Researchers have devised a novel method to rapidly and accurately detect viral nucleic acids – a breakthrough that boasts speed, sensitivity, affordability and adaptability to detect different DNA/RNA targets in viruses like the coronavirus.
With the advent of the coronavirus pandemic, researchers in the biomanufacturing industry are looking for ways to innovate and improve upon methods to detect virus contamination. Presently, the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered to be the gold standard for viral detection. However, they often produce variable results.
To overcome these barriers, researchers have developed the digital PCR method, that offers greater accuracy, and absolute quantification, which means that it can reveal the copy number of virus present in the sample. The digital PCR method allows for the setting of clear thresholds of virus contamination and is not prone to possible fluctuations of reference gene required by standard qPCR methods. But even then, the digital PCR is limited in terms of speed, requiring a relatively long reaction time of around four hours. Furthermore, all current PCR-based methods, in general, require expensive equipment for precise temperature control and cycling.
Addressing the problems of PCR-based methods, researchers from Critical Analytics for Manufacturing Personalized-Medicine (CAMP), an Interdisciplinary Research Group (IRG) at the Singapore-MIT Alliance for Research and Technology (SMART), MIT’s research enterprise in Singapore, have developed a new method for rapid and accurate detection of viral nucleic acids. Their novel technology, called RApid DIgital Crispr Approach (RADICA), is highly versatile and easily adapted to detect different DNA/RNA targets in viruses like the coronavirus. Their findings are fully explained in a paper titled “Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids”, published recently in the prestigious journal Biomaterials.
RADICA is a molecular rapid testing methodology by CAMP, that allows absolute quantification of viral nucleic acids in 40-60 minutes in an isothermal manner in a water bath, a prototypical and inexpensive laboratory equipment. As such, it is not only four times faster but is also significantly less expensive than conventional PCR methods as it does not require costly equipment for precise temperature control and cycling. RADICA is also highly specific, sensitive, and tolerant to contamination or inhibitors that may be present in biological samples, which is often the case with cell therapy products.
The RADICA method has been tested on SARS-CoV-2 synthetic DNA and RNA, Epstein–Barr virus in human B cells and patient serum, demonstrating its ability to be adapted to detect other kinds of viruses, and in other types of samples such as saliva and cell culture media. Viruses can also be distinguished from their close relatives using RADICA.
“This is the first reported method of detecting nucleic acids to utilise the sensitivity of isothermal amplification and specificity of CRISPR based detection in a digital format – allowing rapid and specific amplification of DNA without the time consuming and costly need for thermal cycling,” explained Dr Xiaolin Wu, Postdoctoral Associate at SMART CAMP. “RADICA offers four times faster absolute quantification compared to conventional digital PCR methods.”
The team made use of DNA/RNA extracted from the sample and divided a 15 µL reaction into thousands of independent partitions. In each partition, the DNA/RNA is amplified and identified by Cas12a protein, an enzyme that can turn the target signal into a fluorescent signal. This allows absolute quantification to be achieved by counting the number of partitions that have the target DNA/RNA and are lit up.
“The last year has shown us the importance of detecting viruses quickly and accurately, and RADICA can help fill existing gaps in this area,” said National University of Singapore Professor Hanry Yu, co-corresponding author and Co-Lead Principal Investigator at SMART CAMP. “Cell therapy products have a very short shelf life and patients are usually in need of treatment urgently. Current sterility tests need around 14 days, which is too slow for clinical needs but RADICA shortens the process into hours.”
Although RADICA was initially developed for monitoring cell therapy manufacturing processes and biosafety release testing of cell therapy products, it is expected that this method can also be used to detect DNA/RNA targets of different viruses, and identify how many viruses there are in samples to help clinicians in deciding the course of treatment, as well as production and inventory management of cell therapy products.
Source: Singapore-MIT Alliance for Research and Technology (SMART)