Color-Coding of Microchip RT-PCR Test System for SARS-CoV-2 Detection

An RT-PCR based microchip test system for the detection of SARS-CoV-2 offers pre-loaded and lyophilized reagents in the microchip. However, the 30and 48-microwell formats of the microchip being miniaturized and performing 1.2 μl reaction, seek visual attention during sample addition. Therefore, adding colorants as color indicator in the lyophilized matrix in the microchips or adding to sample or master mix can impart not only user-friendliness to the task of liquid handling but also precision, and color-codes for easy identification of multiple kits in the layout of the microchip without compromising PCR data quality. A panel of colorants was screened for their background intensity, spectral inertness towards detection channels of AriaDNA analyzer, interference with the reporter dyes (FAM, Cy5 and ROX), and visibility of optimal concentration in the microwell. The concentration of the colorant displaying insignificant impact on the quality of the amplification (Ct, fluorescence, and sensitivity) in comparison to no-colorant control was chosen for inclusion in the test kit. Tartrazine, Acid Red, Brilliant Blue and FAST Green colorants lyophilized with the reagents in the SARS-CoV-2 microchips were found to be stable and suitable. Storage of microchips with Fast Green colorant was tested at 40 ̊C, 22 ̊C, 4 ̊C, and −20 ̊C for 70 days and was found to be suitable and compatible with different master mixes available as liquid or lyophilized. Additionally, the microchips pre-loaded with lyophilized reagents in the presence and absence of two colorants Tartrazine and Fast Green were validated with clinical samples of SARS-COV-2. No significant impact of these colorants both intraand inter-microchips was observed on the Ct and intensity of amplification for the tested samples in comparison to no-colorant control. The data suggested that the tested colorants can be used to color the sample, or the master mix or PCR mix for user-friendly liquid handling in empty microchips. For the microchip with How to cite this paper: Gill, R.K., Gill, S.S., Gelimson, I., Slyadnev, M., Martinez, G., Gaines, M., Nunley, R. and Majoros, T. (2021) Color-Coding of Microchip RT-PCR Test System for SARS-CoV-2 Detection. Journal of Biosciences and Medicines, 9, 94-119. https://doi.org/10.4236/jbm.2021.95010 Received: April 16, 2021 Accepted: May 24, 2021 Published: May 27, 2021 Copyright © 2021 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
A microchip based realtime RT-PCR test system consisting of a 1-plex disposable microchip for the detection of SARS-CoV-2 has gone through the validation of this detection assay with clinical samples [1]. This microchip consists of 30 microwells, each of which can accommodate a miniature TaqMan chemistry-based reaction of 1.2 μl. The microchip is preloaded with lyophilized US-CDC recommended N1 and N2 primers and probes for detecting the nucleocapsid (N) gene of this virus, along with HsRPP30 (Hs) as a human specimen control involving 1-step qRT-PCR reactions. Recently, we have developed a 2-plex format of the microchip for SARS-CoV-2 detection in which the N1 gene target has been paired with the Hs target in each microwell of the microchip raising the throughput from 7 samples in 1-plex test kit to 27 samples in addition to the controls performing 45 cycles in 32 minutes. Another 2-plex test system with increased throughput of 45 samples in 32 min has higher density of microwells (48-microwell, columns 8 × rows 6). Apart from SARS-CoV-2 detection, the microchip based realtime PCR test systems are involved in detection of other targets including phytopathogens, GMOs, pashmina fibers, meat identifiers, and others as 1-plex or 2-plex test systems [2] [3] [4] [5] [6].
These next generation test systems are technically optimized for imparting sensitivity, specificity, reproducibility, cost-effectiveness, and rapidity in results [2]- [7]. Most of these advantages are attributed to the miniaturized reaction volume, high heat transfer efficiency offered by the metal plate with etched microwells providing high surface area to volume ratio, low reagent consumption, surface dominant properties, and cost-effective product design [7]. The reduced thermal mass of microchips allows for extremely fast temperature ramping requiring PCR protocols established plate-based reactors to be adjusted to the microchips [8]- [16]. Therefore, the attributes of this technology are expected to meet the current requirements and demands of existing detection assays, giving fast results in under 30 minutes [17].
To ease reagent addition to the microcwells and ensuring surface inertness to the PCR process, the passivation of exposed metal surfaces offers hydrophilicity to the microwells that attracts the liquid reagents to the microwells. This feature of the microchip offers user-friendliness in liquid handling and retention of the reagent mix in the microwells. However, the PCR master mixes being colorless These features of the microchip test systems demand more attention for careful pipetting into the reagent pellet loaded microwells. However, by including PCR compatible colorant(s) as standalone reagent for adding into the PCR reagent mix for running PCR in empty microchips or including the colorant into lyophilized pellet of the reagents in ready-to-run microchips, can prove highly user-friendly (Figure 1(a)). Therefore, in view of the practical considerations involved in adopting the colorants in this technology for routine laboratory use, and for precision of quality control in the manufacturing process, precision in running assays, and as color-code identifier, the validation task was attempted.    Slow or fast thermal settings were applied, including a reverse transcription step at 50˚C for 300 s, followed by a denaturing step at 95˚C for 120 s and 45 cycles of 95˚C for 1 s followed by extension and signal recording at 55˚C for 20 s.
The Ct values were determined as a second derivative maximum (SDM) once fluorescence passed an auto-set SDM threshold. The SDM serves as an automated alternative but manually tweakable threshold setting and confers flexibil-   With the purpose of minimising the dose dependent inhibitory effect of the colorant displayed by the test dilutions of Brilliant Blue and Sunset Yellow, the last three serial dilutions (2-fold) of the remaining colorants (Fast Green, 1 mg/3.33ml 1 mg/6.66ml 1 mg/13.33ml; Acid Red 33, 1 mg/2.5ml 1 mg/5ml 1 mg/10ml, and Tartrazine 1 mg/0.25ml 1 mg/0.5ml 1 mg/1ml) offering visibly apparent color in the PCR reaction were tested. Depending upon the acceptability of the results in comparison to the no colorant control, the least visible concentration or higher concentration of each colorant was selected as working concentration of the colorant leading to the preparation of stock working solutions of the screened colorants (Table 1).
2) Determining background intensity in empty microchips: The background intensity of 1 µl of the working concentration of each colorant in 10 µl of water, in 10 µl of UltraPlex TM 1-Step ToughMix ® , in10 µl of PCR mix containing UltraPlex TM 1-Step ToughMix ® and primer probes of N1-FAM and Hs-Cy5 was determined in empty microchips. The standard thermal PCR profile was run for 45 cycles to determine any impact of temperature in comparison to the no-colorant control. The PCR mix contained UltraPlex TM 1-Step ToughMix ® , and primers and probes of both N1-FAM and Hs-Cy5/Hs-ROX along with N1-RNA and Hs-RNA.
3) Determining spectral interference of colorants in empty microchips: The

Results and Discussion
Screening of colorants for PCR compatibility and inertness: To determine  Figure   3(a), Figure 3     ToughMix ® , and in the PCR mix containing UltraPlex TM 1-Step ToughMix ® , primers and probe, respectively ( Figure 5(b)).

A comparison of the background intensities of two reporter dyes Hs-Cy5 and
Hs-ROX on channel-2 of AriaDNA TM analyzer, were also compared for Tartra Therefore, these background intensities do not interfere in the analysis.
Determining non-interference of colorants during PCR thermocycling: The PCR reporter dyes, and the colorants are known to have their specific absorbance and emission spectra that may interfere during the PCR run [18] [19]. Moreover, the data from screening food colorant formulations on compatibility with realtime PCR, and from the comparison of the intensity of the blank colorants, some food colorants formulations for food applications were not found to be compatible due to high optical absorbance [20]. Therefore, interference of the working solutions of these test colorants with channel-1 and channel-2 of AriaDNA TM analyzer was studied by running PCR with primer and probes of FAM, and Cy5 reporter dyes.
The absence of any signal from all the test colorants added as sample (1 µl in10 µl RT-PCR mix) in the 2-plex Microchip RT-PCR Covid-19 Detection kit preloaded with lyophilized FAM, and Cy5 reporter dyes, did not generate any signal indicating their inertness to the detection channels of the AriaDNA TM analyzer (Figure 6(c)). This data also suggested the compatibility of these colorants in the microchip. Thus, these colorants can be used either for coloring of master mix for precision liquid handling in microchips or to color-coding of lyophilized kits in the microchip.   and IC-ROX (channel-2) of AriaDNA TM in the lyophilized microchip did not indicate any significant impact on activity of the PCR reagents [20]. These observations suggest that the tested colorants can be successfully used to add us- Similarly, the intensity of N1-FAM target was also insignificantly affected by the Tartrazine colorant displaying 6% and Fast Green colorant 8% lower intensity than no-colorant control (Figure 11(a), Figure 11  Acceptable rank order of colorants: Applying acceptability criteria on these colorants, a rank order of the colorants is presented in Table 4 that suggests the applicability of these colorants with insignificant compromise on confidence of the PCR detection of SARS-CoV-2. The colorants or the derivative colorants created from mixing different colorants can generate other color shades for expanding multi color-coding of the kits.

Conclusion
The results obtained in the present studies suggest that the tested colorants can be successfully used to add user-friendliness of the ready-to-use lyophilized microchips to improve liquid handling. The storage at low and high temperatures confirmed that visibility of the colorant in the microchip is not affected for at least 70 days.