Molecular Diagnostic (MDx) Platform for On-Site Testing of Infectious Diseases: SARS-CoV-2
Meya Kuo, Neo Yang, Jerry Liu, Varouj Amirkhanian & Eric Tsai
BiOptic, Inc. (www.bioptic.com.tw)
Background: Nucleic acid detection by RT-PCR method is one of the criteria approved by China FDA and US CDC for diagnosis of COVID-19, which requires highly trained personnel and can create inaccurate test results (for example, high false-negative rate and some false positive rate). Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False-negative detection could subject patients to multiple tests before a diagnosis can be made, which burdens the health care system. The delayed diagnosis could cause infected patients to miss the best treatment time window. False-negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus infecting many others.
Method / Solution: To resolve the existing issues of RT-PCR BiOptic, Inc. (www.bioptic.com.tw) has created a novel solution for a high sensitivity RNA detection method that utilizes a compact field portable thermal cycler (Qamp-mini) and a miniature Capillary Gel Electrophoresis (Qsep1-LiteTM) system (Figure 1) helping to double-check the end-point PCR product, which the Ct was above 40 (Figures 2, 3 & 4). And since the capillary gel electrophoresis system has much higher fluorescence detection sensitivity it can identify lower copies of collected samples (weaker signal) for faster and more accurate COVID-19 diagnosis.
Figure 1. Quick and Easy Steps to Get Results at High Sensitivity Confirming the Real-Time PCR Results
The plasmid containing COVID-19 Nucleoprotein gene was serially diluted into about 3-copies in every reaction for 45 cycles of real-time PCR. Typically, the samples which Ct values that are between 40-45 may not be reliable and need re-testing for more accurate quantification (Figure 2 & 3).
Figure 2. Real-time PCR product of COVID-19 N gene detected by BiOptic’s Capillary Gel Electrophoresis system
Figure 3. RT-PCR results with 3-copies of plasmid in every reaction including a positive control
For the Real-Time PCR (SYBR Green) systems, the BiOptic MDx Platform can help to double check the samples which Ct are above 40, that have weak fluorescence signal utilizing the Qsep1-Lite capillary gel electrophoresis (Figure 4). And it takes only 3-5 mins per sample analysis.
Figure 4. 3-copies of plasmid in every reaction detected by BiOptic capillary gel electrophoresis system in 3-5 minutes
BiOptic MDx Platform:
BiOptic MDx platform utilizes in house developed Direct "RT-PCR" reagents, which eliminates the need for nucleic acid extraction, where it provides a unique solution for rapid pathogen detection with reproducible results by nontechnical operators. In addition, it resolves the false positive issues associated with real-time PCR systems, since our platform has much higher sensitivity and provides better detection capabilities. Furthermore, BiOptic’s MDx platform should be suitable as a reconfirm platform after real-time PCR increasing the reliability of the diagnosis of the disease more efficiently.
1. Direct RT-PCR amplification. There is no need for RNA extractions and purifications. Buccal or nasal swabs are immersed into a lysis buffer that liberates the nucleic acids. The RNA in these lysates can then be used to set up the Reverse Transcriptase PCR Amplifications.
2. Reverse Transcriptase PCR Amplifications are run on the Qampmini PCR System. There is no need for costly Real-Time PCR instruments.
3. PCR products that have been generated after the full number of cycles by the Qampmini are then analyzed by Capillary Gel Electrophoresis (CGE) utilizing the fully automated Qsep1-Lite Analysis System with gel cartridges. Results for up to 8 samples can be obtained quickly.
In a couple of cases, Real-Time PCR assays had been run and had shown questionable results, such as false-negative results. Analysis of the same sample using BiOptic’s technology demonstrated that COVID-19 RNA sequences were present in these samples as shown in the CGE tracings below (Figure 5: A & B). These results clearly indicate that this technology can be more sensitive than standard Real-Time PCR assays.
Figure 5. Quick and Easy Steps to Get Results at High Sensitivity Confirming the Real-Time PCR Results
(A) Covid-19 detection kit (Company A): Red line: Positive control, Blue line: Negative control, Green line: Reconfirm Negative Sample No.1 and Yellow line: Reconfirm Negative Sample No.2. Red arrow indicates the amplicon signals detected by Qsep Series.
(B) Covid-19 detection kit (Company B): Red line: Positive control, Blue line: Negative control, Green line: Reconfirm Negative Sample No.1
The above analysis demonstrates the sensitivity of our detection platform. We used Qsep Series (Qsep1-Lite) to analyze samples that were determined as negative results by real-time PCR. It is obvious that the signal peak can be seen in the corresponding target area. This advantage can effectively reduce the issue of real-time PCR false negatives and each analysis takes only 3-5 minutes.
Field Portable Solution:
Everything that one needs for COVID-19 testing are provided in a portable kit that can be taken into the field. You are not confined to having to run the assays in a fully equipped laboratory. Lysis buffers, PCR primers and reagents, Qampmini PCR Thermal Cycler, and the Qsep1-Lite CGE Analysis system are all included in a carryon case that can be loaded into the trunk of a car or an SUV (Figure 6) for field analysis.
BiOptic’s MDx platform can provide fast, accurate and cost-effective results, which should be suitable for decentralized testing application of COVID-19.
Advantages of BiOptic’s MDx Platform are:
• No DNA/RNA Extraction: saving 30 to 60 mins
• Application Specific Qampmini PCR: One click to go for 8-sample runs
• Post analysis with Qsep1- Lite CGE System: less than 7 min
• Multiple Genes analyzed in one reaction: Multiplex Direct "RT-PCR"
• High Detection Sensitivity: Validation platform for Real-Time PCR
• Cost Effective
• Field Portable
1. Meya Kuo, et all: “Infectious Diseases Detection Platform: COVID-19” Labcompare, May 15, 2020
2. Neo Yang, et all: “Rapid Genetic Identification of Meat” Labcompare, April 25, 2019
3. Neo Yang, et all: “A Portable CGE System for Cell Free DNA Detection” Labcompare, January 29, 2018 4. www.bioptic.com.tw