Fabrication of NEC-SD-LAMP device
Among the components of the NEC-SD-LAMP device, the PS container was fabricated by combining 2.0-cm-thick polystyrene boards. A φ1.0-cm diameter hole was opened in the lid to direct the generated water vapor and hydrogen outside the container. A 2.0-mm-thick chloroprene rubber was placed between the top of the container and lid, providing a seal between the lid and the container. Additionally, two bands were placed around the PS container to fix the lid to the container.
The stand and frame were fabricated using a Mojo 3D printer (Stratasys, Rehovot, Israel) with ABS resin (Mojo P430 QuickPack Print Engine; Stratasys) as the resin material.
The PP container was made from a 200-µm-thick PP film. The aforementioned stand and 80 g of palmitic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were placed inside a container, and a handle made from the PP film was placed on top of the PP container for ease of operation.
An exothermic agent (Morians Heat Pack size L; Morian Heat Pack Co. Ltd., Irima City, Japan) and VI container (SR250; ASVEL, Yamatokoriyama-shi, Japan), which are commercially available products, were used.
Reaction temperature measurement
Temperature measurements during the exothermic agent reaction and the LAMP reaction with palmitic acid were carried out using a previously developed temperature control system18. Each temperature in the system was measured by measuring the resistance of a thermistor (104 JT; SEMITEC, Tokyo, Japan) at 50 Hz and running calculations on a computer. The resistance of the thermistor was inputted into a laptop using an analog I/O PC card (ADA16-8/2(CB)L; CONTEC Co., Ltd., Osaka, Japan), and the temperature calculation program was created using LabVIEW ver8.2 (NI, Austin, TX, USA). During the exothermic reaction, the thermistor was placed on the side of the PS container. During the LAMP reaction, the thermistor was also placed on the side of the PP container after storage in the VI container such that it was in contact with the melted palmitic acid. The averages and deviations of the measured temperatures 3–13 and 3–63 min after commencing measurements during the exothermic and LAMP reactions, respectively, were calculated.
NEC-SD-LAMP Verification Experiment
The following experiments were conducted to verify the functionality of NEC-SD-LAMP.
50 µL of saliva from a healthy person were mixed with AMPLIRUN Adeno Virus DNA (MBC001; Vircell S.L., Grenada, Spain) to a final concentration of 100 copies per µL and added to the PCR tube. The SalivaDirect reaction was performed by mixing MagMAX™ Viral/Pathogen Proteinase K (Thermo Fisher Scientific, Waltham, MA, USA) with a saliva sample to a final concentration of 50 µg per µL and incubating at room temperature for 5 min. After the reaction, 30 µL of mineral oil (M-5904; Sigma-Aldrich, St Louis, MO, USA) was added to prevent sample evaporation, and the PCR tube was placed on a stand. Thereafter, an exothermic agent, VI container without a lid, PP container, and stand with a PCR tube were placed inside the PS container. Subsequently, 150 mL of MilliQ water was added to react with the exothermic agent to heat the inside of the PS container to approximately 95°C for 5 min to inactivate the proteinase K. After the reaction, it was confirmed that all palmitic acid in the PP container had melted, after which it was placed in a VI container.
LAMP reagents were prepared by mixing a 2× reaction mix, enzyme mix, distilled water, fluorescence detection reagent (all from Eiken Chemical Co., Ltd., Tokyo, Japan), and six primers (FI, BI, F3, B3, Loop F, and Loop B; 001-00890; LAMP Primer Design and Synthesis Service; Nippon Gene, Tokyo, Japan) according to the RT-LAMP kit protocol (Eiken Chemical Co., Ltd.). Table.1 shows the primer sequences used to detect adenovirus DNA in this experiment. The final concentration of each primer was adjusted to 2 µM (PI and BI primers), 0.25 µM (F3 and B3 primers), and 1 µM (Loop F and Loop B primers). Thereafter, 20 µL of the prepared LAMP reagent and 5 µL of the post-SalivaDirect sample were added to the PCR tube (final concentration of adenovirus DNA: 20 copies per µL). After adding 20 µL of mineral oil to prevent evaporation, the PCR tube was placed on a stand in a PP container. LAMP was then performed by closing the lid of the VI container and placing it for 1 h on the laboratory table. After the reaction, the PCR tube was removed from the stand, and the color of the solution was observed under natural or excitation light using a SmartBlue Transilluminator (Greiner Bio-One, Frickenhausen, Germany), and each observation was captured using a camera (SO-03j; Sony Corporation, Tokyo, Japan).
Table 1. Primer sequences for detecting adenovirus DNA
As a comparison experiment, the following four reactions were also performed.
As a negative control (NC), a saliva sample without AMPLIRUN adenovirus DNA was used, and NEC-SD-LAMP was performed using the same reaction as above.
As a positive control (PC), a saliva sample containing the same concentration of AMPLIRUN adenovirus DNA was prepared and heated at 95°C for 5 min to stop the SalivaDirect reaction and at 63°C for 1 h for the LAMP reaction using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA).
To verify the effect of the proteinase K residue, we prepared a sample that was not heated by the exothermic agent after the SalivaDirect reaction. LAMP with palmitic acid was used for this sample. The amount of sample and the concentration of reagents in each process were the same as above.
To verify the functionality of the NEC-SD-LAMP device in tropical regions, it was installed in an incubator (EO-450V; AS ONE Corporation, Osaka, Japan) heated to 50°C, and NEC-SD-LAMP was performed. The amount of sample and the concentration of reagents in each process were the same as above.
Verification of detection limit
To verify the limit of detection concentration of NEC-SD-LAMP, the following experiments were performed.
50 µL of saliva from a healthy person were mixed with AMPLIRUN adenovirus DNA to a final concentration of 1,000 copies per µL and added to the PCR tube. The same procedure detailed in the "NEC-SD-LAMP Verification Experiment" was used to perform the SalivaDirect and exothermic agent reactions.
The LAMP method with palmitic acid heating was performed using the same procedure as in the “NEC-SD-LAMP Verification Experiment,” after which the color of the solution was observed under natural or excitation light on a SmartBlue Transilluminator (Greiner Bio-One). For the LAMP method, the post-SalivaDirect reaction sample was diluted using MilliQ water to final concentrations of 1, 2, 10, 20, 100, and 200 copies per µL of adenovirus DNA.
As a comparison experiment, the following two reactions were also performed.
As a negative control (NC), a saliva sample without AMPLIRUN adenovirus DNA was used, and NEC-SD-LAMP was performed using the same reaction as above (final concentration of adenovirus DNA: 0 copy per µL).
As a large-volume NEC-SD-LAMP experiment, NEC-SD-LAMP was performed in a 1.5-mL tube. The reagent concentration was the same as described above, and the reaction was performed using a 4-fold increase in the total solution volume. The final concentration of adenovirus DNA was adjusted to 0 copy per µL (NC), 0.5 copy per µL, and 1 copy per µL.