2.1 Substrate synthesis
In this assay, we use a luciferase-based biochemiluminescent substrate, luciferin-4,7-di-O-methyl-neuraminic acid . In the presence of influenza virus in a reaction, the substrate is cleaved to free luciferin, which becomes an active substrate of firefly luciferase. In the presence of firefly luciferase, the free luciferase is oxidized to oxyluciferin, resulting in a stable luminescent signal that can be detected by instruments [19, 20].
The process of substrate synthesis has been reported previously . The synthesis of luciferin-4,7-di-O-methyl-neuraminic acid is based on NeuAc and D-luciferin. The 4,7-di-O-methyl-2,8,9-tri-O-acetyl NeuAc methyl ester was achieved with optimized reaction conditions. Then the 2-chloro-4,7-di-O-methyl-8,9-di-acetyl NeuAc methyl ester was prepared by chlornation reaction. Finally, the D-luciferin had been linked to 2-chloro-4,7-di-O-methyl-8,9-di-O-acetyl NeuAc methyl ester by Williamson ether synthesis, followed by removing the protection groups. The product purified by HPLC with its yield above 98%.
The test kit of the HBA contains a sample buffer and a master mixture including the NA substrate, lucifery1N-acteyl-neuraminic acid, and luciferase. Innovative reagent production technology makes the reagent as a freeze-dried bead, this allows for room temperature shipment and a longer shelf life.
2.2 Clinical samples preparation and detection
In the clinical study, a total of 389 clinical samples were collected between 26 January 2019 to 7 April 2019 at ZhuJiang Hospital of Southern Medical University, Guangzhou, China. Nasopharyngeal swabs were used to collect samples. The colloidal gold reagents were from Guangzhou Wondfo Biotech Co., Ltd., (registration number: 20153400613, production batch number: W05980202). PCR reagents and instrument were from Shanghai ZJ Bio-Tech Co.,Ltd.(registration number: 20173404656, production batch number: P 20190601)
To perform a clinical sample testing, place the swab into sample buffer for elution. Add 250uL sample buffer to reagent tube to dissolve the reagent beads, and then incubate for 15minutes in the instrument. The instrument will measure the luminescent signal and prints the results automatically. For samples that were not in sample buffer, dilute the samples 1:1 with sample buffer and then test as described above.
2.3 Data Analyses
The Sensitivity and specificity were used to determine the diagnostic yield among difference assays. 95% confidence intervals were calculated according to the Wilson score method. Kappa was used to evaluate the consistency of the assays.
2.4 Design of instrument
Here we designed a compact and reliable instrument to measure luminescent signal emitted by the reaction mixture. The instrument consists of a reaction vessel, a heating apparatus, a measuring module, and a reaction vessel moving mechanism (Figure 1). The reaction vessel has 24 channels for reagents incubation, reaction and luminescence. The moving mechanism moves the vessel and bring particular channel to detection position. A photomultiplier tube (PMT) is used to capture the luminescent light. Optical fiber for transferring the luminescence light to PMT is located below the detection position and forms a confined space shielded from external light with reaction vessel. A metal cover for PMT protects it from vibration, moisture and external bright light, which may cause permanently damages.
The instrument is capable of carrying out measurement of luminescence quickly and effectively. The optical signals measured by the instrument indicated the quantification of influenza virus. The results of the instrument are read as Relative Light Unit (RLU).