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Specificity of the Pcinn13739-

ERA-LFDassay

A specific pair of primers (Pcinn13739-RPA-F and Pcinnl3739-RPA-R) was designed to exclusively and consistently amplify P. cinnamomi isolates by RPA-LFD assays (Table 1). The 5'-end of the reverse primer was labeled with biotin. A Pcinn13739-probe was also designed in accordance with the instructions provided with the GenDx ERA Kit (GenDx Biotech, Suzhou, China). In the RPA-LFD assay, the specificity of Pcinn13739-RPA-F and Pcinn13739-RPA-R was verified using a variety of different species (50 isolates of Phytophthora sPp., 13 isolates of Phytopythium and Pythium spp., 32 isolates of fungi, and 11 isolates of Bursaphelenchus spp. All dipsticks yielded visible control lines, indicating that the tests were valid. No test lines were detected in gDNAs (100 ng) from other species (Phytophthora, Phytopythium, Pythium, fungi, and Bursaphelenchus) or NTCs (Figure 3). Consistent results were obtained for the RPA-LFD assay in three biological replicates.

DOI 10.3389/fcimb.2022.923700