Lyophilized Loop-mediated Isothermal Amplification Reagents for Endpoint Detection of Kaposi's Sarcoma Herpesvirus

Introduction:: Loop-mediated isothermal amplification (LAMP) is a highly specific technique for nucleic acid amplification. LAMP is advantageous over polymerase chain reaction (PCR) for point-of-care testing because it can be conducted isothermally in under an hour, requiring less technical lab equipment. Our lab has deployed portable devices for real-time LAMP testing of Kaposi sarcoma-associated herpesvirus (KSHV) in resource-limited settings, providing an accurate and rapid viral quantification. However, transportation, storage, and pipetting of reagents is still a challenge, requiring -20 C refrigeration and careful assembly of the final assay. We propose a lyophilized master mix, containing hydroxynapthol blue (HNB) as an endpoint colorimetric indicator, to simplify the number of time-consuming steps, specialized equipment, and trained personnel needed in the field. Not only can this one-pot reaction greatly reduce the chance of errors and contamination, but it offers an accessible and affordable option for the point-of-care setting.


Materials and Methods:: The LAMP master mix contains Bst polymerase, dNTPs, KSHV primers, MgSO4, isothermal buffer, trehalose, and HNB. Before lyophilization, 20uL of master mix was adding to either a PCR tube or 384-well plate. The master mix was lyophilized in a freeze dryer for 2.5 hours. The tubes or plate were packaged and stored at 4 C for varying lengths of time. During testing, the tube or well were reconstituted with a total volume of 20uL, 5uL DNA and 15uL DNase & RNase free water. Recombinant plasmid DNA contained the Orf26 gene of KSHV was used as a positive control, while DNA extraction from human tissue was used as a negative control. The reconstituted master mix with DNA was incubated at 65 C for 60 minutes, with spectroscopy measurements taken every 20 minutes during incubation. The fluorescent values of HNB were taken at 540/ 610 nm.


Results, Conclusions, and Discussions:: HNB is a metal ion indicator, changing color from a violet purple in the presence of magnesium to sky blue with the lack thereof (Fig. 1a). As a cofactor to Bst polymerase, magnesium enables dNTP synthesis and then falls out of solution into an insoluble precipitate with pyrophosphate. This drop in magnesium creates the color shift that can be seen in visible light (Fig. 1a) and is typically quantified with absorbance measurements. However, HNB is more rarely measured fluorescently, and here we show the discernable difference in endpoint measurements (Fig. 1b).

Thus, we’ve optimized a lyophilized master mix for seven-day storage at 4 C, showing significant fluorescent difference between positive and negative reactions with target DNA. The presence of viral DNA (KSHV) results in a decrease of HNB fluorescence signal. If measured at 60 minutes (a typical incubation period for LAMP), all positive LAMP reactions containing KSHV (down to ~1,000 copies/ reaction) will dip below an intensity threshold as shown in Figure 2 (horizontal line at 75 au).

Although further optimization is required, we have shown that this colorimetric master mix can remain stable at 4 C for 1 week and requires far fewer pipetting steps at the time of testing, needed only DNase/ RNase free water and the DNA extracted sample. With this technology, tubes with varying primers can easily be distributed for high-throughput screening of varying bacterial and viral diseases. Making LAMP testing simply and cost effective will making early detection more feasible for low-resource settings.


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