Open AccessMultiplex detection of “Candidatus Liberibacter asiaticus” and Spiroplasma citri by qPCR and droplet digital PCR
Author(s) -
Yogita Maheshwari,
Vijayanandraj Selvaraj,
Kristine Godfrey,
Subhas Hajeri,
R. K. Yokomi
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0242392
Subject(s) - diaphorina citri , biology , multiplex , spiroplasma , multiplex polymerase chain reaction , polymerase chain reaction , virology , microbiology and biotechnology , gene , bacteria , genetics , botany , mollicutes , hemiptera
“ Candidatus Liberibacter asiaticus” ( C Las) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S . citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. C Las is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. C Las is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of C Las and S . citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of C Las—RNR (5 copies) and S . citri –SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of C Las and S . citri . ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for C Las detection in California. Moreover, the ddPCR provided unambiguous absolute detection of C Las and S . citri at very low concentrations without any standards for pathogen titer.