Sensitive Nematode Detection Assay

The second stage juveniles (J2s) of M. chitwoodi race 1 were hatched from eggs collected from Rutgers tomatoes grown in greenhouses at Washington State University. Meloidogyne fallax and M. minor J2s were provided by the Wageningen Nematode Collection (National Plant Protection Organization, the Netherlands) and stored in DESS at -20°C. Second stage juveniles were digested using the protocol described in Qiu et al. (2006) (link). In brief, J2s were picked under a dissecting scope and transferred to 15 µL droplets of Milli-Q water. The nematodes were crushed using a pipet tip; 10 µL of the crushed nematode was transferred to a 10 µL solution containing 2 µL AmpliTaq Gold 360 buffer, 2 uL of 600 µg/mL Proteinase K, and 6 µL Milli-Q water. The reaction mixes were incubated at -20°C for 20 minutes, heated for 1 hour at 65°C, and then 95°C for 10 min before cooling to room temperature. The nematode samples were spun at 12,000 rpm for 2 min before storing at -20°C until used in PCR. Five µL of these J2 samples were used as template for molecular beacon RT-PCR, and each sample was run in duplicate or triplicate. The 50 µL reaction mixes contained 1x AmpliTaq Gold 360 buffer, 3 mM MgCl2, 200 µM dNTPs, 200 nM F-HSP90 and R-HSP90 primers, 200 nM target beacon (C1-Hsp90-FAM-6, F1-Hsp90-HEX-5, or M2-Hsp90-Cyan-5) and 1.25 U of AmpliTaq Gold. The reaction conditions were the same as those used for the standard curves. All molecular beacon RT-PCR assays with these three species were repeated at least twice with similar results.
To ensure the specificity of this assay 40 ng of gDNA from M. incognita, M. javanica, M. arenaria, or M. hapla was used as template for molecular beacon RT-PCR assay using C1-Hsp90-FAM-6, F1-Hsp90-HEX-5, and M2-Hsp90-Cyan-5 beacons. Non-template controls containing only Milli- Q water were included as well as positive controls using 4 pg of plasmid HSP90 for M. chitwoodi,
M. fallax, or M. minor. The 50 µL reaction mixes contained 1x AmpliTaq Gold 360 buffer, 3 mM MgCl2, 200 µM dNTPs, 200 nM F-HSP90 and R-HSP90 primers, 200 nM target beacon (C1- Hsp90-FAM-6, F1-Hsp90-HEX-5, or M2-Hsp90-Cyan-5) and 1.25 U of AmpliTaq Gold. The reaction conditions were the same as those used for the standard curves. The molecular beacon RT-PCR assays with non-target species were repeated twice with similar results.
All PCR products in this paper were visualized as follows: 10 µL of each PCR product was loaded onto a 1.5% agarose gel and separated for 45 min at 100 V before visualizing with ethidium bromide under UV light. The Invitrogen 1-kb plus DNA ladder was used as reference for size.