S. aureus is a common clinical pathogen that causes a variety of serious hospital and community-acquired infections (Holland et al. 2014). Rapid and accurate diagnosis of S. aureus is a key component of treatment, prevention, and interruption of transmission. The currently existing diagnostic methods have limitations as they are either time-consuming (microbial culturing) or expensive (PCR). LAMP is a simple and cost-effective molecular diagnostic method with distinct advantages for the rapid diagnosis of pathogens.
In this study, we successfully developed, optimized, and validated a molecular biology diagnostic LAMP assay for the detection of S. aureus. The critical parameters of the LAMP reaction were optimized to achieve optimal amplification and performance by visual inspection. The optimized LAMP assay can accurately differentiate S. aureus from non-S. aureus strains, and sensitively detect trace amounts of S. aureus.
At present, a variety of colorimetric indicators have been developed for LAMP reaction endpoint detection. Although these indicators are reliable and convenient, they have some limitations. Calcein needs to bind the ionic form of manganese to work, but manganese may inhibit the LAMP reaction and reduce the sensitivity of the assay (Tanner et al. 2015). The fluorescent intercalating dye SYBR Green I is expensive, highly toxic, and requires additional testing equipment (Fischbach et al. 2015). Leuco crystal violet (LCV) requires the preparation of proprietary LCV immobilized tubes, which are complicated to operate (Miyamoto et al. 2015). In contrast, HNB is a cheap and stable synthetic dye. It indicates the result of the LAMP reaction by detecting the consumption of free magnesium ions in the reaction system, which is reliable and effective for LAMP. A multi-observer study showed that HNB as an indicator was superior to the other methods (Wastling et al. 2010). The use of the colorimetric indicator HNB makes the judgment of the LAMP amplification results easier, eliminating the need for tedious electrophoresis operations and greatly avoiding cross-contamination caused by aerosols (Nie 2005). Therefore, HNB was selected as the best dye for this study.
We compared the sensitivity of the LAMP assay with that of the PCR assay and found that the LOD of the LAMP was 10-fold lower than PCR, both for the positive plasmid and S. aureus. Several related studies have also confirmed that the sensitivity of LAMP assay was equivalent or higher compared to PCR (Choopara et al. 2021; Maeda et al. 2005; Prusty et al. 2016). In addition, LAMP technology is more convenient, time-saving, and cost-saving. The entire detection process of LAMP, including template preparation (approximately 40 min), isothermal amplification (45 min), and result determination (approximately 2 min), can be completed within 90 min and the cost of analysis is also lower than that of PCR (Zheng et al. 2018). More importantly, LAMP testing is easy to operate, which can reduce labor costs. Finally, LAMP is often described as a less demanding test than PCR in terms of DNA purification. For example, bacterial colonies are added directly to the reaction mixture for the LAMP reaction, or centrifuged pellet samples are boiled in the presence of 1% Triton X-100 and the released DNA can be used directly for the LAMP reaction without purification (Sowmya et al. 2012; Yan et al. 2017).
The clinical diagnostic efficacy of LAMP analysis is shown in Table 3. A Klebsiella pneumoniae strain, isolated from the sputum of a patient with chronic obstructive pulmonary disease, showed a non-specific electrophoresis band by PCR amplification. The electrophoresis results showed that the amplified band size was over 250 bp, while the target fragment band size for S. aureus was 219 bp (Fig. 4c). One possible explanation is that the genome of this strain has some base fragments homologous to the target gene in S. aureus. However, due to the specific amplification mechanism, the LAMP reaction did not produce a false-positive result for this sample. This indicates that the specificity of the LAMP assay (100%) is superior to that of the PCR (99.33%). In other related studies, the specificity of both detection methods for bacterial pathogens was consistent (Thakur et al. 2018; Zhao et al. 2013). Meanwhile, 2 strains of S. aureus were not identified by LAMP, one case isolated from a sputum sample of a patient with cerebral hemorrhage, and the other isolated from the blood of a shock patient. We speculate that this result may be related to a genetic mutation of the strains themselves. Another point worth mentioning is that although the LOD of the LAMP assay was lower than that of the PCR assay, the sensitivity of the PCR assay is equivalent to that of the LAMP assay in clinical strain detection, which may be related to the low number of clinical strains we analyzed. Our study provides a rapid detection protocol for S. aureus. However, for point-of-care testing, further research is still needed. If LAMP technology is integrated into a microfluidic system, DNA extraction, isothermal amplification, and colorimetric analysis could be completed in one step, which will have greater potential for clinical applications (Liu et al. 2020; Wan et al. 2019; Zhang et al. 2019).
Table 3
Diagnostic efficacy of LAMP and PCR in clinical bacteria strains
Method | Specificity | Sensitivity | NPV | PPV |
LAMP | 100% | 99.09% | 98.68% | 100% |
PCR | 99.33% | 99.09% | 98.67% | 99.54% |
Notes: NPV, negative predictive value; PPV, positive predictive value. |