Knowledge, Attidue and Practice of Advanced Trauma Life Suppoort (ATLS) Protocol among House-Officers in Khartoum State Hospitals, Sudan, 2023.

BACKGROUND: Advanced Trauma Life Support was originated mainly to train doctors who don't manage trauma on a regular basis, including junior doctors as it prepares them more efficiently and effectively for treating and managing trauma patients. This study was conducted to study knowledge, attitude and practice of advanced trauma life support protocol among house-officers in Khartoum state hospitals, Sudan 2023. METHODS: This is a cross-sectional descriptive health facility based study conducted in Bahri Teaching Hospital, Omdurman Teaching Hospital and Ibrahim Malik teaching hospital, Khartoum state, Sudan. Data of 151 House-officers of all nationality working in Khartoum state hospitals was collected using a simple random technique, filling questionnaire that was designed especially for this study. Comparison between different variables by Chi-square test and statistical significance difference at P value < 0.05 was done. RESULT: A total of 151 house officers were included in the study. 49% aged between 20 and 25 years, females were the majority 56.3%. About 41.1% have took ATLS course before. 55.21% of the study participants didn't take the course because it was not available, while 35.42% because it was expensive and 29.17% referred it to their busy lifestyle. 91% of the study population think that ATLS course should be compulsory and 85% think that the ATLS protocol should be recommended to both junior and senior doctors. 77% of the study participants stated that their seniors teaching skills affect how they apply ATLS. CONCLUSION: Overall junior doctors at Khartoum state hospitals demonstrated a positive attitude towards ATLS, but they showed poor knowledge regarding the topic. It's advised that an earlier training program is introduced by incorporating ATLS course to be part of all final year medical school's curriculum.

Genetic Analysis of the Emerging Citrus Yellow Vein Clearing Virus Reveals a Divergent Virus Population in American Isolates.

Citrus yellow vein clearing virus is a previously reported citrus virus from Asia with widespread distribution in China. In 2022, the California Department of Food and Agriculture conducted a multipest citrus survey targeting multiple citrus pathogens including citrus yellow vein clearing virus (CYVCV). In March 2022, a lemon tree with symptoms of vein clearing, chlorosis, and mottling in a private garden in the city of Tulare, California, tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and noncitrus species, were collected and tested for CYVCV using conventional reverse transcription polymerase chain reaction, reverse transcription quantitative polymerase chain reaction, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using 17 complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study made up the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, the CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.

Outcomes of a Pharmacy Internship Program at a Quaternary Care Hospital.

OBJECTIVE: To design, implement, and evaluate a Hospital-based Pharmacy Internship Program that meets the educational requirements of  pharmacy graduates to register as competent pharmacists in the United Arab  Emirates. METHOD: The Pharmacy Internship Program was designed as a 6-month, full- time, competency-based program. Intern performance was assessed through  monthly continuous evaluations. Interns who successfully completed the  program were eligible to take the Department of Health Licensing Examination. Pharmacy intern surveys were collected to assess their overall satisfaction with the program. RESULTS: Over the previous 5 years, the program has trained 53 interns. All  interns completed the 6-month training program. Of the 53 graduates, 45  completed the post-internship survey. Interns reported a high level of satisfaction with the program structure and content. CONCLUSIONS: The Pharmacy Internship Program structure was successful in its first 5 years of implementation and was both feasible and sustainable. The  program was viewed as highly beneficial for the professional and personal  growth of pharmacy interns and provided them with the necessary competencies and skills to successfully enter the workforce.

OBJETIVO: Diseñar, implementar y evaluar un programa de prácticas de farmacia dentro de un hospital que cumpliera con los requisitos formativos de los graduados en farmacia y les permitiese posteriormente  acceder al examen de licencia para ejercer como farmacéuticos en los Emiratos Árabes Unidos.Método: El programa de prácticas de farmacia fue diseñado como un programa de 6 meses, a tiempo completo, y enfocado a la adquisición de  competencias. El desempeño de los participantes se evaluó minuciosamente mediante un método de evaluación continua mensual. Los  participantes que completaron con éxito el programa podían presentarse al  Examen del Departamento de Salud (examen de licencia) para obtener  la  licencia de farmacéutico. Se realizaron encuestas a los participantes del programa para evaluar su satisfacción general con el mismo. RESULTADOS: El programa ha capacitado a 53 participantes en los últimos 5  años. Todos los participantes completaron el programa de prácticas de 6  meses. De los 53 graduados que participaron, 45 completaron la encuesta  sobre el programa de prácticas. Los participantes manifestaron una gran  satisfacción con el formato y el contenido del programa. CONCLUSIONES: La estructura del programa de prácticas en farmacia hospitalaria fue un éxito en sus primeros 5 años de existencia y  resultó ser viable y sostenible. El programa se consideró altamente beneficioso para el crecimiento profesional y personal de los participantes y les proporcionó las competencias y habilidades necesarias para incorporarse con  éxito al mundo laboral.

Resultados de un programa de prácticasde farmacia en un hospital terciario de referencia / Outcomes of a Pharmacy Internship Program at a Quaternary Care Hospital

Objetivo: Diseñar, implementar y evaluar un programa de prácticas defarmacia dentro de un hospital que cumpliera con los requisitos formativosde los graduados en farmacia y les permitiese posteriormente accederal examen de licencia para ejercer como farmacéuticos en los EmiratosÁrabes Unidos.Método: El programa de prácticas de farmacia fue diseñado como unprograma de 6 meses, a tiempo completo, y enfocado a la adquisiciónde competencias. El desempeño de los participantes se evaluó minuciosamente mediante un método de evaluación continua mensual. Los participantes que completaron con éxito el programa podían presentarse alExamen del Departamento de Salud (examen de licencia) para obtener lalicencia de farmacéutico. Se realizaron encuestas a los participantes delprograma para evaluar su satisfacción general con el mismo.Resultados: El programa ha capacitado a 53 participantes en los últimos 5 años. Todos los participantes completaron el programa de prácticas de 6 meses. De los 53 graduados que participaron, 45 completaronla encuesta sobre el programa de prácticas. Los participantes manifestaron una gran satisfacción con el formato y el contenido del programa.Conclusiones: La estructura del programa de prácticas en farmaciahospitalaria fue un éxito en sus primeros 5 años de existencia y resultóser viable y sostenible. El programa se consideró altamente beneficiosopara el crecimiento profesional y personal de los participantes y les proporcionó las competencias y habilidades necesarias para incorporarsecon éxito al mundo laboral. (AU)

Objective: To design, implement, and evaluate a Hospital-based PharmacyInternship Program that meets the educational requirements of pharmacy graduates to register as competent pharmacists in the United Arab Emirates.Method: The Pharmacy Internship Program was designed as a 6-month,full-time, competency-based program. Intern performance was assessedthrough monthly continuous evaluations. Interns who successfully completed the program were eligible to take the Department of Health LicensingExamination. Pharmacy intern surveys were collected to assess their overall satisfaction with the program.Results: Over the previous 5 years, the program has trained 53 interns.All interns completed the 6-month training program. Of the 53 graduates,45 completed the post-internship survey. Interns reported a high level ofsatisfaction with the program structure and content.Conclusions: The Pharmacy Internship Program structure was successfulin its first 5 years of implementation and was both feasible and sustainable. The program was viewed as highly beneficial for the professional andpersonal growth of pharmacy interns and provided them with the necessary competencies and skills to successfully enter the workforce. (AU)

Transcriptome Analysis of Citrus Dwarfing Viroid Induced Dwarfing Phenotype of Sweet Orange on Trifoliate Orange Rootstock.

Dwarfed citrus trees for high-density plantings or mechanized production systems will be key for future sustainable citrus production. Citrus trees consist of two different species of scion and rootstock. Therefore, any observed phenotype results from gene expression in both species. Dwarfed sweet orange trees on trifoliate rootstock have been produced using citrus dwarfing viroid (CDVd). We performed RNA-seq transcriptome analysis of CDVd-infected stems and roots and compared them to non-infected controls. The identified differentially expressed genes validated with RT-qPCR corresponded to various physiological and developmental processes that could be associated with the dwarfing phenotype. For example, the transcription factors MYB13 and MADS-box, which regulate meristem functions and activate stress responses, were upregulated in the stems. Conversely, a calcium-dependent lipid-binding protein that regulates membrane transporters was downregulated in the roots. Most transcriptome reprogramming occurred in the scion rather than in the rootstock; this agrees with previous observations of CDVd affecting the growth of sweet orange stems while not affecting the trifoliate rootstock. Furthermore, the lack of alterations in the pathogen defense transcriptome supports the term "Transmissible small nuclear ribonucleic acid," which describes CDVd as a modifying agent of tree performance with desirable agronomic traits rather than a disease-causing pathogen.

High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids.

High-throughput nucleic acid extraction is critical for the implementation of modern viroid detection assays. Successful large-scale nursery, field surveys, and other regulatory, quarantine, or research diagnostic programs are increasingly dependent on high-throughput tissue pulverization and nucleic acid extraction protocols. Magnetic bead-based approaches using semi-automated robotic equipment allow high-throughput extraction and purification of high-quality uniform total nucleic acids for each individual sample. Here, we describe a streamlined and optimized protocol for citrus tissue processing and RNA extraction that can be used for downstream applications such as viroid detection by reverse transcription-quantitative polymerase chain reaction.

Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction.

Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction.

Complete Nucleotide Sequence, Genome Organization, and Comparative Genomic Analyses of Citrus Yellow-Vein Associated Virus (CYVaV).

Citrus yellow-vein disease (CYVD) was first reported in California in 1957. We now report that CYVD is associated with a virus-like agent, provisionally named citrus yellow-vein associated virus (CYVaV). The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acid (aa) whereas ORF2 is presumably generated by a -1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.

Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors.

BACKGROUND: Spiroplasma citri comprises a bacterial complex that cause diseases in citrus, horseradish, carrot, sesame, and also infects a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen's wide host range serve as drivers of genetic diversity. This diversity was examined in silico by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC-2, C5, C189, LB 319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas. RESULTS: Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-2 and C5; C189 and R8-A2; BR12, BLH-MB, BLH-13 and LB 319. Strain GII3-3X remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish, and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. CONCLUSIONS: The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1597-2232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

The fundamentals of health economics and its application in the provision of vascular surgery in the UK.

Health economics offers a lifeline to policymakers as a way of improving health outcomes in the face of increased monetary constraints. Doctors are uniquely placed in healthcare delivery where they have a pertinent influence on both supply and demand for healthcare provisions. Every clinical decision made by doctors is also an economic decision, and the true cost of offering a scarce healthcare resource includes not being able to fund alternative therapies and hence, foregoing its benefit. Technology and innovation in medicine is seeing an increase in potential therapies; however, how well do they perform against the current gold standard and are they worth the additional cost? A personalized and patient-centered approach to medicine has paved the way for a holistic health outcome measure, quality-adjusted life years, which is predominately used by United Kingdom resource allocators. Aortic surgical interventions are resource-intensive, and recent trends have shown the growing economic burden as yearly costs continue to climb. Health economic models are not without their weaknesses and it is important that future analyses assess the impact on society, distributional consequences, and the value of collecting more information to reduce the uncertainty of the economic result.

COVID-19: The rising cost of cardiac surgery and disease.

The coronavirus disease 19 (COVID-19) pandemic has resulted in widespread economic, health and social disruptions. The delivery of cardiovascular care has been stifled during the pandemic to adhere to infection control measures as a way of protecting patients and the workforce at large. This cautious approach has been protective since individuals with COVID-19 and cardiovascular disease are anticipated to have poorer outcomes and an increased risk of death. The combination of postponing elective cardiovascular surgeries, reduced acute care and long-term cardiac damage directly resulting from COVID-19 will likely have increased the demand for cardiac care, particularly from patients presenting with more severe symptoms. The combination of increased demand and inhibited supply will likely result in huge backlog of unmet patients' needs. The novelty, virulence and infectivity of severe acute respiratory syndrome coronavirus 2 has caused substantial morbidity and mortality, thus necessitating modifications to the UK government's healthcare strategy. Without improving cost efficiency, the UK's ageing population will likely need an increasing spend on cardiac surgery simply to maintain the same level of service. However, the government's short-term increase in spending is unsustainable especially in the face of ongoing economic uncertainty. This means that the long-term impact of COVID-19 will only increase the need to find innovative ways of delivering equivalent or superior cardiac care at a reduced unit cost.

Undergraduate exposure to patient presentations on the acute medical placement: a prospective study in a London teaching hospital.

OBJECTIVES: To identify the availability and variability of learning opportunities through patient presentations on an acute medical placement at a teaching hospital. DESIGN: A prospective study evaluating all acute admissions to the Acute Medical Unit over 14 days (336 hours). Clinical presentations and the day and time of admission were recorded and compared with the learning outcomes specified in the medical school curriculum. SETTING: An Acute Medical Unit at a London teaching hospital. OUTCOMES: (1) Number of clinical presentations to the Acute Medical Unit over 14 days and (2) differences between the availability and variation of admissions and presentations between in-hours and out-of-hours. RESULTS: There were 359 admissions, representing 1318 presentations. Of those presentations, 76.6% were admitted out-of-hours and 23.4% in-hours. Gastrointestinal bleeding, tachycardia, oedema and raised inflammatory markers were over three times more common per hour out-of-hours than in-hours. Hypoxia was only seen out-of-hours. Important clinical presentations in the curriculum such as chest pain and hemiparesis were not commonly seen. CONCLUSIONS: There is greater availability of presentations seen out-of-hours and a changing landscape of presentations seen in-hours. The out-of-hours presentation profile may be due to expanded community and specialist services. Medical schools need to carefully consider the timing and location of their clinical placements to maximise undergraduate learning opportunities.

Breast ultrasound region of interest detection and lesion localisation.

In current breast ultrasound computer aided diagnosis systems, the radiologist preselects a region of interest (ROI) as an input for computerised breast ultrasound image analysis. This task is time consuming and there is inconsistency among human experts. Researchers attempting to automate the process of obtaining the ROIs have been relying on image processing and conventional machine learning methods. We propose the use of a deep learning method for breast ultrasound ROI detection and lesion localisation. We use the most accurate object detection deep learning framework - Faster-RCNN with Inception-ResNet-v2 - as our deep learning network. Due to the lack of datasets, we use transfer learning and propose a new 3-channel artificial RGB method to improve the overall performance. We evaluate and compare the performance of our proposed methods on two datasets (namely, Dataset A and Dataset B), i.e. within individual datasets and composite dataset. We report the lesion detection results with two types of analysis: (1) detected point (centre of the segmented region or the detected bounding box) and (2) Intersection over Union (IoU). Our results demonstrate that the proposed methods achieved comparable results on detected point but with notable improvement on IoU. In addition, our proposed 3-channel artificial RGB method improves the recall of Dataset A. Finally, we outline some future directions for the research.

Whole genome sequence of five strains of Spiroplasma citri isolated from different host plants and its leafhopper vector.

OBJECTIVES: Spiroplasma citri is a bacterium with a wide host range and is the causal agent of citrus stubborn and brittle root diseases of citrus and horseradish, respectively. S. citri is transmitted in a circulative, persistent manner by the beet leafhopper, Neoaliturus (Circulifer) tenellus (Baker), in North America. Five strains of S. citri were cultured from citrus, horseradish, and N. tenellus from different habitats and times. DNA from cultures were sequenced and genome assembled to expand the database to improve detection assays and better understand its genetics and evolution. DATA DESCRIPTION: The whole genome sequence of five strains of S. citri are described herein. The S. citri chromosome was circularized for all five strains and ranged from 1,576,550 to 1,742,208 bp with a G + C content of 25.4-25.6%. Characterization of extrachromosomal DNAs resulted in identification of one or two plasmids, with a G + C content of 23.3 to 27.6%, from plant hosts; and eight or nine plasmids, with a G + C content of 21.65 to 29.19%, from N. tenellus. Total genome size ranged from 1,611,714 to 1,832,173 bp from plants and 1,968,976 to 2,155,613 bp from the leafhopper. All sequence data has been deposited in DDBJ/ENA/GenBank under the accession numbers CP046368-CP046373 and CP047426-CP047446.

Genome Sequence Resource for Spiroplasma citri, Strain CC-2, Associated with Citrus Stubborn Disease in California.

Spiroplasma citri is a bacterium that causes stubborn disease of citrus and infects other crops, ornamentals, and weeds. It is transmitted by leafhoppers in a circulative manner. Due to limited sequence data on S. citri, the bacterium was isolated from naturally infected Chinese cabbage grown on a farm in Fresno County, CA. DNA from S. citri CC-2 was extracted from a pure culture in LD8 and subjected to PacBio sequencing. Four contigs were obtained with a single circular chromosome of 1,709,192 bp and three plasmids of 40,210, 39,313, and 2,921 bp in size. The genome developed herein extends the sequence database of S. citri and is the first whole-genome sequence record of S. citri from California.

Full genome characterization of 12 citrus tatter leaf virus isolates for the development of a detection assay.

Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.

Breast ultrasound lesions recognition: end-to-end deep learning approaches.

Multistage processing of automated breast ultrasound lesions recognition is dependent on the performance of prior stages. To improve the current state of the art, we propose the use of end-to-end deep learning approaches using fully convolutional networks (FCNs), namely FCN-AlexNet, FCN-32s, FCN-16s, and FCN-8s for semantic segmentation of breast lesions. We use pretrained models based on ImageNet and transfer learning to overcome the issue of data deficiency. We evaluate our results on two datasets, which consist of a total of 113 malignant and 356 benign lesions. To assess the performance, we conduct fivefold cross validation using the following split: 70% for training data, 10% for validation data, and 20% testing data. The results showed that our proposed method performed better on benign lesions, with a top "mean Dice" score of 0.7626 with FCN-16s, when compared with the malignant lesions with a top mean Dice score of 0.5484 with FCN-8s. When considering the number of images with Dice score > 0.5 , 89.6% of the benign lesions were successfully segmented and correctly recognised, whereas 60.6% of the malignant lesions were successfully segmented and correctly recognized. We conclude the paper by addressing the future challenges of the work.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs.

Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701: Citrus Nursery Stock Pest Cleanliness Program". Adopting a compatible multiplex RT-qPCR testing protocol for these viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for virus detection and efficient program implementation.

Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D.

A single real-time multiplex quantitative PCR (qPCR) assay for the simultaneous detection of Grapevine virus A, B and D (GVA, GVB and GVD) was developed, using three different fluorescently labeled minor groove binding probes. This multiplex RT-qPCR was compared to singleplex RT-qPCR designed specifically for each virus and a conventional multiplex RT-PCR. The capacity of the multiplex RT-qPCR assay in detecting the three vitiviruses in mixed infections from a range of virus concentrations in the host was assessed. A series of cDNA derived from 48 different grapevine cultivars obtained from diverse geographical regions infected with various isolates and strains of GVA, GVB and GVD were subjected to singleplex, multiplex RT-qPCR, and conventional multiplex RT-PCR testing. The results showed that the developed multiplex RT-qPCR assay was a cost-effective diagnostic tool that could streamline the testing of grapevine vitiviruses, and replace the singleplex RT-qPCR assays, thus reducing time and labor while retaining the same sensitivity and specificity. In particular, no significant differences in detection limits were found between singleplex and multiplex RT-qPCR and specificity was not affected by the inclusion of the three primer/probe combinations within a multiplex RT-qPCR. Comparing the viral load for each virus using singleplex and multiplex RT-qPCR assays revealed no significant differences between the two assays in detecting GVB and GVD. However, while in detecting GVA using singleplex RT-qPCR assay, viral load was higher. Finally, the multiplex RT-qPCR assay was also more sensitive and time efficient than the conventional multiplex RT-PCR that was designed using degenerate primers to detect GVA, GVB and GVD. This multiplex RT-qPCR method could detect viruses in 95.83% of mixed infected samples as compared to 77.08% for multiplex RT-PCR.