Huifang Liu

Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGT→GAT) and HSSP-G13D (GGC→GAC)) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold (C_t) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) was 5–10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for G12D and 92% for G13D) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.

The spin-column system for the isolation of nucleic acids (NAs) from multiple samples presents the inconvenience of repeated experimentation, time-consumption, and the risk of contamination in the process of the spin-column exchange. Herein, we propose a convenient and universal assay that can be used to diagnose multiple pathogens using a multi-sample preparation assay. The multi-sample preparation assay combines a 96-well filter/membrane plate, a bio-micromaterial lattice-like micro amine-functional diatomaceous earth (D-APDMS), and homobifunctional imidoesters (HI) for the processing of pathogen enrichment and extraction for multiple samples simultaneously. The purity and quantity of the extracted NAs from pathogens (E. coli and Brucella) using the proposed assay is superior to that of the commercialized spin-column kit. The assay also does not require the replacement of several collection tubes during the reaction processing. For the multi-sample testing, we used as many as six samples simultaneously with the proposed assay. This assay can simultaneously separate up to 96 NAs from one plate, and the use of multichannel pipettes allows faster and simpler experimentation. Therefore, we believe it is a convenient and easy process, and can be easily integrated with other detection methods for clinical diagnostics.

Invasive aspergillosis (IA) is an important cause of morbidity and mortality among immunocompromised people. Imaging and specimen tests used in the clinical diagnosis of aspergillosis with weak and indistinct defects leads to delay in the treatment of early aspergillosis patients. The developing molecular techniques provide a new method for the aspergillosis diagnosis. However, the existing methods are complex, time-consuming and may even be potentially hazardous. In this study, we developed a simple and rapid Aspergillus fumigatus spores DNA isolation assay using synthesized zinc oxide (ZnO). ZnO nanoparticles were used to take the place of the traditional commercial lysis buffer. The quality and quantity of the extracted DNA were sufficient for further diagnostics with polymerase chain reaction (PCR) analysis. This method offers easy, green, and economic alternative DNA isolation for the diagnosis of invasive aspergillosis.

Infectious diseases, especially pathogenic infections, are a growing threat to public health worldwide. Since pathogenic bacteria usually exist in complex matrices at very low concentrations, the development of technology for rapid, convenient, and biocompatible sample enrichment is essential for sensitive diagnostics. In this study, a cucurbit[6]uril (CB) supermolecular decorated amine-functionalized diatom (DA) composite was fabricated to support efficient sample enrichment and in situ nucleic acid preparation from enriched pathogens and cells. CB was introduced to enhance the rate and effectiveness of pathogen absorption using the CB–DA composite. This novel CB–DA composite achieved a capture efficiency of approximately 90% at an Escherichia coli concentration of 10⁶ CFU/mL within 3 min. Real-time PCR analyses of DNA samples recovered using the CB–DA enrichment system showed a four-fold increase in the early amplification signal strength, and this effective method for capturing nucleic acid might be useful for preparing samples for diagnostic systems.