Journal of Virological Methods Minor Groove Binder Modification of Widely Used Taqman Hydrolysis Probe for Detection of Dengue V
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Journal of Virological Methods 268 (2019) 17–23 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Minor groove binder modification of widely used TaqMan hydrolysis probe for detection of dengue virus reduces risk of false-negative real-time PCR T results for serotype 4 ⁎ Eleanor R. Graya,b, , Judith Heaneyc, R. Bridget Fernsb,d, Patricia C. Sequeirae, Eleni Nastoulic,f, Jeremy A. Garsonb,g a London Centre for Nanotechnology, Faculty of Maths and Physical Sciences, University College London, London, United Kingdom b Department of Clinical Virology, University College London Hospitals NHS Foundation Trust, London, United Kingdom c Advanced Pathogen Diagnostics Unit, University College London Hospitals NHS Foundation Trust, London, United Kingdom d Division of Infection and Immunity, University College London, London, United Kingdom e Flavivirus Laboratory, Instituto Oswaldo Cruz/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil f Department of Population, Policy and Practice, UCL GOS Institute of Child Health, London, United Kingdom g National Transfusion Microbiology Laboratories, NHS Blood and Transplant, London, United Kingdom ARTICLE INFO ABSTRACT Keywords: Dengue is a vector-transmitted viral infection that is a significant cause of morbidity and mortality in humans Dengue worldwide, with over 50 million apparent cases and a fatality rate of 2.5 % of 0.5 million severe cases per annum TaqMan in recent years. Four serotypes are currently co-circulating. Diagnosis of infection may be by polymerase chain False-negative reaction, serology or rapid antigen test for NS1. Both pan-serotype and serotype-specific genome detection as- Sensitivity says have been described, however, achieving adequate sensitivity with pan-serotype assays has been challen- Serotype 4 ging. Indeed, as we show here, inspection of components and cycling parameters of a pan-serotype RT-qPCR Minor groove binder assay in use in laboratories worldwide revealed insufficient probe stability to accommodate potential nucleotide mismatches, resulting in false-negatives. A minor-groove binder (MGB)-modified version of the probe was de- signed and its performance compared with that of the original probe in 32 samples. Eight of the samples were undetected by the original probe but detected by the MGB modified probe and six out of seven of these that could be serotyped belonged to serotype 4. Sequencing of the region targeted by the probe in these samples revealed two mismatches which were also universally present in all other serotype 4 sequences in a public database. We therefore recommend adoption of this MGB modification in order to reduce the risk of false-negative results, especially with dengue serotype 4 infections. 1. Introduction et al., 2016; Halstead, 2017; World Health Organization, 2017). A vaccine would need to prevent infection in the infection-naïve, such as Dengue, caused by a member of the family Flaviviridae, is the most travellers to dengue-endemic regions, and neonates in endemic coun- common arboviral infection worldwide. Globally, case incidence is tries, as well as those who have experienced primary infection. Re- thought to be 50–100 million per annum, though the true number may commendations for the only licensed vaccine currently available (the be higher as not all infections are apparent and misclassification may Pasteur CYD-TDV) are limited to those aged 9–45, who are likely to occur in regions with a high burden of febrile illness (Bhatt et al., 2013; have previously been exposed to infection (Sridhar et al., 2018; Wilder- Stanaway et al., 2016). Clinical infections range widely in severity from Smith et al., 2018). asymptomatic to dengue haemorrhagic fever and death. Other than Four serotypes co-circulate in most endemic countries, and these are supportive therapy no specific treatment is available and as yet there is genetically variable (Costa et al., 2012). Initial infection with any ser- no effective vaccine universally recommended for those at risk (Aguiar otype gives type-specific lifelong immunity, however, prior infection Abbreviations: MGB, minor groove binder; qPCR, quantitative polymerase chain reaction; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Cq, quantification cycle (sometimes referred to as Ct or threshold cycle) ⁎ Corresponding author at. London Centre for Nanotechnology, 17-19 Gordon St, London, WC1H 0AH, United Kingdom. E-mail address: [email protected] (E.R. Gray). https://doi.org/10.1016/j.jviromet.2019.03.006 Received 31 May 2018; Received in revised form 8 March 2019; Accepted 10 March 2019 Available online 11 March 2019 0166-0934/ © 2019 Elsevier B.V. All rights reserved. E.R. Gray, et al. Journal of Virological Methods 268 (2019) 17–23 Fig. 1. Representatives of the four dengue serotypes showing primer/probe binding sites. The sequences are: serotype 1 Hawaii (EU848545); serotype 2 New Guinea C (AF038403); serotype 3 Philippines H87 (M93130); serotype 4 Philippines H241 (AY947539). F = forward primer; P = probe; R1 = reverse primer 1; R2 = reverse primer 2. The genomic numbering refers to AF038403 (serotype 2). The black shading represents the single nucleotide overlap between the probe and the reverse primer. with one serotype can predispose to a more serious episode on sub- requirements for diagnostic assay development for Samples #1-21 and sequent infection with a different serotype (Halstead et al., 1970; the Ethical Screening Committee of Fiocruz (CAAE): Katzelnick et al., 2017). 90249218.6.1001.5248 (2.998.362) for Samples #22-32. Diagnosis of dengue is on clinical suspicion based on symptoms and travel history; in addition a wide variety of laboratory methods are in 2.3. Nucleic acid extraction current use. Blood samples may be analysed for RNA, IgM and IgG or NS1 antigen; the presence of RNA and/or IgM suggesting acute infec- RNA was extracted from plasma samples manually using the Qiagen tion. Point of care diagnostic tests are also available, although the viral RNA mini kit (Qiagen, Hilden, Germany) according to the manu- fi sensitivity of these tests when performed in the eld has been variable facturer’s instructions (except volumes as follows) or the Qiagen EZ1 (Pang et al., 2017; Sa-ngamuang et al., 2018). Molecular techniques are BioRobot. Sample input and elution volumes for manual extraction widely used, though the resource requirements for these tests mean that were matched to the BioRobot for the samples from UCLH: 200 μl their implementation may not be feasible in resource-limited settings. sample input and 90 μl elution. For samples from Brazil, only 100 μl was Molecular tests to detect viraemic infections include qualitative/nested available, which was supplemented with 100 μl negative normal human PCR, reverse-transcription quantitative PCR (RT-qPCR) and isothermal serum, and extracted as described. Extracted RNA was stored at -80°C. amplification methods (Domingo et al., 2010; Poersch et al., 2005). Some tests seek to differentiate serotypes for epidemiological purposes, 2.4. Reverse-transcription quantitative polymerase chain reaction (RT- while others are intended to be pan-serotype. Serotyping of dengue qPCR) infection has limited applicability in immediate clinical management. However, epidemiological surveys are important to assess whether a Real-time RT-qPCR was performed in accordance with the MIQE predominant serotype in a locale is supplanted, bringing with it the risk guidelines (Bustin et al., 2009) using TaqMan Fast Virus 1-Step Master of secondary infections and more severe consequences. Mix (cat. no. 4444432, Life Technologies, Paisley, UK), primers at A widely cited pan-serotype RT-qPCR assay published in 2002 600 nM and probe at 100 nM (sequences as in (Drosten et al., 2002) and (Drosten et al., 2002) that is apparently used by laboratories in many Fig. 1, synthesized by Sigma, Haverhill, UK); either a 6-carboxy- countries (Domingo et al., 2010) was identified in a literature search fluorescein (FAM)/ tetramethylrhodamine (TAMRA) probe for the and evaluated using archived samples. An initial failure to detect one of ‘original’ assay (Drosten et al., 2002) or a FAM/ minor groove binder four samples tested led to a closer inspection of this assay and, as a (MGB)-non-fluorescent quencher probe (synthesized by Life Technolo- result, to its improvement through minor groove binder (MGB) mod- gies) for the MGB-modified assay (Fig. 1). Each well contained 15 μl ification of its TaqMan probe (i.e. 5’ hydrolysis probe). The MGB- total reagents with 1 μl RNA extract, and each condition was run in modified and un-modified probe (herein designated the ‘original’ triplicate wells. Cycling conditions (ABI 7500 thermal cycler, Applied probe) assays were tested on a larger group of samples and two se- Biosystems) were: 50 °C 5 min, 95 °C 20 s, then 45 cycles of 95 °C 3 s, 60 quence mismatches between the ‘original’ probe and dengue serotype 4 °C 1 min. Negative controls (H O) were run on every plate. Data were sequences were identified. The MGB modification was thus shown to 2 analysed using the ABI 7500 software v2.0.6. prevent false-negative results being generated with serotype 4 samples. 2.5. Synthesis of RNA by in vitro transcription 2. Materials and methods Transcripts used to generate the dengue RNA dilution series were 2.1. Samples synthesised using the T7 MegaScript kit (Life Technologies) and PCR amplicons as template, amplified from Sample #14 (serotype 2). RNA Samples #1-21: Archived EDTA-plasma samples from University from this clinical sample was initially reverse transcribed using College London Hospital that had been submitted for arbovirus testing Superscript IV and random hexamers (both Life Technologies). between January and August 2016 were reviewed. 21 samples that had Amplified DNA template was produced by nested PCR using primers been reported elsewhere as positive for dengue virus RNA were iden- EG273/EG250 then EG295/EG296 (Table 1) and Platinum SuperFi fi ti ed and retrieved from storage at -20 °C The total storage time at polymerase (cat.