Reverse transcription-duplex-polymerase chain reaction for simultaneous detection of Citrus tristeza virus and ‘Candidatus Liberibacter’ from citrus plants

Adkar-Purushothama, Charith Raj; Quaglino, F.; Casati, P.; Bianco, P.A.

doi:10.1007/bf03356367

Cited by 8 publications

(3 citation statements)

References 17 publications

(15 reference statements)

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“…Misdiagnosis of either of these diseases may be catastrophic and involve costly regulatory actions. RNA and DNA targets multiplexing by qPCR analysis has been shown for citrus pathogens [19][20][21][22][23][24]. Duplex ddPCR has been reported for simultaneous detection of two gene targets for unambiguous detection of CLas [14] and S. citri [10].…”

Section: Discussionmentioning

confidence: 99%

Multiplex detection of “CandidatusLiberibacter asiaticus” andSpiroplasma citriby qPCR and droplet digital PCR

Maheshwari

Selvaraj

Godfrey³

et al. 2020

Preprint

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“ 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. Consequently, a multiplex qPCR and duplex droplet digital PCR (ddPCR) were developed for simultaneous detection of C Las and S. citri . 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 resilience 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.

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Section: Discussionmentioning

confidence: 99%

Multiplex detection of “CandidatusLiberibacter asiaticus” andSpiroplasma citriby qPCR and droplet digital PCR

Maheshwari

Selvaraj

Godfrey³

et al. 2020

Preprint

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show abstract

“…In other assays, a reverse transcription step is included, allowing detection of RNA viruses as well. For instance, Adkar-Purushothama et al. (2010) developed such a test for the simultaneous detection of Citrus tristeza virus (CTV) and Candidatus Liberibacter species from citrus plants.…”

Section: Nucleic Acid-based Assaysmentioning

confidence: 99%

Current and emerging trends in techniques for plant pathogen detection

Venbrux,

Crauwels,

Rediers

2023

Front. Plant Sci.

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Plant pathogenic microorganisms cause substantial yield losses in several economically important crops, resulting in economic and social adversity. The spread of such plant pathogens and the emergence of new diseases is facilitated by human practices such as monoculture farming and global trade. Therefore, the early detection and identification of pathogens is of utmost importance to reduce the associated agricultural losses. In this review, techniques that are currently available to detect plant pathogens are discussed, including culture-based, PCR-based, sequencing-based, and immunology-based techniques. Their working principles are explained, followed by an overview of the main advantages and disadvantages, and examples of their use in plant pathogen detection. In addition to the more conventional and commonly used techniques, we also point to some recent evolutions in the field of plant pathogen detection. The potential use of point-of-care devices, including biosensors, have gained in popularity. These devices can provide fast analysis, are easy to use, and most importantly can be used for on-site diagnosis, allowing the farmers to take rapid disease management decisions.

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“…regulatory actions and impact the grower. RNA and DNA targets multiplexing by qPCR analysis has been shown for citrus pathogens [19][20][21][22][23][24]. Duplex ddPCR has been reported for simultaneous detection of two gene targets for unambiguous detection of CLas [14] and S. citri [10].…”

Section: Plos Onementioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

“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.

show abstract

Reverse transcription-duplex-polymerase chain reaction for simultaneous detection of Citrus tristeza virus and ‘Candidatus Liberibacter’ from citrus plants

Cited by 8 publications

References 17 publications

Multiplex detection of “CandidatusLiberibacter asiaticus” andSpiroplasma citriby qPCR and droplet digital PCR

Multiplex detection of “CandidatusLiberibacter asiaticus” andSpiroplasma citriby qPCR and droplet digital PCR

Current and emerging trends in techniques for plant pathogen detection

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

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