Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Background: Respiratory Syncytial Virus (RSV) is the leading cause of severe lower
respiratory tract infection in infants and children worldwide. Early diagnosis of RSV infection
is associated with shorter periods of hospitalisation and decreased mortality. Current point of
care (PoC) tests for RSV is less sensitive than molecular methods. Reverse transcription
loop-mediated isothermal amplification (RT-LAMP), is a novel method of nucleic acid
detection which allows for rapid, robust amplification, and visual detection of infectious
agents.
Aim: The objective of this study was to develop a novel, rapid, and sensitive multiplex RSV
RT-LAMP assay for PoC diagnosis of RSV A and B.
Methods: Preparation of a quantitative RSV standard for assay optimisation was done using
a rapid hypotonic burst recovery method of infective virus during sub-passaging, and a shell
vial fluorescent focus assay for titration of culture-derived viral stock. We designed a single
set of eight primers targeting the large polymerase gene of both RSV A and B, and
developed a novel single-step multiplex RSV RT-LAMP assay, using an in-house reaction
mix and the Rotor-Gene Q real-time thermocycler (Qiagen, Hilden, Germany). The metal ion
indicator hydroxy naphtol blue (HNB) was added to the multiplex RSV RT-LAMP assay for
visual detection of RSV.
Results: The final optimised multiplex RSV RT-LAMP assay had an analytical detection
sensitivity of <10 focus forming units (FFU) per reaction for both RSV A and B, with a mean
time to positivity of 21.85 minutes (95% CI 19.2-24.5 minutes), compared to 90-120 minutes
for conventional PCR. Evaluated against the Seeplex RV15 multiplex PCR (Seegene, Seoul,
Korea) by testing 44 (22 RSV A/22 RSV B) nasopharyngeal specimens, the multiplex RSV
RT-LAMP assay had a sensitivity of 100%, and a specificity of 100% when screened against
nine common respiratory viruses. Visual detection of RSV using HNB as colorimetric reagent
was equivalent to the analytical sensitivity (10 FFU/reaction) and specificity (100%) of the
multiplex RSV RT-LAMP assay.
Conclusion: Compared with conventional PCR, our novel single-step multiplex RSV RTLAMP
assay had excellent sensitivity, specificity, and when combined with HNB dye could
provide accurate visual diagnosis within 1 hour. We envisage that this multiplex RSV RTLAMP
assay will be used for rapid and sensitive RSV detection at the PoC. / AFRIKAANSE OPSOMMING: Agtergrond: Respiratoriese Syncytial Virus (RSV) is die hoof oorsaak van erge laer
lugweginfeksie in babas en kinders wêreldwyd. Vroeë diagnose van RSV infeksie word
geassosieer met korter periodes van hospitalisasie en verlaagde mortaliteit. Huidige punt van
sorg (PoC) toetse vir RSV is minder sensitief as molekulêre metodes. Omgekeerde
transkripsie lus-gemedieerde isotermiese amplifisering (RT-LAMP), is 'n nuwe metode van
nukleïensuur opsporing wat voorsiening maak vir vinnige, doeltreffende amplifisering, en
visuele bevestiging van aansteeklike agente.
Doel: Die doel van hierdie studie was om 'n nuwe, vinnige en sensitiewe multipleks RSV RTLAMP
toets te ontwikkel wat PoC diagnose van RSV A en B in staat stel.
Metodes: Voorbereiding van 'n kwantitatiewe RSV standaard vir toets optimisering is gedoen
met behulp van 'n hipotoniese sel-lise metode van infektiewe virus tydens sub-kultuur, en 'n
“shell-vial” kultuur en fluorosensie fokus toets vir titrasie van kultuur-geproduseerde virus
voorraad. Ons het 'n enkele stel van agt inleiers ontwerp wat gebaseer is op die groot
polimerase geen van beide RSV A en B, en 'n nuwe enkel-stap multipleks RSV RT-LAMP
toets ontwikkel, met gebruik van 'n in-huis reaksie mengsel en die Rotor-Gene Q “real-time”
thermocycler (Qiagen, Hilden, Duitsland). Die metaalioon aanwyser hidroksi naphtol blou
(HNB) is bygevoeg in die multipleks RSV RT-LAMP toets vir visuele bevestiging van RSV.
Resultate: Die finale geoptimiseerde multipleks RSV RT-LAMP toets het 'n analitiese
sensitiwiteit van <10 fokus vormende eenhede (FFU) per reaksie vir beide RSV A en B
gehad, met 'n gemiddelde tyd tot positiwiteit van 21.85 minute (95% CI 19.2-24.5 minute) , in
vergelyking met 90-120 minute vir konvensionele PCR. Geëvalueer teen die Seeplex RV15
multipleks PCR (Seegene, Seoul, Korea) deur 44 (22 RSV A/22 RSV B) nasofaringeale
monsters te toets, het die multipleks RSV RT-LAMP toets 'n sensitiwiteit van 100% getoon,
en 'n spesifisiteit van 100% wanneer getoets teen nege algemene respiratoriese virusse.
Visuele bevestiging van RSV met gebruik van HNB as kolorimetriese reagens was
gelykstaande aan die analitiese sensitiwiteit (10 FFU/reaksie) en spesifisiteit (100%) van die
multipleks RSV RT-LAMP toets.
Gevolgtrekking: In vergelyking met konvensionele PCR, het ons nuwe enkel-stap multipleks
RSV RT-LAMP toets uitstekende sensitiwiteit, spesifisiteit, en wanneer dit gekombineer word
met HNB kleurstof kon dit akkurate visuele diagnose voorsien binne 1 uur. Ons verwag dat
hierdie multipleks RSV RT-LAMP toets gebruik sal word vir vinnige en sensitiewe RSV
bevestiging by die PoC.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96670 |
| Date | 12 February 2015 |
| Creators | Hart, Dirk |
| Contributors | van Zyl, Gert U., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | x, 66 pages : colour illustrations |
| Rights | Stellenbosch University |
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