Polymerase chain reaction detection of candidatus liberibacter asiaticus associated with citrus huanglongbing (greening) of sweet orange
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Date
2011-08-08
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Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani
Abstract
Polymerase chain reaction diagnosis of disease is molecular technique
which is used for detection of disease when pathogen present is very low
concentration in disease sample. Before molecular technique use, first survey
of the Marathwada region for greening disease, then confirm the presence of
disease in infected samples. Use three different method for DNA isolation
from infected sample i.e. commercial kit method, sodium sulphite method,
nucleic acid membrane method. Among these three DNA isolation method is
sodium sulphide is cost effective for commercial use. In nucleic acid
membrane method for DNA extraction isolation there is no use of liquid
nitrogen.
Polymerase chain reaction detection of disease is based on principal of
thermal cycling in which PCR instrument allow to run generally 60-65 thermal
cycle, during PCR operation it allow different stages of cycle at different
temperatures for different period of time i.e. initiation (94°C), denaturation
(94°C), primer annealing (60°C), extension/elongation step (72°C), final
elongation (72°C) and holding temperature ( 4°C). After all completion of
procedure and cost effective analysis of different DNA extension and isolation
method. Final following conclusions are drawn 1) PCR based diagnosis
system is developed for detection of greening bacteria 2) The comparative cost
of detection by various combination of reagent and sampling time was
determined and cost effective technology was standardized and validated.