Back to Search
Start Over
Evaluation of a simple membrane-based nucleic acid preparation protocol for RT–PCR detection of potato viruses from aphid and plant tissues
- Source :
- Journal of Virological Methods. 121:163-170
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- A rapid and simple protocol for preparing viral RNA from aphids ( Myzus persicae ) and potato ( Solanum tuberosum L.) tissue (leaves, sprouts, and tubers) for reverse transcription–polymerase chain reaction (RT–PCR) was developed. The four-step method involves: (1) preparing plant crude sap or aphid macerates in a buffered detergent (Triton XL-80N) solution; (2) immobilizing clarified sap on a nitrocellulose membrane; (3) performing reverse transcription using eluted water extract from cut-out spot from membrane; and (4) amplifying cDNA through PCR. The entire procedure from tissue grinding to RT–PCR can be completed within 6 h. The protocol was applied successfully for the detection of individual potato viruses: carlavirus Potato virus S (PVS), potexvirus Potato virus X (PVX), potyvirus Potato virus Y (PVY), and polerovirus Potato leafroll virus (PLRV). PLRV was also detected from individual viruliferous aphids or composites of viruliferous and healthy aphids. PVY and PLRV were detected from extracts immobilized on nitrocellulose membranes, stored for more than 65–273 days at room temperature (25 °C). The protocol was companed with the ‘long protocols’ involving enzyme and phenol extraction for aphids or sodium sulfite and phenol extraction for tubers. The simplified protocol was found comparable in sensitivity to these long procedures, and is especially suitable for those regions where specialized PCR laboratories are only available in centralized locations. Viral RNA immobilized membranes can be mailed out for detection by RT–PCR to these centralized laboratories from remote areas of the country.
- Subjects :
- Carlavirus
Time Factors
food.ingredient
Potyvirus
Buffers
Polyethylene Glycols
Specimen Handling
Polerovirus
food
Virology
Luteovirus
Animals
Solanum tuberosum
Potato leafroll virus
biology
Reverse Transcriptase Polymerase Chain Reaction
fungi
Temperature
Water
food and beverages
Membranes, Artificial
Potato virus S
biology.organism_classification
Potato virus X
Potexvirus
Plant Leaves
Plant Tubers
Potato virus Y
Aphids
RNA, Viral
Myzus persicae
Subjects
Details
- ISSN :
- 01660934
- Volume :
- 121
- Database :
- OpenAIRE
- Journal :
- Journal of Virological Methods
- Accession number :
- edsair.doi.dedup.....0f44f0427717a49e49f841d4bc764512