Legend: Graphical abstract showing real time/quantitative PCR (qPCR) melt curves (left, https://commons.wikimedia.org/wiki/File:QPCR_results_-_amplification_plot_and_melting_curves.png), and a typical 96-well plate used for qPCR (right, https://www.flickr.com/photos/iaea_imagebank/49869473421).
Introduction/Background
The SmartChip real-time PCR system is a great example of a novel high throughput technology, specifically in the realm of high throughput quantitative PCR (HT-qPCR). While standard quantitative PCR or digital PCR are widely used microbiological techniques, they are not high throughput because researchers are limited to screening for a specific amplicon (4). Screening for one amplicon at a time is not efficient when studying metagenomic samples, which can require around 10000 reads to detect a single copy of the 16S rRNA gene (3). The main advancement of HT-qPCR is that it allows reactions to be performed at the nanoliter scale, which consumes only a minute amount of DNA, leading to significant cost savings (3). The SmartChip real-time PCR system requires a reaction volume of 100 nL in comparison to the Bio-Rad CFX real-time PCR system, which requires 3000 nL (3). This allows for a much higher throughput capacity when using the SmartChip real-time PCR system (54x) versus the Bio-Rad system (4x) (2). The SmartChip real-time PCR system allows for 5184 simultaneous reactions on a single chip, which allows multiple primer sets to be tested from multiple samples, and the platform has had many diverse applications in remediation and detecting disease biomarkers and antibiotic resistance genes (1, 2, 3, 4).
There are many advantages common to HT-qPCR systems such as the SmartChip real-time PCR system in comparison to well-known metagenomic shotgun sequencing techniques such as those offered by Illumina. HT-qPCR ideally can preserve the specificity, sensitivity, and simple data interpretation of single species approaches such as qPCR while allowing for efficient, parallel analysis for detection of multiple different microbial taxa, with greater detection limits than metagenomic shotgun sequencing (4). In general, as the throughput capacity of a technology increases and the reaction volume decreases, the specificity of an assay is reduced, and the SmartChip real-time PCR system offers greater analytical sensitivity due to a good balance between these two factors in comparison to other HT-qPCR technologies (3). Reagent and run costs of HT-qPCR for covering large numbers of potential taxa are significantly lower than single-species standard PCR and are potentially lower than shotgun metagenomic sequencing depending on the number of taxa quantified (2, 3). Lastly, HT-qPCR in common with standard PCR has a lower learning curve than metagenomic shotgun sequencing as the results generated do not require complex data analysis using bioinformatics tools and pipelines (3).
The main disadvantage of HT-qPCR is an inability to analyze unknown sequences as with metagenomic shotgun sequencing (2, 3). This is because as with standard qPCR, HT-qPCR requires the preparation of specific primers for each microbial taxa of interest and can only quantify amplicons generated from these primers (2, 3). Reaction conditions are also fixed for each run, which means that the number of cycles and temperatures cannot be optimized for individual primer pairs as with standard qPCR using a single primer pair (2, 3). Lastly, in common with many high throughput technologies, startup costs for HT-qPCR and the SmartChip real-time PCR system are prohibitive for labs based in developing countries although future advances in high throughput capacity, automation, and fieldable devices may reduce this barrier to entry in the future (3).
A great example of the real-world applications of HT-qPCR and the SmartChip real-time PCR system is found in the field of bioremediation. A study published by Collier et al. (1) used the SmartChip real-time PCR system to study biodegradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), which is a common contaminant at many military sites. The researchers used HT-qPCR to quantify the six functional genes linked to RDX biodegradation (diaA, nfsI, pnrB, xenA, xenB, xplA) in RDX-contaminated groundwater and sediment as well as uncontaminated control samples to determine the RDX biodegradation potential at contaminated sites (1). The SmartChip real-time PCR system was chosen for this experiment because of its ability to run multiple primer pairs corresponding to the six known RDX degrading microorganisms at once from each of the sample types while allowing for absolute quantification of the taxa of interest (1). The researchers ran 3 chips, each containing 2 functional genes, and using 24 assays by 216 samples format, which allowed for 64 samples to be tested with each chip (1). Interestingly, the researchers mentioned the possibility of using one chip for the entire experiment if funding was only available for one chip, although this would have required a modified format of 12 assays by 384 samples, allowing for 232 samples to be tested (1). The results of this assay were that xenA and xenB were the most prominent in the environmental samples, with all functional genes except diaA detected, which helped establish HT-qPCR and the SmartChip real-time PCR system as a comprehensive tool for evaluating RDX biodegradation potential at contaminated sites (1).
Overall, it appears that the SmartChip real-time PCR system and other HT-qPCR technologies are ideal in situations where there are multiple known functional genes or taxa of interest, and an accurate quantification of these populations is desired. This technology is particularly useful in situations where it is desirable to monitor microbial populations over time as with many bioremediation projects (1) or in evaluating antimicrobial resistance over time (2, 3). The agricultural sector may also benefit from this technology as it may provide a way to monitor indicator populations to monitor the overall soil health (3). With advances in affordability and practicality of HT-qPCR likely to occur as the technology matures, and with the SmartChip real-time PCR system as the most used system (4), HT-qPCR may be used to improve the lives of people and animals around the world as a powerful public health tool for monitoring significant changes in important microbial species over time.
References
1.) Collier, J. M., Chai, B., Cole, J. R., Michalsen, M. M. & Cupples, A. M. High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system. Appl. Microbiol. Biotechnol. 103, 7161-7175 (2019).
2.) Kang, W., Zhang, Y., Shi, X., He, J. & Hu, H. Short-term copper exposure as a selection pressure for antibiotic resistance and metal resistance in an agricultural soil. Environ. Sci. Pollut. Res. Int. 25, 29314-29324 (2018).
3.) Waseem, H. et al. Contributions and Challenges of High Throughput qPCR for Determining Antimicrobial Resistance in the Environment: A Critical Review. Molecules (Basel, Switzerland) 24, 163 (2019).
3.) Wilcox, T. M. et al. Parallel, targeted analysis of environmental samples via high‐throughput quantitative PCR. Environmental DNA (Hoboken, N.J.) 2, 544-553 (2020).