Parameters for specific detection of Clavibacter michiganensis subsp. sepedonicus in potato stems and tubers by multiplexed PCR-ELISA

American Journal of Potato Research, Jul/Aug 2003 by Mills, Dallice, Russell, Brian W

Combining the three primer sets in a single reaction tube did not result in primer combinations that foster the priming and amplification of other fragments that could have limited the amplification of the expected PCR products. Although various annealing conditions and primer concentrations were used together with a new reverse primer for CmsSO to establish an optimal condition for multiplex PCR, the end-point dilutions of the PCR products indicated that the amplification of Cms72 was twofold and fourfold greater than Cms85 and Cms50, respectively (Table 2). At the dilution end point of 300 cfu/mL of potato tuber core fluid, amplification of Cms72 and Cms50 DNA was detected (Figure 1). This level of sensitivity represents 3 cfu in 10 [mu]L of potato core fluid used for PCR analysis, which approximates the sensitivity previously observed by PCR of single fragments (1 X 10^sup 2^ cfu/mL) (Mills et al. 1997; Baer et al. 2001), and is approximately 30-fold more sensitive than detection by immunodetection tests (Li and De Boer 1995). However, it should be noted that with the conditions defined here, amplification of either Cms50 or Cms85 may not always occur at the limits of detection (see Figures 1 and 7). The ability to detect the DIG-11-dUTP labeled PCR product by ELISA provided a 10-fold greater level of sensitivity than by gel electrophoresis and should provide a similar increased level of sensitivity with other DNA-based methods such as TaqMan probes and molecular beacons.

This multiplex PCR detection system has two additional features that can eliminate the possibility of false negative readings. First, the inclusion of SA72 template DNA in the reaction mix, which is amplified from primers that are used to amplify Cms72, ensures that primers that specifically amplify Cms template DNA function properly for each sample. Second, the addition of 0.05% (wt/v) casein hydrolysate to the reaction mix proved to be effective for relieving the occasional inhibition of PCR presumed to be caused by plant cell components, soil, and debris. Previously De Boer et al. (1995) had shown that BLOTTO, a non-fat milk cocktail typically used to reduce non-specific binding of proteins and nucleic acids to Southern and Western blots, also relieved PCR inhibition at concentrations of 1-5 % (v/v), and the precise concentration was adjusted with each polymerase batch. They also determined that similar concentrations of hydrolyzed casein were not effective. As shown in Figure 4, concentrations of casein above 0.20% (wt/v) inhibited PCR amplification of purified Cms template DNA, indicating that it is effective at only very low concentrations. Moreover, 0.05% casein did not inhibit different brands and batches of DNA polymerase. For uninfected plants, the amplification of SA72 template DNA was an absolute requirement for assurance that PCR inhibition was not a factor. In other multiplex PCR detection systems, different forward and reverse primers were used to amplify plant DNA (Weller et al. 2000) to monitor for PCR inhibition. Amplification of a heterologous fragment (SA72) with primers that amplify a pathogen target sequence (Cms72) guarantees that the reaction conditions are appropriate when the target PCR product is not detected in a sample.