Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules

Abstract

Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

Rapid discrimination between clinical Clostridioides difficile infection and colonization by quantitative detection of TcdB toxin using a real-time cell analysis system

“…Clinical stool samples were collected from June to September in 2021 at the First Affiliated Hospital of Zhejiang University School of Medicine, and were stored at −80°C for subsequent detection within 48 h. Patients with diarrhea were defined as CDI when their stool samples tested positive for toxigenic C. difficile using the GeneXpert C. difficile assay combined with the TC assay ( Lin et al, 2022 ). Individuals without diarrhea were characterized as having CDC when toxigenic C. difficile were detected in their stool samples using the GeneXpert C. difficile assay ( Lin et al, 2022 ). Clinical information including gender, age, type of underlying medical condition, presence of diarrhea and fever, history of antimicrobial use within 8 weeks, and CDI severities determined according to the guideline ( Cohen et al, 2010 ), was documented.…”

Application of 3D printing technology in tumor diagnosis and treatment

“…The use of 3D printing technology significantly simplifies the processing of microfluidic chips and is more flexible in the selection of printing materials. The chips constructed using 3D printing microfluidic approach have attracted more and more attention in early cancer diagnosis thanks to its remarkable advantages including LOD, repeatable mass production, and low cost [86].…”

Integration of CRISPR/Cas13a and V-Shape PCR for Rapid, Sensitive, and Specific Genotyping of CYP2C19 Gene Polymorphisms

“…37 It is able to save about 2/3 of the amplification time compared to conventional PCR, showing a promising future in achieving fast molecular detection. 38,39 Taking advantage of the rapid amplification of VPCR and the excellent SNP recognition ability of CRISPR-Cas13a, we developed for the first time a rapid, specific, and sensitive genotyping method for CYP2C19. We term this approach "V" shape PCR-coupled CRISPR/Cas13a (VPC).…”

“…To address these challenges, the “V” shape polymerase chain reaction (VPCR) can complete three procedures of PCR during the dynamic heating and cooling process, greatly shortening the amplification time . It is able to save about 2/3 of the amplification time compared to conventional PCR, showing a promising future in achieving fast molecular detection. , …”