Since prevention and early detection are the most logical strategies for pathogen control, the most effective method of disease control is routine screening for pathogens . Sensitive and rapid methods are required for the detection of PCMV under field conditions. However, thus far, there is no practical, simple and rapid method for the diagnosis of PCMV under field conditions.
The detection of PCMV DNA is often performed using PCR-based assays, and the majority of these assays are developed in-house. The individual laboratory determines the performance, verification and validation of such assays. As a result, these assays may vary with regard to specimen type, target DNA, nucleic acid extraction method, or detection method. There is a need for a standardized assay to detect PCMV DNA that can be broadly applied in clinical practice and enable the establishment of clinically significant cutoffs .
LAMP has successfully been used to diagnose pathogenic infections in humans and animals [35, 36]. In our study, no cross-reaction with the other viruses tested was noted in the LAMP assay, similar to the case with PCR. Furthermore, the specificity of LAMP was not affected by non-target genomic DNA in the reaction mixture, which is a highly desirable trait in a diagnostic system . As shown by the results of the present study, the LAMP method was highly sensitive for the detection of PCMV. Consistent with previous reports, LAMP showed the same level of sensitivity as PCR [37, 38].
The optimal conditions for PCMV detection by LAMP were determined to be 63–65°C for 40 min. However, the LAMP assay involves fewer steps than the PCR assay, and does not require expensive equipment to attain a high level of precision . The LAMP assay is more rapid than PCR for the detection of animal pathogens, which requires at least 2–3 h for detection . The time required for diagnosis is considered crucial for the diagnosis of pathogenic infections, making LAMP the obvious choice for diagnosing PCMV. In addition, LAMP is ideal for on-site testing, particularly in situations where time is a critical factor, such as when material is subject to quarantine controls.
Furthermore, in the LAMP assay, amplification can be detected as fluorescence by the naked eye, indicating that this assay can be applied in the field. The appearance of color change indicating a positive result occurs after the addition of SYBR Green I; this is a simple and effective method of detecting LAMP amplification products, eliminating the need for gel electrophoresis and ethidium bromide staining . The orange color of the dye changes to green under natural light in the case of a positive reaction . The sensitivity of detection based on the presence of white turbidity was inferior to that based on the color change with SYBR Green I or electrophoresis (Figures 3 and 4); ten-fold more copies of template DNA are required so that a positive reaction in terms of white turbidity can be visually detected. Quantitative detection is difficult in the LAMP assay, but inspection with the naked eye is simple and rapid. LAMP can potentially be used under field conditions even by non-specialists (for example, to carry out surveillance at ports of entry or in the nursery industry) and in small or regional laboratories where nucleic acid-based testing is not currently performed and equipment is limited . No expensive equipment is necessary to obtain a high level of precision equivalent to or greater than that obtained with other PCR techniques. Therefore, this method of detection may facilitate the application of LAMP, especially in the field, where the availability of equipment and expertise may be limited.
A previous study has reported that earlier detection of infection results in earlier treatment and consequently, earlier recovery . Our final goal was to establish a simple and rapid diagnostic method for the specific detection of PCMV under field conditions. To perform LAMP under field conditions, it is crucial to have a suitable nucleic acid extraction technique that requires minimal equipment and can produce sufficient DNA within a short time. The only equipment required in the LAMP assay is a water bath; this is essential for both DNA preparation and nucleic acid amplification. Thus, the LAMP assay can be adopted in most situations where a rapid diagnosis is required, without the need for complicated equipment and technical training. Thus, LAMP is a more rapid method of detection than PCR, even when the PCMV titers are very low. This is due to the high sensitivity of LAMP and its detection limit of approximately 10 copies. We recommend that this technique be applied routinely for the early detection of PCMV, so that adequate countermeasures can be adopted before infections become epizootic . Nevertheless, it must be clarified that the viral copy numbers in this study are not accurate, because we did not use a purified recombinant plasmid containing the target gene for these assays. Further studies with a purified recombinant plasmid are required to improve this assay technique.
In summary, the LAMP protocol described here is a new, inexpensive, and rapid method with high sensitivity and specificity for the detection of PCMV. No complicated technical operations, experimental conditions, or special equipment is required for this technique; only a simple water bath is necessary. Therefore, LAMP is an advantageous diagnostic tool for the specific detection of PCMV infection in animals under laboratory and field conditions.