Improved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR assay

La caña de azúcar es uno de los cultivos industriales importantes a nivel mundial y es afectado por diversas enfermedades virales, incluido el sugarcane yellow leaf virus (SCYLV). En México este virus se ha detectado basado en sintomatología y °Brix; sin embargo, el diagnóstico basado en estos parámetros no es concluyente. El objetivo del estudio fue detectar la presencia y distribución del SCYLV en zonas cañeras del Occidente de México (Colima, Jalisco y Nayarit) y determinar el origen filogenético de un aislado de Colima. El trabajo se desarrolló en el Campo Experimental Tecomán durante 2013-2014. Se aisló el RNA total a partir de hojas colectadas y se realizaron las RT-PCR con oligonucleótidos específicos para SCYLV. Se analizaron 233 muestras y la incidencia de plantas portadoras del SCYLV fue 14.6%, afectando a los híbridos CP 72-2086, Mex 69-290 y Atemex 96-40. El análisis BLAST mostró que la secuencia parcial CP de Colmex-317 (512 pb) es homóloga con secuencias de Brasil, China, India, Kenia y USA, además comparte porcentajes de identidad superior a 99% con otras secuencias provenientes de varios países. El análisis filogenético de Colmex-317 con secuencias parciales y genomas completos del SCYLV aislados de diversas partes del mundo reveló que el aislado mexicano pertenece al genotipo de Brasil (BRA) y se agrupó con secuencias de Brasil, China, Kenia y Sudáfrica. Sin embargo, es necesario evaluar un mayor número de aislados y secuencias de mayor longitud para determinar si el genotipo BRA es el único presente en México.

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Detection of Sugarcane yellow leaf virus in Quarantine and Production of Virus-free Sugarcane by Apical Meristem Culture

Plant Disease ◽

10.1094/pdis.2001.85.11.1177 ◽

2001 ◽

Vol 85 (11) ◽

pp. 1177-1180 ◽

Cited By ~ 42

Author(s):

M. Chatenet ◽

C. Delage ◽

M. Ripolles ◽

M. Irey ◽

B. E. L. Lockhart ◽

...

Keyword(s):

Apical Meristem ◽

False Negative ◽

Hot Water ◽

Meristem Culture ◽

Vascular Bundles ◽

Yellow Leaf ◽

Rt Pcr ◽

Sugarcane Yellow Leaf Virus ◽

Leaf Virus

Sugarcane yellow leaf virus (SCYLV) was detected for the first time in 1996 in the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) sugarcane quarantine at Montpellier by reverse transcription-polymerase chain reaction (RT-PCR) in varieties from Brazil, Florida, Mauritius, and Réunion. Between 1997 and 2000, the virus was found by RT-PCR and/or tissue-blot immunoassay (TBIA) in additional varieties from Barbados, Cuba, Guadeloupe, Indonesia, Malaysia, Philippines, Puerto Rico, and Taiwan, suggesting a worldwide distribution of the pathogen. An excellent correlation was observed between results obtained for the two diagnostic techniques. However, even though only a few false negative results were obtained by either technique, both are now used to detect SCYLV in CIRAD's sugarcane quarantine in Montpellier. The pathogen was detected by TBIA or RT-PCR in all leaves of sugarcane foliage, but the highest percentage of infected vascular bundles was found in the top leaves. The long hot water treatment (soaking of cuttings in water at 25°C for 2 days and then at 50°C for 3 h) was ineffective in eliminating SCYLV from infected plants. Sugarcane varieties from various origins were grown in vitro by apical bud culture and apical meristem culture, and the latter proved to be the most effective method for producing SCYLV-free plants.

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Prevalence of Sugarcane yellow leaf virus in Sugarcane-Producing Regions in Kenya Revealed by Reverse-Transcription Loop-Mediated Isothermal Amplification Method

Plant Disease ◽

10.1094/pdis-05-15-0602-re ◽

2016 ◽

Vol 100 (2) ◽

pp. 260-268 ◽

Cited By ~ 10

Author(s):

Ruth L. Amata ◽

Emmanuel Fernandez ◽

Denis Filloux ◽

Darren P. Martin ◽

Philippe Rott ◽

...

Keyword(s):

Reverse Transcription ◽

Lamp Assay ◽

Isothermal Amplification ◽

Yellow Leaf ◽

Rt Pcr ◽

Pairwise Identity ◽

Sugarcane Yellow Leaf Virus ◽

Actual Distribution ◽

Loop Mediated Isothermal Amplification ◽

Leaf Virus

Yellow leaf (YL) is a disease of sugarcane that is currently widespread throughout most American and Asian sugarcane-producing countries. However, its actual distribution in Africa remains largely unknown. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to facilitate and improve the detection of Sugarcane yellow leaf virus (SCYLV), the causal agent of YL. The RT-LAMP assay was found to be comparable with or superior to conventional RT-polymerase chain reaction (PCR) for the detection of SCYLV genotypes CUB and BRA in infected sugarcane ‘C132-81’ and ‘SP71-6163’, respectively. Additionally, 68 sugarcane samples that tested negative by RT-PCR were found positive by RT-LAMP, whereas the RT-LAMP assay failed to detect SCYLV in only 5 samples that tested positive by RT-PCR. Combining RT-PCR and RT-LAMP data enabled the detection of SCYLV in 86 of 183 Kenyan sugarcane plants, indicating high SCYLV prevalence throughout the country (ranging from 36 to 64% in individual counties). Seminested PCR assays were developed that enabled the amplification of a fragment encompassing the capsid protein coding region gene and its flanking 5′ and 3′ genomic regions. Sequences of this fragment for four Kenyan SCYLV isolates indicated that they shared 99.2 to 99.6% pairwise identity with one another and clearly clustered phylogenetically with SCYLV-BRA genotype isolates. To our knowledge, this is the first report of SCYLV in Kenya.

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Geographical Distribution of Four Sugarcane yellow leaf virus Genotypes

Plant Disease ◽

10.1094/pd-90-1156 ◽

2006 ◽

Vol 90 (9) ◽

pp. 1156-1160 ◽

Cited By ~ 38

Author(s):

Youssef Abu Ahmad ◽

Monique Royer ◽

Jean-Henrich Daugrois ◽

Laurent Costet ◽

Jean-Michel Lett ◽

...

Keyword(s):

Pcr Primers ◽

Yellow Leaf ◽

Rt Pcr ◽

Sugarcane Yellow Leaf Virus ◽

On This Island ◽

Virus Genotypes ◽

Polymerase Chain ◽

Leaf Virus ◽

Geographical Locations ◽

Local Transmission

Specific primer pairs were designed to distinguish four genotypes (BRA for Brazil, CUB for Cuba, PER for Peru, and REU for Réunion Island) of Sugarcane yellow leaf virus (SCYLV) by reverse transcription-polymerase chain reaction (RT-PCR). A unique genome fragment was amplified from each genotype, with the exception of genotypes BRA and PER that are phylogenetically relatively close and were designated genotype BRA-PER. These RT-PCR primers were then used to identify the SCYLV genotype(s) present in 18 different sugarcane growing locations in the world, and 245 leaf samples infected by the virus were analyzed. Most samples were infected by only one of the three genotypes, but mixed infections occurred. Genotype BRA-PER was found in all sugarcane growing locations, whereas genotypes CUB and REU were each found in four geographical locations only. Genotypes BRA-PER, CUB, and REU were all three detected in locally bred sugarcane cultivars in Guadeloupe, indicating local transmission of these genotypes. In contrast, only genotypes BRA-PER and CUB were found in locally bred cultivars in Brazil, whereas genotype REU was detected in this country in cultivar R570 imported from Réunion. Similarly, genotypes BRA-PER and REU are both present in Réunion, but genotype BRA-PER has not, as of yet, spread on this island. Presence of several SCYLV genotypes in Brazil, Colombia, Guadeloupe, Mauritius, and Réunion suggests different virus introductions and/or different evolution histories of the virus after its introduction into a new environment.

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Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638721994810 ◽

2021 ◽

pp. 104063872199481

Author(s):

Yixin Xiao ◽

Fan Yang ◽

Fumin Liu ◽

Hangping Yao ◽

Nanping Wu ◽

...

Keyword(s):

Avian Influenza ◽

Real Time ◽

Influenza A ◽

Minor Groove Binder ◽

Rt Pcr ◽

Influenza A Viruses ◽

Pcr Assay ◽

Infectious Dose ◽

The Matrix ◽

Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

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