A microfluidic system integrated one-step PCR and high-resolution melting analysis for rapid rice mutant detection

Abstract

Rice is a crucial crop to meet global food demands, therefore, it is necessary to develop rapid, efficient and inexpensive methods for screening mutants of important genes in molecular breeding. In this research, a simple microfluidic chip-integrated one-step polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis were designed for the rapid screening of rice mutants. In order to complete the above processes, we also installed a self-developed functional instrument integrated with a temperature control system and a fluorescence detection department. This microfluidic platform was validated by analysing genes related to insect resistance, phenotype and yield of two important varieties of rice, Oryza sativa L. ssp. japonica. and Oryza sativa L. ssp. indica (93-11). The results showed that the platform could detect singlenucleotide mutations between the wild and the mutant rice plants in 1 h and the accuracy was consistent with gene sequencing regarded as the golden standard of the mutants screening. Compared to the traditional method (requiring approximately 3 h), this platform can distinguish mutants from wild types more rapidly. This system is a simple, integrated, disposable and lowcost tool for high-throughput screening of mutants in molecular breeding. More important, this portable instrument can be used as a point-of-care platform for nucleic acid diagnostics in many scientific fields. ARTICLE HISTORY

A Lab-on-a-Chip Device Integrated DNA Extraction and Solid Phase PCR Array for the Genotyping of High-Risk HPV in Clinical Samples

“…To perform the various reactions on the integrated chip, a system with a thermoelectric unit to provide thermal cycling for SP-PCR and a fluorescence-detecting system based on a charge coupled device (CCD) camera was also set up (Figure S1). These features were designed according to our previous research [24,25]. Briefly, the fluorescence detection system contained of two parts: A light-emitting structure consisting of two light-emitting diodes (LEDs, 475 ± 10 nm, CREE, Research Triangle Park, NC, USA) with a two-filter set (excitation wavelength: 470 ± 25 nm; emission wavelength: 525 ± 25 nm, Bdoptic, Beijing, China) and an optical imaging structure system based on a CCD camera (MC25, Mshot, Guangzhou, China).…”

DNA-Based Tools to Certify Authenticity of Rice Varieties—An Overview

“…High Resolution melting (HRM) is a post-PCR method that does not require separation or post-processing of the samples [117]. The method measures the rate of double strand DNA dissociation when raising the temperature to obtain single-stranded DNA [118].…”

“…The method measures the rate of double strand DNA dissociation when raising the temperature to obtain single-stranded DNA [118]. The amplicons are distinguished based on their melting profiles [117]. HRM was already used in rice to perform rapid screening of rice mutants, being able to detect single nucleotide mutations [117].…”

“…The amplicons are distinguished based on their melting profiles [117]. HRM was already used in rice to perform rapid screening of rice mutants, being able to detect single nucleotide mutations [117]. This method was also used to uncover adulteration in Basmati rice samples, proving to be a highly sensitive tool able to detect a ratio of 1:100 of non-Basmati contamination in Basmati rice [59].…”