Periodic Reporting for period 1 - NORAMID SE (A Novel Rapid Nucleic Acid Isolation Microchip Intergrated in the FASTGENE PCR Microfluidics Device for Point of Care Diagnosis of Infectious Diseases)
Periodo di rendicontazione: 2021-12-01 al 2023-11-30
Infectious diseases are a major challenge to global health. With the emergence of disease outbreak such as coronavirus (covid-19) which is a global pandemic, there is need for early and prompt diagnosis for the effective management of infectious diseases. Conventional polymerase chain reaction (PCR) based devices that detect microbial nucleic acids are the most sensitive, specific and reliable diagnosis tools for microbial pathogens but are time-consuming and requires expertise making them not suitable for Point of Care (POC) diagnosis. More so, nucleic acid extraction is primary to PCR detection of pathogens but most nucleic acid extraction methods are labour-intensive and time consuming, limiting the prompt detection of infectious pathogens. Recently, BforCure developed an ultrafast, sensitive and small-volume (25 µl) real-time PCR system based on microfluidic thermalization known as FASTGENE technology device to molecularly detect bacterial and viral pathogens including covid-19 directly from nasal swap samples and maintains a good thermal homogeneity throughout the reaction to ensure the best PCR efficiency and specificity. However, one of the challenges of this device is that it is less sensitive to detect viruses when the viral load is low since it directly amplify samples without extraction of nucleic acid for optimal detection. Recent reports suggest that microfluidic-based nucleic acid device can speed up nucleic acid isolation from pathogens. Hence,the aim of this project is to develop a simple, rapid and cost-effective novel NA isolation microchip and integrate it in the FASTGENE quantitative PCR device by BforCure for POC diagnosis of infectious diseases.
In this project we designed a rapid microfluidic device for the isolation and concentration of nucleic acids called Fastprep. Following the design of Fastprep A2B4 and A2B6 prototypes, we fabricated and assembled its parts, then evaluated its functionality. Its performance to isolate and concentrate nucleic acid was assessed using various nucleic acids. This was done by filtration and elution of nucleic acids through fastprep using lysis buffer, wash buffer 1 and 2 and elution buffers prepared by the ER. The eluate containing the nucleic acid was then amplified by PCR amplification to assess the efficacy by comparing it with standard control. Our findings showed that the Fastprep A2B4 and A2B4 prototypes were successful in the isolation of nucleic acids within 2 minutes after sample preparation of 5 minutes and the A2B6 prototype had a high efficacy to isolate some nucleic acid than the A2B4 prototype. However, the efficacy was variable for various nucleic acids and was not reproducible for some nucleic acids. Also, the device could not completely be decontaminated of nucleic acids thereby liable to sample contamination. Another limitation of fastprep was that due to its size and accompanied accessories, we were unable to integrate it directly into chronos, the fastgene quantitative device by bforCure. Based on these limitation, we developed a new design called smart concentrator which is disposable to prevent nucleic acid contamination. The smart concentrator showed upto 25% efficacy to extract/concentrate nucleic acid following PCR amplification with chronos. Smart concentrator was indirectly integrated with chronos since extracted nucleic acid directly reacts with PCR mix in the smart concentrator which is then transferred to a microprep chip within a short time interval and amplified in chronos. This gives an advantage of smart concentrator over fastprep which could not be integrated with chronos and also had challenges in decontamination whereas smart concentrator is disposable and cannot be reused avoiding contamination of samples.
Almthough we successful developed a smart concentrator, disposable device for the isolation of nucleic acid, its performance is not satisfactory enough to meet the market demands. As such, we were unable to patent the product. The project has opted out from open research data publication and communication since the beneficiary is a private SME and we would like to keep the research data confidential without publishing the findings until the device has a satisfactory performance before patents can be filed to protect the intellectual property.