Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.
Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA; Amity Institute of Virology & Immunology, Amity University, Noida 201303, UP, India.
Nandlal
N
Choudhary
US Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
G
G
Wei
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
A
A
Govindarajulu
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
Avijit
A
Roy
US Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
Wenbin
W
Li
US Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
Deric D
DD
Picton
US Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Center for Plant Health Science and Technology, Beltsville Laboratory, Beltsville, MD 20705, USA.
M K
MK
Nakhla
US Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Center for Plant Health Science and Technology, Executive Director's Office, Riverdale, MD 20737, USA.
L
L
Levy
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, Lake Alfred, FL 33850, USA. Electronic address: rhby@ufl.edu.
R H
RH
Brlansky
D002489:Central America; D002957:Citrus / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000821:virology*; D053830:Plant Viral Movement Proteins / Q000235:genetics; D010942:Plant Viruses / Q000302:isolation & purification*; D012328:RNA Viruses / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015345:Oligonucleotide Probes; D053830:Plant Viral Movement Proteins
CiLV-C; Citrus leprosis disease; One-step qRT-PCR assays; Virus detection