A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection

Abstract

BackgroundCrimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.Methodology/principle findingsAn isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.Conclusions/significanceThe assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

Diagnostic tests for Crimean-Congo haemorrhagic fever: a widespread tickborne disease

“…Quantitative real-time RT-PCR (qRT-PCR) has better performance over conventional RT-PCR or nested RT-PCR, with a lower contamination rate, higher sensitivity and specificity, and better time-effectiveness59 65; laboratories performing only nested or conventional RT-PCR have been advised to implement qRT-PCR to improve assay performance and viral load determination. An isothermal NAAT test has been developed based on recombinase polymerase amplification, enabling amplification at a single temperature in a more ‘crude’ sample which may prove more amenable as a field diagnostic or in low-resource laboratories 82. Next-generation sequencing (NGS) enables comprehensive genome analysis and has been used for CCHFV phylogeny83 84; however, this complex and expensive approach is not currently practical for diagnostic screening.…”

Rapid molecular detection of macrolide resistance

“…Eukaryotic pathogens detected with RPA include the blood-fluke Schistosoma japonicum [15] and the diarrheal protozoan pathogens Giardia, Cryptosporidium, and Entamoeba [17,18]. Viral pathogens detected by RPA include HIV [19,20], Chikungunya virus (CHIKV) [14], Rift Valley Fever virus [21,22], Middle East respiratory syndrome coronavirus [23], foot-and-mouth disease virus (FMDV) [24], Bovine Coronavirus [25], and Crimean-Congo Haemorrhagic fever Virus (CCHFV) [26]. Bacterial pathogens detected by RPA include Mycoplasma tuberculosis [27,28], Neisseria gonorrhoeae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus (MRSA) [29], Chlamydia trachomatis [30], Francisella tularensis [31], Group B Streptococci [32], Orientia tsutsugamushi (scrub typhus), and Rickettsia typhi (murine typhus) [16].…”

Evaluación de la técnica de amplificación por recombinasa y polimerasa (RPA) para la detección de begomovirus presentes en cultivos de soja y poroto en Argentina

“…El RPA ha sido ampliamente usado para la detección de numerosos patógenos en humanos y animales (Bonney et al, 2017;Boyle et al, 2013;Gao, Jiang, Wang y Wei, 2018;Kim y Lee, 2017;Law et al, 2018;Wang, Liu, Wang, Pang y Yuan, 2018), y más recientemente para enfermedades en plantas, incluyendo la detección de begomovirus (Londoño et al, 2016) igura 1. Alineamiento de la cápside proteica de diferentes especies de begomovirus detectadas en soja y poroto.…”