RT-PCR DETECTION of BBrMV

Published by:

Research and Development Directorate DETECTION

624bp MARIANO MARCOS STATE UNIVERSITY
125bp
324 bp Batac City 2906 Ilocos Norte, Philippines OF
Website: http://research.mmsu.edu.ph
August 2011 PLANT VIRUSES

BULLETIN
Disease symptoms:
Lane 1: MW Marker
IN BANANA USING
Spindle-shaped lesions
on the leaves, red
Lane 2: Uninfected Tissue Polymerase Chain Reaction
Lane 3: BBrMV-infected Tissue
Source of Technology
streaking of the
pseudostem
Lane 4: BBrMV-infected Tissue
(PCR)
PCR amplified the product using Project: ‘Detection of Plant Viruses in
Forward primer for BBrMV
Banana and Garlic’
which generated 324 bp in size.

Researchers: Paul D. Kohnen

Paul D. Kohnen Marissa I. Atis
Marissa I. Atis Maura Luisa S. Gabriel

INFORMATION
CONCLUSION Maura Luisa S. Gabriel
Virus detections in banana, such as Banana Bunchy Top
Virus, Banana Streak Virus and Banana Bract Mosaic Virus
had been successful using PCR technique. The techniques Implementing Agency:
on PCR and RT-PCR may eventually, offer diagnostic Mariano Marcos State University
services to the general public. Batac City, Ilocos Norte
A cost-efficient protocol in the detection of Banana
Bunchy Top Virus simultaneous with Banana Streak Virus
was formulated. This can result in much savings in terms of
time and resources.

For more information, contact :

Ms. Marissa I. Atis

BENEFIT Research and Development Directorate
MARIANO MARCOS STATE UNIVERSITY
The protocols developed on virus Batac City 2906 Ilocos Norte
Telefax No. (077)791-3131
detection in banana allows the possibility of E-mail ad: rddirectorate@yahoo.com MARIANO MARCOS STATE UNIVERSITY
providing banana growers of virus-free planting Batac City 2906 Ilocos Norte
materials. Philippines
 INTRODUCTION METHODOLOGY RESEARCH HIGHLIGHTS
is a very important fruit crop in the Collection of leaf samples is the newest technique to detect plant viruses, which is
Philippines. It ranked second, in terms of more accurate than ELISA. Virus detection protocol for
area, volume and value of production, next to coconut Leaf samples of banana banana using this technique was developed for three major plant
(BAS, 2006). It is also the second leading export crop in the Lakatan and Cardava banana viruses in banana.
country. were collected from plants
showing symptoms of BBTV, BSV PCR DETECTION of BBTV
Banana is propagated
and BBrMV from banana plants
vegetatively through
growing in the campus of MMSU, 2,027 bp
suckers and in vitro. Like ~1,100 bp
Batac City.
any other crop, it can be
564 bp
Virus diseases in easily infected with viruses
banana reduce yield
from 40 to 100%
through mechanical Nucleic acid extraction
transmission of insects.
Most farmers do not renew their planting materials and 1. Homogenize the leaf midrib by
grinding with liquid nitrogen, adding Disease symptoms: Lane 1:Molecular Weight Marker
they are not aware that their plants are already infected
3 ml of CTAB; Internode shortening Lane 2: BBTV
with virus diseases. As a consequence, yield reduction is
observed. To date, degeneration of planting materials 2. Incubate the mixture at 650C for 45 with a bunchy
PCR amplified the product using primer
through the accumulation of viruses is ongoing. Continuous minutes then, centrifuge; appearance at the top
pair for BBTV which generated ~1100 bp.
spread of diseases is observed not only in the Philippines, 3. Clarify the supernatant with phenol, of the plant
but also in other countries. precipitate with isopropanol and
resuspend in quality water.
Detection and identification of plant viruses in banana PCR DETECTION of BSV
is important in addressing the problem. Symptoms of PCR and analysis of products 2,027 bp
virus infection in banana are frequently inadequate to
conclude to a positive identification. Hence, the biological 564 bp
activity of a certain virus in banana must be understood ~221 bp
and identified. 125 bp
The application of molecular techniques gives a large Lane 1:Molecular Weight Marker
‘tool-bag’ for detection and diagnosis of plant viruses. A
Gel electro- Stain- Disease symptoms: Lane 2: BSV
scientific technique through Polymerase Chain Reaction
Yellow striate mosaic, broken lines and
(PCR) can be used. This technique amplifies a single piece PCR amplified the product
diamond-shaped patterns along the leaf
of DNA and generates into thousands and millions of using primer pair for BSV
veins; reduced plant growth, vigor, bunch
copies of a particular DNA sequence. Using this method is which generated a 221 bp.
size and yield; and fruit distortion
more accurate than any other techniques of detections.
PCR ma-
Image of analy- SIMULTANEOUS PCR DETECTION of BBTV AND BSV
OBJECTIVES 1. Perform PCR for BBTV and BSV using 50µl of reaction mixture;
2. Perform RT-PCR for BBrMV using 20µl for reverse transcription
To establish a protocol in the detection of banana and 30µl of reaction mixture for polymerase chain reaction Lane 1: Molecular Weight Marker 2,027 bp
bunchy top virus (BBTV), banana streak virus (BSV) analysis; Lane 2: BBTV
Lane 3: BBTV & BSV 564 bp
and banana bract mosaic virus (BBrMV) using PCR 3. Analyze the products by electrophoresis using 1.4% agarose
Lane 4: BSV
technique; gels;
4. Stain the gel with ethidium bromide, visualize and analyze by 125 bp
To formulate a cost-efficient protocol in the
Doc-ItR LS Image Acquisition Software.
simultaneous detection of BBTV and BSV.