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Article

A Novel RT-LAMP for the Detection of Different Genotypes of Crimean–Congo Haemorrhagic Fever Virus in Patients from Spain

by
Begoña Febrer-Sendra
1,
Pedro Fernández-Soto
1,*,
Juan García-Bernalt Diego
1,
Beatriz Crego-Vicente
1,
Anabel Negredo
2,
Juan Luis Muñor-Bellido
3,
Moncef Belhassen-García
4,
María Paz Sánchez-Seco
2 and
Antonio Muro
1
1
Infectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca-Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, Spain
2
Laboratorio de Arbovirus, Centro Nacional de Microbiología, CIBER de Enfermedades Infecciosas (Instituto de Salud Carlos III), 28222 Madrid, Spain
3
Microbiology and Parasitology Service, Complejo Asistencial Universitario de Salamanca, University of Salamanca, 37007 Salamanca, Spain
4
Internal Medicine Service, Infectious Diseases Section, Complejo Asistencial Universitario de Salamanca, University of Salamanca, 37007 Salamanca, Spain
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(7), 6411; https://doi.org/10.3390/ijms24076411
Submission received: 9 February 2023 / Revised: 25 March 2023 / Accepted: 27 March 2023 / Published: 29 March 2023
(This article belongs to the Special Issue Application of Advanced Molecular Methods to Study Infections)
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Abstract

Crimean–Congo haemorrhagic fever (CCHF) is a potentially lethal tick-borne viral disease with a wide distribution. In Spain, 12 human cases of CCHF have been confirmed, with four deaths. The diagnosis of CCHF is hampered by the nonspecific symptoms, the high genetic diversity of CCHFV, and the biosafety requirements to manage the virus. RT-qPCR and serological tests are used for diagnosis with limitations. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) could be an effective alternative in the diagnosis of the disease. However, none of the few RT-LAMP assays developed to date has detected different CCHFV genotypes. Here, we designed a RT-LAMP using a degenerate primer set to compensate for the variability of the CCHFV target sequence. RT-LAMP was performed in colorimetric and real-time tests on RT-qPCR-confirmed CCHF patient samples notified in Spain in 2020 and 2021. Urine from an inpatient was analysed by RT-LAMP for the first time and compared with RT-qPCR. The amplicons obtained by RT-qPCR were sequenced and African III and European V genotypes were identified. RT-LAMP amplified both genotypes and was more sensitive than RT-qPCR in urine samples. We have developed a novel, rapid, specific, and sensitive RT-LAMP test that allows the detection of different CCHFV genotypes in clinical samples. This pan-CCHFV RT-LAMP detected viral RNA for the first time in urine samples. It can be easily performed as a single-tube isothermal colorimetric method on a portable platform in real time and without the need for expensive equipment, thus bringing molecular diagnostics closer to rural or resource-poor areas, where CCHF usually occurs.
Keywords: Crimean–Congo haemorrhagic fever virus; CCHFV; RT-LAMP; genotypes; Spain Crimean–Congo haemorrhagic fever virus; CCHFV; RT-LAMP; genotypes; Spain

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MDPI and ACS Style

Febrer-Sendra, B.; Fernández-Soto, P.; García-Bernalt Diego, J.; Crego-Vicente, B.; Negredo, A.; Muñor-Bellido, J.L.; Belhassen-García, M.; Sánchez-Seco, M.P.; Muro, A. A Novel RT-LAMP for the Detection of Different Genotypes of Crimean–Congo Haemorrhagic Fever Virus in Patients from Spain. Int. J. Mol. Sci. 2023, 24, 6411. https://doi.org/10.3390/ijms24076411

AMA Style

Febrer-Sendra B, Fernández-Soto P, García-Bernalt Diego J, Crego-Vicente B, Negredo A, Muñor-Bellido JL, Belhassen-García M, Sánchez-Seco MP, Muro A. A Novel RT-LAMP for the Detection of Different Genotypes of Crimean–Congo Haemorrhagic Fever Virus in Patients from Spain. International Journal of Molecular Sciences. 2023; 24(7):6411. https://doi.org/10.3390/ijms24076411

Chicago/Turabian Style

Febrer-Sendra, Begoña, Pedro Fernández-Soto, Juan García-Bernalt Diego, Beatriz Crego-Vicente, Anabel Negredo, Juan Luis Muñor-Bellido, Moncef Belhassen-García, María Paz Sánchez-Seco, and Antonio Muro. 2023. "A Novel RT-LAMP for the Detection of Different Genotypes of Crimean–Congo Haemorrhagic Fever Virus in Patients from Spain" International Journal of Molecular Sciences 24, no. 7: 6411. https://doi.org/10.3390/ijms24076411

APA Style

Febrer-Sendra, B., Fernández-Soto, P., García-Bernalt Diego, J., Crego-Vicente, B., Negredo, A., Muñor-Bellido, J. L., Belhassen-García, M., Sánchez-Seco, M. P., & Muro, A. (2023). A Novel RT-LAMP for the Detection of Different Genotypes of Crimean–Congo Haemorrhagic Fever Virus in Patients from Spain. International Journal of Molecular Sciences, 24(7), 6411. https://doi.org/10.3390/ijms24076411

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