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

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0242392
Author(s):  
Yogita Maheshwari ◽  
Vijayanandraj Selvaraj ◽  
Kristine Godfrey ◽  
Subhas Hajeri ◽  
Raymond Yokomi
Keyword(s):  
Simultaneous Detection ◽  
Digital Pcr ◽  
Positive Control ◽  
Droplet Digital Pcr ◽  
Qpcr Assay ◽  
Absolute Quantitation ◽  
Spiroplasma Citri ◽  
Multiplex Qpcr ◽  
Liberibacter Asiaticus ◽  
Imminent Threat

“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.

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

2020 ◽  
Author(s):  
Yogita Maheshwari ◽  
Vijayanandraj Selvaraj ◽  
Kristine Godfrey ◽  
Subhas Hajeri ◽  
Raymond Yokomi
Keyword(s):  
Simultaneous Detection ◽  
Digital Pcr ◽  
Positive Control ◽  
Droplet Digital Pcr ◽  
Qpcr Assay ◽  
Absolute Quantitation ◽  
Spiroplasma Citri ◽  
Multiplex Qpcr ◽  
Liberibacter Asiaticus ◽  
Imminent Threat

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

scholarly journals Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus"

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197184 ◽  
Author(s):  
Vijayanandraj Selvaraj ◽  
Yogita Maheshwari ◽  
Subhas Hajeri ◽  
Jianchi Chen ◽  
Thomas Greg McCollum ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Detection ◽  
Candidatus Liberibacter Asiaticus ◽  
Pcr Assay ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

scholarly journals Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1462
Author(s):  
Ricardo Maggi ◽  
Edward B. Breitschwerdt ◽  
Barbara Qurollo ◽  
Jennifer C. Miller
Keyword(s):  
Animal Cell ◽  
Simultaneous Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Pcr Assay ◽  
Theileria Equi ◽  
Cytauxzoon Felis ◽  
Vector Borne ◽  
Type Water

We describe the development, optimization, and validation of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of Babesia, Bartonella, and Borrelia spp. DNA from several sample matrices, including clinical blood samples from animals and humans, vectors, in-vitro infected human and animal cell lines, and tissues obtained from animal models (infected with Bartonella and/or B. burgdorferi). The multiplex ddPCR assay was able to detect 31 Bartonella, 13 Borrelia, and 24 Babesia species, including Theileria equi, T. cervi, and Cytauxzoon felis. No amplification of Treponema or Leptospira spp. was observed. Sensitivity of 0.2–5 genome equivalent DNA copies per microliter was achieved for different members of the Bartonella and Borrelia genus, depending on the species or matrix type (water or spiked blood DNA) tested. The ddPCR assay facilitated the simultaneous detection of co-infections with two and three vector-borne pathogens comprising four different genera (Babesia, Bartonella, Borrelia, and Theileria) from clinical and other sample sources.

scholarly journals Droplet Digital PCR for the Detection of Plasmodium falciparum DNA in Whole Blood and Serum: A Comparative Analysis with Other Molecular Methods

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 478
Author(s):  
Elena Pomari ◽  
Ronaldo Silva ◽  
Lucia Moro ◽  
Giulia La Marca ◽  
Francesca Perandin ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Dna Analysis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Clinical Parameters ◽  
Serum Samples ◽  
Absolute Quantitation ◽  
Blood Samples ◽  
Paired Samples

Background: The estimation of Plasmodium falciparum parasitaemia can vary according to the method used. Recently, droplet digital PCR (ddPCR) has been proposed as a promising approach in the molecular quantitation of Plasmodium, but its ability to predict the actual parasitaemia on clinical samples has not been largely investigated. Moreover, the possibility of applying the ddPCR-sensitive method to serum samples has never been explored. Methods: We used, for the first time, ddPCR on both blood and serum to detect the DNA of P. falciparum in 52 paired samples from 26 patients. ddPCR was compared with loop-mediated isothermal amplification (LAMP) and rtPCR. The correlation between the ddPCR results, microscopy, and clinical parameters was examined. Results: ddPCR and microscopy were found to be strongly correlated (ρ(26) = 0.83111, p < 0.0001) in blood. Samples deviating from the correlation were partially explained by clinical parameters. In serum samples, ddPCR revealed the best performance in detecting P. falciparum DNA, with 77% positive samples among malaria subjects. Conclusion: Absolute quantitation by ddPCR can be a flexible technique for Plasmodium detection, with potential application in the diagnosis of malaria. In particular, ddPCR is a powerful approach for Plasmodium DNA analysis on serum when blood samples are unavailable.

scholarly journals Multiplex qPCR Assay for Direct Detection and Quantification of Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum in Soybean Seeds

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 3002-3009
Author(s):  
Maísa Ciampi-Guillardi ◽  
Juliana Ramiro ◽  
Maria Heloisa Duarte de Moraes ◽  
Marina Coan Goldoni Barbieri ◽  
Nelson S. Massola
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Internal Control ◽  
Direct Detection ◽  
Simultaneous Detection ◽  
Plant Diseases ◽  
Qpcr Assay ◽  
Soybean Seeds ◽  
Corynespora Cassiicola ◽  
Colletotrichum Truncatum ◽  
Multiplex Qpcr

Precise diagnosis of plant diseases is one of the most effective tools to minimize yield losses. Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum are common soilborne pathogens that affect soybeans all over the world. We developed a multiplex quantitative real-time polymerase chain reaction (qPCR) assay to simultaneously detect and quantify the three pathogens in soybean seeds and to survey their occurrence in the main soybean production areas in Brazil. Species-specific primers and probes for C. truncatum and C. cassiicola were designed based on GAPDH and TEF1 genes, respectively, to be combined with qPCR detection of S. sclerotiorum previously reported. The multiplex qPCR assay was successful in the simultaneous detection of C. truncatum, C. cassiicola, and S. sclerotiorum, along with a host internal control. The four pathogens were detected and quantified in artificially and naturally infested soybean seeds, even in the lowest incidence level tested of 0.0625% or 1 infected seed out of 1,599 healthy ones. From 81 seed samples tested, C. truncatum was the most frequently detected pathogen and with higher incidence levels (0.25 to 0.125%), followed by S. sclerotiorum and C. cassiicola, both with lower incidence levels (0.125 to 0.0625%). Together, the results evidenced the high sensitivity of the multiplex qPCR assay, indicating its usefulness for a quick and reliable diagnosis of soybean diseases in seeds.

scholarly journals Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

PLoS ONE ◽  
2017 ◽  
Vol 12 (9) ◽  
pp. e0184751 ◽  
Author(s):  
Yogita Maheshwari ◽  
Vijayanandraj Selvaraj ◽  
Subhas Hajeri ◽  
Raymond Yokomi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Detection ◽  
Spiroplasma Citri

scholarly journals Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy

2018 ◽  
Vol 64 (12) ◽  
pp. 1753-1761 ◽  
Author(s):  
Noemi Vidal-Folch ◽  
Dimitar Gavrilov ◽  
Kimiyo Raymond ◽  
Piero Rinaldo ◽  
Silvia Tortorelli ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Newborn Screening ◽  
Copy Number ◽  
Neuronal Degeneration ◽  
Muscular Atrophy ◽  
Simultaneous Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Carrier Status ◽  
Survival Motor Neuron Gene

Abstract BACKGROUND Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were &lt;7.1% CV for individuals with ≥1 SMN1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity. CONCLUSIONS This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.

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