PJB-1999-15
RAPID MULTIPLEX PCR FOR THE SPECIFIC DETECTION OF TWO WHITEFLY-TRANSMITTED GEMINIVIRUS SPECIES ASSOCIATED WITH COTTON LEAF CURL DISEASE IN PAKISTAN
SHAHID MANSOOR, AFTAB BASHIR, SULTAN H. KHAN, MAZHAR HUSSAIN, M. SAEED, YUSUF ZAFAR, PETER G. MARKHAM*, AND KAUSER A. MALIK
Abstract
Cotton leaf curl disease in Pakistan is associated with two whitefly-transmitted geminivirus species named cotton leaf curl virus Pk1 (CLCuV-Pk1) and cotton leaf curl virus Pk2 (CLCuV-Pk2). PCR is a highly specific and reliable technique for the detection of geminiviruses. A protocol has been developed for rapid isolation of a suitable template for PCR. The method is based either on the adsorption of DNA template from cleared lysate on PCR tubes or a rapid minipreparation of total DNA by CTAB method. Similarly, a simplified protocol is used for the isolation of total DNA from individual whitefly which is suitable for PCR amplification. Primers have been designed in such a way that the two geminivirus species are amplified in a single tube by multiplex PCR. In this PCR virus sense primer is common to both viruses in the rep gene whereas the complementary sense primer is specific for either of the two viruses. For CLCuV-Pk1, the reverse primer is designed at the start of C4 ORF gene whereas for CLCuV-Pk2 the primer is designed at the start of rep gene. The two PCR products of about 360 bp (CLCuV-Pk1) and 510 bp (CLCuV-Pk2) are resolved on an agarose gel. A rapid profIle for multiplex PCR was used and completed in 2 hr. The whole process of template preparation, PCR and the detection of PCR product by agarose gel electrophoresis is completed in a single day. The protocol has been used reliably for the detection of cotton geminiviruses in plant and whitefly samples collected from the field.
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