Development and comparison of a loop mediated isothermal amplification assay for the rapid diagnosis of lumpy skin disease

Edna W. Macharia; Yatinder S.  Binepal; Justus  Onguso; Roy G. Kiambi; Bramwel W. Wanjala

doi:10.4314/jagst.v23i3.4

Authors

Edna W. Macharia Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
Yatinder S. Binepal Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, Nairobi, Kenya
Justus Onguso Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
Roy G. Kiambi Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, Nairobi, Kenya
Bramwel W. Wanjala Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, Nairobi, Kenya

DOI:

https://doi.org/10.4314/jagst.v23i3.4

Keywords:

Capripoxvirus, LAMP, Lumpy skin disease, PEG extraction, Pen-side test

Abstract

Lumpy skin disease virus is a poxvirus in the genus Capripoxvirus and is closely related to sheeppox virus and goatpox virus. It’s economically important in cattle and a notifiable disease by World Organization for Animal Health. Lumpy skin disease (LSD) is endemic in most parts of Africa with small-scale farmers experiencing the highest loss during outbreaks due to restricted animal trade and costly control and eradication measures. Serological methods of LSD detection are sensitive, inexpensive but can be laborious and time-consuming while, molecular methods such as Polymerase chain reaction (PCR), and real-time PCR/quantitative PCR (qPCR) are sensitive but require expertise and sophisticated laboratories. Loop-mediated isothermal amplification (LAMP) molecular method is advantageous, as it does not require expertise or sophisticated equipment.

This study aimed to develop a rapid, simple, specific, and sensitive detection method for LSD. Sixty-two samples that included skin biopsies, whole blood, serum, and cell cultures were used. New LAMP primer (10_LSD) that could detect lumpy skin disease virus, was designed using Genome based LAMP primer designer (GLAPD) software. Samples were analyzed by LAMP assay and a gold standard (real-time PCR). A LAMP field-based extraction method using polyethylene glycol (PEG) was developed and used for the detection of lumpy skin disease virus. The 10_LSD had a kappa value of 0.32 against the qPCR gold standard. In terms of limit of detection, qPCR had a detection limit of 10-3 ng/µl while 10_LSD had a limit of detection of 1 ng/µl and. The 10_LSD assay showed sensitivity of 60% and a specificity of 86 %. The LAMP assay did not cross-react with closely related viruses like camelpox, Orf virus, and Pestes des Petit Ruminants but could amplify sheeppox virus and goatpox virus. The average time to positivity was 14-28 minutes. The study supports the adoption of the LAMP assay for rapid Capripoxvirus diagnosis as a simpler, effective, and rapid method of detection, monitoring, and controlling outbreaks and the spread of disease in a field set up.