| Pratylenchus coffeae is one of the most economically damaging plant-parasitic nematodes and is found on a wide variety of crops.Accurate identification and quantification of this nematode are necessary for providing advice to farmers,but are not easily obtained through traditional approach of microscopic observation.We developed a PCR assay to detect and quantify P.coffeae in a rapid but accurate manner.A set of PCR primer was designed based on the sequence of the 28 S.The assay was optimized by using the primers in a PCR assay setting the PCR program to different annealing temperatures ranging from 56°C to 61 °C.Based on the Ct-values,we retained the program with an annealing temperature of60 °C.The assay with the primer was very sensitive as it was able to detect a single individual of P.coffeae and P.loosi.The main result are organized as below:(1)The specificity of the reaction was confirmed by the amplification of 28 S gene from22 populations of 16 other Pratylenchus species and plant-parasitic nematodes in other general.(2)A set of primer was used to identify and detect P.coffeae and P.loosi,using multiPCR.This method does not require expertise in nematode taxonomy and morphology and can be used as a rapid diagnostic tool in research projects,diagnostic labs and extension services advising farmers for pest management. |
