A team of scientists from the University of Connecticut, USA, has developed a low-cost lab-on-paper technology to rapidly and easily detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The work has recently been published in a Royal Society of Chemistry’s Lab on a Chip journal.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of the coronavirus disease 2019 (COVID-19) pandemic, is an enveloped RNA virus of the human beta-coronavirus family. Being a respiratory virus, SARS-CoV-2 primarily attacks the upper respiratory tract and gradually propagates to the lower respiratory tract to cause mild to severe infection.
Reverse transcription-polymerase chain reaction (RT-PCR)-based amplification of viral RNA in respiratory samples is considered as the golden standard method to diagnose COVID-19. However, despite high accuracy, RT-PCR is not always convenient for mass detection of SARS-CoV-2 infection at the community level because of its high turnaround time. In contrast, despite a shorter turnaround time, rapid antigen testing largely suffers from a lower accuracy level. Thus, for the better management of the COVID-19 pandemic, it is necessary to develop rapid, high accuracy diagnostic technologies that can be used for large-scale detection of SARS-CoV-2 infection.
In the current study, the scientists have developed an autonomous lab-on-paper device for multiplex gene detection of SARS-CoV-2. The method, which combines reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR–Cas12a detection, can simultaneously detect nucleoprotein and spike genes of SARS-CoV-2 in a single respiratory swab sample. As an internal control, the device uses human housekeeping RNAse P gene.
Regarding potential advantages, the device is capable of detecting 102 copies of viral RNA within one hour. Since two viral genes are simultaneously detected instead of a single gene, the device is expected to provide high accuracy information. Moreover, because of its simple and easy operational technique, testing with the device can be performed by any healthcare professional, making it suitable for large-scale detection of SARS-CoV-2 infection at the community level.
To determine the performance of the device, the scientists collected 21 nasal swab samples from patients. Using commercially available kits, they processed these samples to isolate and purity nucleic acids. Afterward, they transferred the nucleic acid preparations to the cellulose-based paper membrane used in the device to detect SARS-CoV-2 infection. The results obtained from the device were comparable to those obtained from conventional RT-PCR testing.
The CRISPR technology used in the device is a highly sensitive and specific gene-editing technology that can alter the genome of an organism by targeting and cutting specific nucleic acid segments, such as DNA or RNA segments.
In the lab-on-paper method, CRISPR first locates the nucleoprotein and spike genes of SARS-CoV-2 and subsequently cuts them. Because of the cut, a fluorescent signal is produced on the device paper, indicating a positive test result. In addition, the device detects the human housekeeping RNAse P gene in order to validate the quality of the sample and the reliability of the result.
The specialized cellulose-based paper membrane used in the device is hydrophilic in nature. Because of this property, the paper can transport samples that contain nucleic acids. This is highly advantageous as the test can be performed automatically once the sample is loaded. Moreover, because of certain specific formulations, the paper does not interfere with the biochemical reaction required for CRISPR to locate and cut the viral genes. This further reduces the risk of false negative/positive results.
By selecting specific genes of interest, the device can be used for detecting various other pathogens, including human immunodeficiency virus (HIV), human papillomavirus (HPV), or influenza virus.
The scientists have already applied for a provisional patent for the invention through the University of Connecticut Technology Commercialization Services. They are currently looking for an industrial partner to commercialize the device and expand its usage.