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      Screening nested-PCR primer for ‘Candidatus Liberibacter asiaticus’ associated with citrus Huanglongbing and application in Hunan, China

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          Abstract

          Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated “ Candidatus Liberibacter spp”. “ Candidatus Liberibacter spp” that infect Citrus includes “ Candidatus Liberibacter asiaticus” (Las), “ Candidatus Liberibacter africanus” (Laf) and “ Candidatus Liberibacter americanus” (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.

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          Current epidemiological understanding of citrus Huanglongbing .

          Huanglongbing (HLB) is the most destructive citrus pathosystem worldwide. Previously known primarily from Asia and Africa, it was introduced into the Western Hemisphere in 2004. All infected commercial citrus industries continue to decline owing to inadequate current control methods. HLB increase and regional spatial spread, related to vector populations, are rapid compared with other arboreal pathosystems. Disease dynamics result from multiple simultaneous spatial processes, suggesting that psyllid vector transmission is a continuum from local area to very long distance. Evolutionarily, HLB appears to have originated as an insect endosymbiont that has moved into plants. Lack of exposure of citrus to the pathogen prior to approximately 100 years ago did not provide sufficient time for development of resistance. A prolonged incubation period and regional dispersal make eradication nonviable. Multiple asymptomatic infections per symptomatic tree, incomplete systemic distribution within trees, and prolonged incubation period make detection difficult and greatly complicate disease control.
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            Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes.

            A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids (RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasite/μl using cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 parasite/μl were detected when analyzing clinical samples. Analysis of clinical samples showed that detection of 18S rRNA genes from total nucleic acids increased the analytical sensitivity of the assay by more than 1 log unit compared to DNA only. When clinical samples with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were positive for the presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-PCR using total nucleic acid versus DNA was compared to microscopy. There was a significantly greater correlation of parasite density to microscopy when DNA alone was used than with total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a suitable assay for detection of low parasite levels in patients with early-stage malaria and/or submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, and malaria control and elimination efforts.
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              Rapid multiplex nested PCR for detection of respiratory viruses.

              Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Writing – original draft
                Role: Data curationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: Investigation
                Role: Investigation
                Role: SupervisionRole: Visualization
                Role: ConceptualizationRole: Funding acquisition
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 February 2019
                2019
                : 14
                : 2
                : e0212020
                Affiliations
                [1 ] Hunan Provincial Key Laboratory for Biology and Control of Plant Pests, College of Plant Protection, Hunan Agricultural University, Changsha, Hunan province, China
                [2 ] College of life Science and Technology, Beijing University of Chemical Technology, Beijing, China
                Fujian Agriculture and Forestry University, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                ‡ These authors also contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-7520-6527
                http://orcid.org/0000-0002-4658-780X
                Article
                PONE-D-18-19707
                10.1371/journal.pone.0212020
                6386535
                30794562
                fc7308c5-9595-422f-b5c8-92072fe0581c
                © 2019 Hong et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 July 2018
                : 26 January 2019
                Page count
                Figures: 6, Tables: 3, Pages: 15
                Funding
                Funded by: Research and demonstration of prevention and control technology in central China citrus Huanglongbing interdiction belt and low epidemic areas
                Award ID: 2018YFD0201505
                Award Recipient :
                Funded by: Research and application of key technologies for green control of citrus pests and diseases
                Award ID: 2018NK2012
                Award Recipient :
                The project was supported by National key Research and development project of China "Research and demonstration of prevention and control technology in central China citrus Huanglongbing interdiction belt and low epidemic areas (2018YFD 0201505) " and Hunan province key Research and development project:" Research and application of key technologies for green control of citrus pests and diseases (2018NK 2012)" Tuyong Yi received the first fund and Yanyun Hong received the second fund.
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Nested Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Nested Polymerase Chain Reaction
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Fruits
                Citrus
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Fruits
                Biology and Life Sciences
                Genetics
                DNA
                Biology and Life Sciences
                Biochemistry
                Nucleic Acids
                DNA
                Biology and life sciences
                Genetics
                DNA
                DNA electrophoresis
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA electrophoresis
                Research and analysis methods
                Electrophoretic techniques
                DNA electrophoresis
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Research and analysis methods
                Database and informatics methods
                Bioinformatics
                Sequence analysis
                BLAST algorithm
                Custom metadata
                All relevant data are contained in the manuscript/or Supporting Information files.

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