Simultaneous detection of four pathogens in Dendrobium officinale by nested multiplex PCR assay

Dendrobium officinale is a valuable and rare Chinese herb, which is easy to suffer from various diseases during its growth. Thus early diagnosis of those diseases is crucial to increase the yield of D. officinale. In this study, we compared the internal transcribed spacer regions of the ribosomal DNA sequences of four widely disseminated pathogens in D. officinale (Sclerotium delphinii, Colletotrichum gloeosporioides, Alternaria alternata, and Cladosporium cladosporioides), designed specific primers for A. alternata and C. cladosporioides, and established a four-seat nested multiplex PCR assay. The sensitivity for S. delphinii, C. cladosporioides, A. alternata was found to be 10 fg/μL, and 10 pg/μL for C. gloeosporioides. In the actual application test, our established nested multiplex PCR can detect pathogens more quickly and accurately than conventional culture method. This method combines the advantages of nested and multiplex techniques, and it considerably improves the sensitivity and specificity of the detection results. Therefore, we hope that this method can provide a tool for diagnosing D. officinale diseases. •Simultaneous detection of 4 pathogens in Dendrobium officinale by nested multiplex PCR assay.•This method considerably improves the sensitivity and specificity of the detection results.•This method can provide a tool for diagnosing D. officinale diseases.•We designed the species-specific primers of A. alternata and C. cladosporioides.•This method is not only more sensitive but also faster than conventional culture.