Evaluation and Optimization of Loop-Mediated Isothermal Amplification (LAMP) Technique for Capripoxvirus Diagnosis and Its Comparison with PCR Method

Abstract

Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD) are caused by subspecies of the capripox virus (CaPVs). They are significant pathogens of sheep, goats, and cattle. The causative agent is the capripox virus (CaPV), which was first isolated in South Africa. The viruses responsible for sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD) are morphologically indistinguishable and have been adapted to different host species (4). Serologically, distinguishing between these viruses is challenging, and cross-immunity exists among them (2). The present study reports the evaluation and optimization of a novel loop-mediated isothermal amplification (LAMP) technique for the rapid detection of capripox viruses (CaPVs) and compares it with the polymerase chain reaction (PCR) method. LAMP primers were selected from the P32-protected gene of CaPV. The Safe-Red fluorescent dye was used to monitor the color change from red to bright yellow at a wavelength of 320 nm in positive cases, and the final results were documented through electrophoresis. The proposed LAMP test for the capripox virus demonstrated high specificity and did not cross react with other viruses in the Poxviridae family that present similar clinical symptoms. The optimized LAMP test was then compared with the PCR. The diagnostic sensitivity of LAMP and PCR was found to be identical (100%). The specificity of the LAMP test was evaluated using 30 samples of cow skin that were suspected of lumpy skin disease, along with16 additional samples, including nine positive references, fivenegative references, and two negative controls. A negative reference sample was used to assess the diagnostic sensitivity of LSDV. The proposed LAMP test is simple to implement, cost-effective, and highly sensitive, making it particularly well-suited for the detection of the capripox virus in less developed regions, laboratories, and facilities with limited resources.

Keywords: Capripoxvirus; LAMP; LSDV; PCR.

Figure 1

LAMP at different dilutions 1- 0.1ng dilution, 2- marker 100 kb, 3- 4ng dilution, 4- undiluted, 5- 0.4ng dilution, 6-0.2ng dilution 7-negative control, and 8- 0.05ng dilution.

Figure 2

Perform PCR with different dilutions: 1) negative control, 2) markers kb100, 3) dilutions 0.1 ng, 4) Not diluted, 5) 4 ng dilution, 6) 0.4 ng dilution, 7) 0.2 ng dilution, 8) 0.05 ng dilution, and 9) 0.001 ng dilution

Figure 3

The LAMP test was conducted with varying dilutions: 1) negative control, 2) 0.05 ng dilution, 3) 0.1 ng dilution, 4) 0.2 ng dilution, 5) 0.4 ng dilution, 6) 4 ng dilution, and 7) undiluted.

Figure 4

The LAMP test was conducted on a series of samples, arranged from left to right in the following order: 1) negative control, 2) marker 100 kb, 3) sheep pox RM/65 strain, 4) goat pox Gorgan strain, 5) LSD Neethling strain, 6) camelpox, 7) ecthyma, and 8) healthy cattle skin sample.