近日浙江省農(nóng)科院在《Journal of Advanced Research》發(fā)表文獻(xiàn)《Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops》。文獻(xiàn)中使用LUYOR-3415RG便攜式雙波長(zhǎng)熒光蛋白激發(fā)光源檢測(cè)轉(zhuǎn)基因作物。

文獻(xiàn)摘要:

開發(fā)一種新型 Cas13a/Cas12a 介導(dǎo)的轉(zhuǎn)基因作物“一鍋”雙重檢測(cè)方法

轉(zhuǎn)基因（GM）作物已在世界各地廣泛種植，開發(fā)適合田間部署的快速、超靈敏、視覺(jué)多重檢測(cè)平臺(tái)對(duì)于轉(zhuǎn)基因生物調(diào)控至關(guān)重要。在這項(xiàng)研究中，我們開發(fā)了一種新型一鍋法系統(tǒng)，稱為 MR-DCA（多重 RPA 和雙重 CRISPR 檢測(cè)），用于同時(shí)檢測(cè)轉(zhuǎn)基因作物中的基因靶標(biāo)。這種創(chuàng)新方法將多重 RPA（重組酶聚合酶擴(kuò)增）與雙 CRISPR（成簇規(guī)則間隔短回文重復(fù)）測(cè)定技術(shù)相結(jié)合，為轉(zhuǎn)基因作物檢測(cè)提供了一種簡(jiǎn)化且高效的方法。用于擴(kuò)增和靶標(biāo)的 RPA 反應(yīng)包含在管底座中，而雙 CRISPR 酶則放置在管蓋中。離心后，啟動(dòng)雙 CRISPR (Cas13a/Cas12a) 檢測(cè)系統(tǒng)。使用熒光可視化通過(guò) FAM 通道和 HEX 通道進(jìn)行測(cè)量。使用側(cè)流試紙條時(shí)，使用兔抗地高辛（藍(lán)線）進(jìn)行檢測(cè)，同時(shí)使用抗小鼠 FITC（紅線）進(jìn)行識(shí)別。使用 Image J 量化線強(qiáng)度并以圖形方式描繪。 35分鐘內(nèi)完成目標(biāo)檢測(cè)，檢測(cè)限低至20個(gè)拷貝。此外，還開發(fā)了兩種分析系統(tǒng)，它們?cè)?MR-DCA 測(cè)定中表現(xiàn)良好。在對(duì)來(lái)自廣泛基因組范圍的轉(zhuǎn)基因作物的24個(gè)盲樣進(jìn)行分析時(shí)，MR-DCA給出了與定量PCR方法一致的結(jié)果，表明了較高的準(zhǔn)確性、適用性和半定量能力。 MR-DCA的發(fā)展代表了GM檢測(cè)領(lǐng)域的重大進(jìn)步，為可在資源有限的環(huán)境中使用的多目標(biāo)檢測(cè)提供了快速、靈敏和便攜的方法。

Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops

Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of and genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. The RPA reaction used for amplification and targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure through the FAM channel and through the HEX channel. When using lateral flow strips, was detected using rabbit anti-digoxin (blue line), whilst was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

MR-DCA combined the RPA reaction with Cas13a/Cas12a digestion in a one-pot reaction system. The 40 μL one-pot reaction assay consisted of 15 μL multiplexed-

RPA-MIX, 2 μL DNA, 1 μL B buffer, 1×Cas-reaction buffer, 100 nM 13-35S-crRNA, 100 nM 12-NOS-crRNA, 1 μM quenched fluorescent ssRNA reporter, 1 μM quenched

fluorescent ssDNA reporter, 0.1×Transcription buffer (contain rNTP MIX) and 0.5 μL RNase inhibitor, enzyme MIX (100 nM Cas13a, 100 nM Cas12a and 0.5 μL T7 RNA

polymerase) was initially added on the tube cap and add nuclease-free water to 40 μL. After RPA reaction for 15 min, enzyme mix was centrifuged into the reaction solution, then the reaction was performed at 37 °C for 20 min on CFX96 Real-Time PCR system with fluorescence measurements taken every 30s. In System 1, the ChemiDoc Touch Imaging System (Bio Rad, Hercules, CA, USA) was used to observe the green-redyellow fluorescence. For on-site detection, a handheld LUYOR-3415RG instrument (LUYOR, USA) can be equipped with a 494-nm excitation filter for the FAM reporter (green) and a 535-nm excitation filter for the HEX reporter (red).

DOI: 10.1016/j.jare.2024.07.027