of 1154-base pairs encoding a putative RNA-dependent RNA polymerase was identified in PMeV-Mx. Primers designed against this sequence detected both virus isolates, 2 amplicons of 173 and 491 base pairs from PMeV-Br and PMeV-Mx, and shared 100 and 98% identity, respectively. PMeV-Mx was found in the latex of fruits, in seedlings, and in the leaves, flowers, petioles, and seeds of mature plants. PMeV-Mx was more abundant in the latex of fruits than in the leaves. The limit of detection of the CB38/CB39 primer pair was 1 fg and 1 pg using total RNA extracted from the latex of fruits and from seedlings, respectively. A sensitive and early diagnosis protocol was developed; this method will enable the certification of seeds and seedlings prior to transplantation to the field.
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México.
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Zamudio-Moreno
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México.
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Ramirez-Prado
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México.
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Moreno-Valenzuela
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México luisa_lopez@cicy.mx.
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Lopez-Ochoa
D001483:Base Sequence; D001938:Brazil; D029441:Carica / Q000821:virology; D003001:Cloning, Molecular; D005638:Fruit / Q000821:virology; D015723:Gene Library; D023281:Genomics; D008800:Mexico; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D010942:Plant Viruses / Q000235:genetics*; D012328:RNA Viruses / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D036226:Seedlings / Q000821:virology; D017423:Sequence Analysis, RNA
D018749:RNA, Plant; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase