Enhanced detection of a plant pathogenic mycoplasma-like organism by polymerase chain reaction.

According to the sequence of cloned genomic DNA of clover proliferation (CP) MLO, two polymerase chain reaction (PCR) primer pairs were synthesized to specifically amplify two CP MLO DNA fragments from crude nucleic acids containing CP MLO DNA and host plant DNA. The first primer pair guided the amplification of a 196-bp DNA fragment of CP MLO. The second primer pair directed the amplification of a 109-bp DNA fragment of CP MLO. PCR products were identified by liquid hybridization using 5' end-labeled sequence-specific internal probe followed by 8% polyacrylamide gel electrophoresis and direct sequencing of PCR products. A minimum of 2.5ng nucleic acids were needed to detect CP MLO when PCR was not applied, whereas a minimum of only 2.5×10-5ng nucleic acids were needed when the 109-bp CP MLO DNA fragment was amplified by PCR, and a minimum of 2.5×10-8ng nucleic acids were needed to detect CP MLO when the 196-bp CP MLO DNA fragment was amplified through PCR. No DNA fragments were amplified when nucleic acids from healthy periwinkle plants were used as PCR templates.