Point-of-care (POC) molecular diagnostics play a crucial role in the prevention and treatment of infectious diseases. It is necessary to develop portable, easy-to-use, inexpensive and rapid molecular diagnostic tools. In this study, we proposed a lab-on-a-chip device that integrated DNA extraction, solid-phase PCR and genotyping detection. The ingenious design of the pneumatic microvalves enabled the fluid mixing and reagent storage to be organically combined, significantly reducing the size of the chip. The solid oligonucleotide array incorporated into the chip allowed the spatial separation of the primers and minimized undesirable interactions in multiplex amplification. As a proof-of-concept for POC molecular diagnostics on the device, five genotypes of high-risk human papillomavirus (HPV) (HPV16/HPV18/HPV31/HPV33/HPV58) were examined. Positive quality control samples and HPV patient cervical swab specimens were analyzed on the integrated microdevice. The platform was capable of detection approximately 50 copies of HPV virus per reaction during a single step, including DNA extraction, solid-phase PCR and genotype detection, in 1 h from samples being added to the chip. This simple and inexpensive microdevice provided great utility for the screening and monitoring of HPV genotypes. The sample-to-result platform will pave the way for wider application of POC molecular testing in the fields of clinical diagnostics, food safety, and environmental monitoring.
The outbreak of Zika virus (ZIKV) has posed a great challenge to public health in recent years. To address the urgent need of ZIKV RNA assays, we integrate the microfluidic chip embedded with chitosan-modified silicon dioxide capillaries, smartphone-based detection unit to be a C3-system for the rapid extraction and detection of ZIKV RNA. The C3-system is characterized by: (1) four chitosan-modified silicon dioxide capillaries integrated in the microfluidic chip for target ZIKV RNA enrichment and “in situ PCR” (polymerase chain reaction) amplification; (2) smartphone-based point of care (POC) device consisting of a pneumatic subsystem for controlling the nucleic acid extraction processes in the microfluidic chip, a heating subsystem for sample lysis and PCR amplification, and an optical subsystem for signal acquisition. The entire detection processes including sample lysis, ZIKV RNA enrichment, and reverse-transcription polymerase chain reaction (RT-PCR) is achieved in the microfluidic chip. Moreover, PCR buffers can be directly loaded into the chitosan-modified silicon dioxide capillaries for “in situ PCR”, in which the captured ZIKV RNA is directly used for downstream PCR without any loss. ZIKV RNA extracted by the C3-system can be successfully recovered at very low concentrations of 50 transducing units (TU)/mL from crude human saliva. This means that our method of detecting viremia in patients infected with ZIKV is reliable.
Background: Circulating microRNAs (miRNAs) have proved to be promising biomarkers for early diagnosis and therapeutic monitoring in cancers. Particularly for hepatocellular carcinoma (HCC), detection of circulating miRNA biomarkers as a new diagnostic approach has been written into the latest Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China (2019 edition). However, no general consensus on an ideal endogenous normalizer for circulating miRNAs quantification has been reached, so it will affect the accuracy of quantitative results. In this study, we aim to identify a stable endogenous normalizer for analyzing circulating miRNAs. Methods: Candidate miRNAs were selected by screening dataset GSE104310, as well as data statistics and analysis. Five commonly reference genes were chosen for further comparison and verification. Then, the expression levels of these genes in serum were analyzed by quantitative reverse transcription PCR (RT-qPCR) among four groups, including patients diagnosed with HCC, chronic hepatitis B (CHB), liver cirrhosis, and healthy subjects. Furthermore, the stability of target genes was evaluated using geNorm, NormFinder, comparative ΔCq programs, and validated by database. We also explored the availability of the miRNA combination, and compared the performance difference between combination and individuals, as well as the selectivity of miRNA references in the combinations. Results: 11 candidate miRNAs were obtained, and miR-4644 stood out among these miRNAs, and proved to be much more stable than other endogenous miRNAs. Further study showed that miR-4644 exhibited higher stability and expression abundance than other commonly miRNA reference controls. Finally, we discovered the combination of miR-4644 and miR-16 revealed high performance in stability when compared to miRNA individuals. Furthermore, the combination consisted of references with closer nature could give rise to amplification effects in stability. Conclusions: Our findings demonstrated that miR-4644 is an ideal endogenous normalizer for circulating microRNA quantification in hepatocellular carcinoma. Besides, combining miR-4644 with miR-16 into a whole as a reference control would greatly improve the accuracy of quantification.
Mobile sensing based on the integration of microfluidic devices and smartphones, so-called MS2 technology, has enabled many applications over recent years and continues to stimulate growing interest in both research...
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