Multiplex detection of 'Candidatus Liberibacter asiaticus' and Spiroplasma citri by qPCR and droplet digital PCR

Autor: Kristine Godfrey, Vijayanandraj Selvaraj, Raymond Yokomi, Subhas Hajeri, Yogita Maheshwari
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Spiroplasma citri
0301 basic medicine
Citrus
Positive control
Artificial Gene Amplification and Extension
Plant Science
Pathology and Laboratory Medicine
Polymerase Chain Reaction
Biochemistry
Liberibacter
Nucleic Acids
Medicine and Health Sciences
Multiplex
Digital polymerase chain reaction
DNA extraction
Pathogen
Volume concentration
Multidisciplinary
biology
Eukaryota
food and beverages
Plants
Beet leafhopper
Medicine
Pathogens
Research Article
DNA
Bacterial
Spiroplasma
Science
030106 microbiology
Mollicutes
Plant Pathogens
DNA replication
Real-Time Polymerase Chain Reaction
Research and Analysis Methods
Fruits
03 medical and health sciences
Extraction techniques
stomatognathic system
Genetics
Molecular Biology Techniques
Molecular Biology
Plant Diseases
Bacteria
Candidatus Liberibacter asiaticus
Organisms
Reproducibility of Results
Biology and Life Sciences
DNA
Plant Pathology
biology.organism_classification
Virology
030104 developmental biology
Multiplex Polymerase Chain Reaction
Zdroj: PLoS ONE, Vol 16, Iss 3, p e0242392 (2021)
PLoS ONE
ISSN: 1932-6203
Popis: “Candidatus Liberibacter asiaticus” (CLas) 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. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas 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 CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas—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 CLas 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 CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.
Databáze: OpenAIRE
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