Orf is distributed throughout many countries [2, 17–23], and ORFV infections can cause weight loss and poor development as the disease prevents host animals from feeding. Therefore, the development of a rapid, simple and sensitive detection method for ORFV infections is required. For the diagnosis of ORFV infections, the LAMP assay has many advantages, such as simplicity, rapidity and inexpensiveness, compared with other nucleic acid-based tests [8, 9]. In addition, previous reports have demonstrated that the sensitivity of the LAMP is higher than that of conventional PCR or nested PCR for detecting ORFV infections [8, 9]. Therefore, this method shows great potential for use in resource-limited veterinary laboratories in developing countries (e.g., China), where many endemic diseases exist. In the present study, we evaluated the sensitivity and specificity of the rapid diagnostic LAMP method.
The simplest way of detecting LAMP products is to visually inspect the white turbidity that results from magnesium pyrophosphate accumulation as a by-product of the reaction . However, a small amount of this white precipitate is not always distinguishable from other white precipitates, such as proteins or carbohydrates that can be derived from the templates. Therefore, existing “field-friendly” LAMP-based detection systems are still imperfect. Previous report has demonstrated that amplified DNA can be stained (using PicoGreen or ethidium bromide) and visualized in solution with the same sensitivity as agarose gel electrophoresis . In this study, we used the dye SYBR Green I to detect the amplified DNA products. Positive and negative reactions could be differentiated by distinctly different colors when viewed under daylight conditions against a black background (Figure 1). Consistent with previous report, this color inspection method was found to have the same detection limit as agarose gel electrophoresis (Figure 2), which should facilitate the rapid screening of samples without the need for gel electrophoresis. These results indicate that visual color inspection of LAMP products using the SYBR Green I dye should be practical under field conditions.
The reliability of a LAMP assay depends largely on the specificity of the primer sets being used. These primer sets in this study—two outer primers (F3 and B3) and two inner primers (FIP and BIP)—allow for the recognition of six sites in the target sequence that are specific to the F1L gene and that are necessary for the LAMP reaction to occur. Based on the sequences of the ORFV F1L gene available in GenBank and the sequences of ORFV isolates from China, we designed and tested several sets of primers using comparative experiments, and the primer sets that yielded the highest specificity and sensitivity in the LAMP assay is reported here. Our LAMP assay showed high specificity and sensitivity, as it yielded positive results for all 10 of the known ORFV isolates but did not amplify the negative control samples (Figure 3).
Samples from experimentally infected goats were tested using both the LAMP and real-time PCR assays. The LAMP assay correctly identified 41 out of 46 samples as positive (89.13%), and the real-time PCR assay correctly identified 42 out of 46 samples as positive (91.30%). ORFV DNA could be detected in most of the intracutaneous- and cohabitation-infection samples from the time of lesion presentation to recovery (Table 1). Therefore, we observed a very good agreement between the LAMP and real-time PCR results. Only one sample was identified as negative by LAMP (both by visual inspection and agarose gel electrophoresis) but as positive by real-time PCR. This discrepancy was most likely due to very low ORFV levels in this sample, which may have been beyond the detection limit of LAMP. This result suggests that the sensitivity of LAMP is slightly lower than that of real-time PCR. However, it is important to consider that real-time PCR is a time-consuming procedure that requires expensive and relatively complicated equipment, including a thermal cycler with real-time monitoring and data-analysis systems. Therefore, this LAMP-based assay has clear advantages over real-time PCR in terms of practicality, and it can be easily used in any standard diagnostic laboratory, particularly in developing countries where the disease is prevalent.