N-Cap RNA: protected RNAs for in-tube diagnostic controls and inter-laboratory standards

Lead Research Organisation: John Innes Centre
Department Name: Biological Chemistry

Abstract

Amplification of RNA from crude samples for diagnostic or screening purposes can fail at many stages during the isolation, clean-up and amplification processes. Such failures produce false negative results, which are not acceptable when screening critical samples, for instance during outbreaks of epidemic viral disease in animals or humans. It is therefore highly desirable, if not essential, to include control RNA at the earliest possible step in the process, so that a positive control signal can validate every step from sample acquisition to final amplification and detection. However RNA is a fragile molecule and an unprotected RNA standard is very rapidly degraded. We have shown that this degradation can be prevented by encapsulating the RNA within a plant virus particle (N-cap RNA) and, furthermore, that such particles can act as highly effective in-tube controls. We now need to develop methods for the rapid production of such controls for a wide range of target diseases.

Publications

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Madi M (2015) Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus. in Journal of virological methods

 
Description We have developed an efficient way of producing positive in-tube controls for real-time PCR-based diagnostic tests. These controls are based on a disabled version of cowpea mosaic virus (CPMV). The use of a disabled virus means that the controls can be deployed without the need for containment. Several commercial organisations have expressed an interest in adopting this technology.
Exploitation Route Originally the controls were intended for use in conjunction with veterinary diagnostic reactions but it is now clear they can be deployed in the human health sector. It is also apparent that the technology could be used as a means of delivering bespoke RNA molecules.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description The findings have been used to develop standards for use in diagnostic PCR reactions and for proficiency testing. They are making a vital contribution to the in-field diagnosis of infectious diseases. They have also been used as generic method for the specific encapsidation of bespoke RNA molecules into CPMV capsids and the general approach developed during the project has helped unravel the link between RNA encapsidation and replication in the order Picornavirales. The principles derived from this work were used to develop Covid-19 diagnostic reagents and a potential method for the delivery of mRNA into cells.
First Year Of Impact 2014
Sector Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description BBSRC Responsive mode
Amount £800,000 (GBP)
Funding ID BB/L020955/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description Institute Development Grant (IDG) Funding
Amount £12,000 (GBP)
Organisation John Innes Centre 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 01/2015
 
Description Trojan Horse: Using virus-like particles as RNA delivery devices in invertebrates as a pest-control strategy
Amount £553,762 (GBP)
Funding ID BB/V009087/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2021 
End 03/2024
 
Title Encapsidated mimics 
Description A method for producing in-tube positive control reagents for diagnosis or proficiency testing. The method is based on using capsids of cowpea mosaic virus (CPMV) to protect RNAs from degradation in field samples, The encapsidated mimic RNA consists of a version of CPMV RNA-2 containing a deletion in the region encoding the viral movement protein (48K protein) plus sequences from the target pathogen which can be detected by PCR-based methods. The deletion in the 48K protein prevents spread of the virus in the environment. The method can be adapted as detection method for a wide variety of pathogens. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The detection of foot and mouth disease virus (FMDV) and proficiency testing in sub-Saharan Africa 
 
Description Encapsiidate viral mimics 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The team at JIC produced the virus particles containing deleted versions of cowpea mosaic virus (CPMV) RNA-2. The RNA within these particles is also modified to contain test sequences derived from animal virus pathogens that can be detected using PCR-based methods. The encapsidated mimic RNAs can act as in-tube positive controls for diagnostic and proficiency testing applications.
Collaborator Contribution The Pirbright Instituted tested encapsidated mimics containing sequences specific for foot and mouth disease virus (FMDV) and showed that they were effective in-tube positive controls for the diagnosis of FMDV in crude samples.
Impact Publication (Madi et al., 2015) Design of further mimic constructs based on the technology developed during this project. Use of the FMDV construct for diagnosis of infection and proficiency testing in sub-Saharan Africa.
Start Year 2014
 
Description Talk at APHA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Deliver presentation entitled "A Leaf Expression platform for the production of vaccine candidate materials, mAbs and for in vitro diagnosis" at the Animal and Plant Health Agency (APHA), Weybridge, UK.
Year(s) Of Engagement Activity 2017