Numerical and experimental investigation on the performance of rapid ultrasonic-assisted nucleic acid extraction based on dispersive two-phase flow.

BACKGROUND: Nucleic acid extraction (NAE) is an essential step in the whole process of nucleic acid detection (NAT). Traditional manual extraction methods are time-consuming and laborious, unfavorable to the point-of-care testing of nucleic acids. Ultrasound has been emphasized due to its noncontact and easy-to-manipulate characteristics, and integration with microfluidic chip can realize rapid NAE through acoustic streaming effect. The uniformity of magnetic bead mixing in this process is a critical factor affecting the extraction effect. In this study, we developed an ultrasound-assisted NAE technique based on the magnetic bead method and optimized the chip structure to achieve rapid NAE. RESULT: We use ultrasonic-assisted coupled with magnetic bead method for ultra-fast NAE. The mixing process of magnetic beads driven by acoustic streaming is simulated by a dispersive two-phase flow model, and the ultrasonic incidence angle (θin), cone structure aspect ratio (Dc/Hc) and sheet structure thickness (Hp) are optimized to enhance the mixing performance. Furthermore, the effectiveness of NAE is validated by utilizing quantitative real-time PCR (qPCR) detection. The findings reveal that a θin value of 10° yields superior mixing performance compared to other incidence angles, resulting in a maximum increase of 84 % in mixing intensity. When Dc/Hc = 0.5 and Hp = 0.5 mm, the maximum mixing index in the localized region of the chamber after 1 s of ultrasound action can reach 83.6 % and 92.5 %, respectively. Compared to the original chamber, the CT values extracted after 5 s of ultrasound action shifted forward by up to 1.9 ct and 4.1 ct, respectively. SIGNIFICANCE: The dispersed two-phase flow model can effectively simulate the mixing process of magnetic beads, which plays an important role in assisting the structural design of chip extraction chambers. The single-step mixing of ultrasound-assisted NAE takes only 15s to achieve an extraction performance comparable to manual extraction. The extraction process can be completed within 7 min after integrating this technology with microfluidic chips and automated equipment, providing a solution for automated and efficient NAE.

Haplotype mapping of H3K27me3-associated chromatin interactions defines topological regulation of gene silencing in rice.

Histone modification H3K27me3 is an important chromatin mark that plays vital roles in repressing expression of developmental genes. Here, we construct high-resolution 3D genome maps using long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and characterize H3K27me3-associated chromatin interactions in an elite rice hybrid, Shanyou 63. We find that many H3K27me3-marked regions may function as silencer-like regulatory elements. The silencer-like elements can come into proximity with distal target genes via forming chromatin loops in 3D space of the nuclei, regulating gene silencing and plant traits. Natural and induced deletion of silencers upregulate expression of distal connected genes. Furthermore, we identify extensive allele-specific chromatin loops. We find that genetic variations alter allelic chromatin topology, thus modulating allelic gene imprinting in rice hybrids. In conclusion, the characterization of silencer-like regulatory elements and haplotype-resolved chromatin interaction maps provide insights into the understanding of molecular mechanisms underlying allelic gene silencing and plant trait controlling.

Development of amplification system for point-of-care test of nucleic acid.

Nucleic acid testing (NAT) has been widely used in many fields such as medical diagnosis, food safety testing and forensic identification. However, it can only be carried out in professional laboratory because the test process is complicated and rigorous. In this paper, a nucleic acid amplification system based on polymerase chain reaction (PCR) was developed to meet the requirements of point-of-care testing (POCT) for nucleic acids. Firstly, the mechanical structure and electronic control system were designed and constructed. Secondly, an integral separation PID algorithm for temperature control and an intelligent temperature compensation method based on support vector regression (SVR) were proposed. Finally, temperature measurement and biological experiments were performed to prove the stability and availability of the nucleic acid amplification system. The results showed that the system achieved a rapid temperature change velocity of 4.5 °C/s, and the steady-state error was within ± 0.5 °C. The nucleic acids in samples of different concentrations were well amplified, the system can be used for quantitative detection of nucleic acid with the help of a fluorescence detection system, and has higher sensitivity than Tianlong PCR instrument.

Accurate nucleic acid quantification in a single sample tube without the need for calibration.

Convenient and accurate nucleic acid quantification (NAQ) is crucial to clinical diagnosis, forensic medicine, veterinary medicine and food analysis. However, traditional NAQ relies on the preparation of a laborious, time-consuming and expensive calibration curve, which would also propagate pipette errors through serially dilutions. Besides, traditional NAQ is run in different tubes, which introduces bias from random tube-to-tube variations and is unable to detect inhibitors from biological samples. To solve these problems, a single-tube quantitative PCR (stqPCR) technique is proposed which enables accurate quantification without the need for a calibration curve. In this method, an internal quantitative standard DNA (IQS-DNA) for quantification was screened out by co-amplification with the target DNA. Then the difference between the quantification cycle value (ΔCq) of the IQS-DNA and the target DNA was used for NAQ. The method permitted high accuracy quantification with reliable data for concentrations in plasmid, serum standard, and clinical samples being confirmed (R2 values of 0.9951, 0.9889, and 0.9727, slope values of 1.011, 1.028, and 0.9327, and intercept values of -0.06037, -0.1486, and 0.3325, respectively). Accurate NAQ could also be achieved by stqPCR even though inhibitors were present in a sample; however, in the case of using a commercial assay kit, satisfactory performance was only attained after the same sample was diluted some 32-fold. Moreover, integration of the present method into a microfluidic system could achieve super-fast NAQ in less than 30 min and achieve super-fast "sample in, quantitative answer out" testing in less than 40 min. Thus, the stqPCR method present here would promote the development of NAQ in the laboratory and on site.