Development Of Real-time RT-PCR Assay Research Articles

Development of real-time RT-PCR assay for diagnosis of viral enteritis in neonatal goat kids

Rotavirus gastroenteritis is a worldwide disease affecting primarily infants, young children and young ones of wide variety of mammalian and avian species. Diarrhoea in goat kids is most frequently found associated with Group A rotavirus (GARV) and another enteric pathogen bovine coronavirus (BCoV), a major viral pathogen associated with neonatal diarrhoea. Enteric BCoV replicates in epithelial cells of gut, destroying villi, resulting in severe, often bloody diarrhoea in calves. It requires highly sensitive and specific assays to diagnose the disease at field level. In the present study, a real-time reverse-transcriptase (RT) polymerase chain reaction (PCR) were developed and validated for specific detection and quantification of GARV and BCoV with high sensitivity and specificity. For real-time RT-PCR, primers were designed to target nucleocapsid gene for BCoV; NSP4 gene and VP6 gene were designed for GARV using discontiguous conserved sequences. Real-time RT-PCR assay was standardized by serial dilution of positive GARV and BCoV RNA. The rotavirus real-time RT-PCR assay was found to be specific to rotavirus, but broadly reactive to GARV. The sensitivity of the assay for detecting rotavirus and BCoV in faecal samples and tissue sample was found to be high in such reactions. The real-time RT-PCR assay was effective in detecting GARV and BCoV in all positive samples obtained from sheds, farms and outbreaks. The results of this study demonstrate that the real-time RT-PCR assay for viral enteritis is broadly reactive, specific, and sensitive for detection of GARV and BCoV in faecal sample and tissue samples.

Development of real-time RT-PCR assays for two viruses infecting pome fruit

Apple stem grooving virus (ASGV) and Apple green crinkle-associated virus (AGCaV) negatively impact production, maintenance, and distribution of apples and other Malus species world-wide. Due to the increasing diversity of isolates found by high-throughput sequencing, we have developed real-time RT-qPCR assays for these two viruses. Primers and probes were designed against alignments of representative extant sequences from around the world, and reaction conditions optimized for sensitivity and specificity. Assays were validated against a panel of virus isolates, and compared to extant endpoint RT-PCR and ELISA assays. The new real-time RT-qPCR assays showed greater detection sensitivity than extant assays and were able to detect their target viruses from different host tissues.

Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus) by qRT-PCR.

Investigation of the immune response of the koala (Phascolarctos cinereus) is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV), which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala’s susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1) and Th2 lymphocyte responses are important to an individual’s susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala’s adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNγ) along with CD4 and CD8β. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A). Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not consistently up-regulated by any of the protocols. Expression of CD4 and CD8β was down-regulated by mitogen stimulation. We found that the reference genes GAPDH and 28s are valid for normalising cytokine expression by koala lymphocytes after mitogen stimulation.

Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conserved fragments of the polymerase region of each virus and were effective for the detection of different isolates tested in this study. The RBDV assay amplified a 94bp amplicon and was able to detect as few as 30 viral copies. Whereas the RLMV assay amplified a 180bp amplicon and detected as few as 300 viral copies from plant and aphid RNA extracts. Both assays were significantly more sensitive than their corresponding conventional RT-PCR methods. The sensitivity of the RLMV assay was also tested on single aphids after a fixed acquisition access period (AAP). In addition, the assays revealed a novel synergistic interaction between the two viruses, where the concentration of RBDV was enhanced ∼400-fold when it occurred in combination with RLMV compared to its concentration in single infections. The significance of this finding and the importance of the development of real-time RT-PCR assays for the detection of RBDV and RLMV are discussed.

Development of real-time RT-PCR assays for the detection of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in the whitefly vector Bemisia tabaci

Reverse transcription followed by real-time PCR assays based on TaqMan ® chemistry have been developed for the detection and quantification of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci. The method includes an internal control for the detection of a gene from B. tabaci to compensate for variations in extraction efficiency. The assays designed were used to estimate proportions of viruliferous whiteflies collected from commercial greenhouse-grown crops in Spain. In a significant number of whiteflies, both viruses were detected and their amounts were estimated. The assays could be used to assist risk assessment of CVYV and CYSDV which constitute limiting factors in cucurbit crops. They are also suited to investigating the epidemiology and plant–virus–vector relationships in these diseases.

Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are key factors in the brain-pituitary-gonad axis and understanding their regulation remains essential for future management of eel reproduction. In this regard, we developed quantitative real-time RT-PCR (qrtRT-PCR) assays for the expression of European eel LHβ, FSHβ and GPα subunits, using the Light Cycler system. The qrtRT-PCR was adapted to permit detection of the three gonadotropin subunit mRNAs in individual pituitaries and in dispersed pituitary cells. The validated assays were applied to investigate the effects of sex steroids (estrogens and androgens) on gonadotropin subunit expression, in vivo in steroid-injected eels, and in vitro by steroid treatments of primary cultures of eel pituitary cells. In vivo, a stimulation of LHβ mRNA was observed after estradiol (E2) treatments, while testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT) had no effect. Concerning FSHβ expression, slight but non-significant decreases were observed after both E2 and androgen treatments. Different results were obtained in vitro: E2 induced an increase in FSHβ mRNA levels but had no effect on LHβ expression. In contrast, androgens (T and DHT) stimulated LHβ expression while no significant variation was observed on FSHβ mRNA levels following androgen treatment. Concerning the GPα mRNA, no significant effect of sexual steroids was observed in vivo or in vitro. This demonstrated specific direct actions of steroids on gonadotropin subunit expression. The differences observed between in vivo and in vitro experiments may be explained by the involvement of cerebral control, including GnRH and dopamine neurons, and their specific regulation by sex steroids. The data indicate that sex steroid feedbacks on gonadotropins are exerted via multiple pathways, indirectly at the brain level and directly on pituitary gonadotrope cells.

Novel one-step real-time RT-PCR assay for rapid and specific diagnosis of Crimean-Congo hemorrhagic fever encountered in the Balkans

Early and accurate diagnosis of Crimean-Congo hemorrhagic fever (CCHF) is essential for the treatment and outcome of the disease and prevention of its further transmission. Molecular-based diagnostic assays now serve as the front-line tool in the diagnosis of CCHF. However, the development of real-time RT-PCR assay for the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) has been hampered by a virus strain variation. The development of a one-step real-time RT-PCR assay for the detection of CCHFV is described herein. The technique is based on the fluorescence resonance energy transfer probe technology employing the endonuclease activity of Taq polymerase enzyme. The assay was designed to detect specifically the strains from a phylogenetic cluster of CCHFV which encompasses the known CCHFV strains circulating in the Balkan region. The detection system was tested using CCHFV strain Kosovo Hoti, clinical serum samples and ticks. The real-time assay described is rapid, specific and sensitive. Since the Balkan peninsula is also an endemic region for hemorrhagic fever with renal syndrome (HFRS), this method is suggested as convenient for early differential diagnosis of suspected viral hemorrhagic fever patients.