OMNI Bead Ruptor Elite应用 人类冠状病毒中提取RNA-自主发布-资讯-生物在线

OMNI Bead Ruptor Elite应用 人类冠状病毒中提取RNA

作者:北京平利洋经贸有限公司 2022-02-21T17:01 (访问量:5207)

从组织样本中提取病毒 RNA 是用于确认是否存在疑似病毒感染的关键方法。随着全球病毒感染的增加,来自各行各业的研究人员和临床医生已将注意力转向增加对病毒引起的病理学的研究。在该协议中,我们已经证明 OMNI 通用 RNA 试剂盒可用于从受感染的人肺成纤维细胞组织培养瓶上清液中的人冠状病毒 229e (HCoV-229e) 中提取病毒 RNA。
此外,这些样品使用 Bead Ruptor Elite 进行处理,将 RNA 试剂盒提取的初始均质化步骤所需的时间减少了一半。这种半自动化的样品制备步骤提高了传统离心柱 RNA 提取方案的通量,允许在 30 秒内处理多达 24 个样品,而传统方法需要长达 24 分钟才能完成相同的程序。
使用 Bead Ruptor Elite 进行 RNA 试剂盒处理可提高 RT-qPCR 检测到的病毒 RNA 产物产量,这表明可能检测到低浓度样品。通过从组织培养上清液中扩增 HCoV-229e 的核衣壳基因,我们证明了在使用传统的离心柱试剂盒进行病毒 RNA 提取时,珠磨机均质比涡旋的效率和功效更高。

Materials and Methods

Materials

Bead Ruptor Elite (PN 19-040E)

2mL Tube Carriage Kit (PN 19-010-310)

Universal RNA Purification Kit (PN 26-010V), includes:

Universal Microbial Homogenizing Mix, Nuclease Free (PN 19-632)

2mL Reinforced Tubes with Screw Caps (PN 19-649)

Methods

Cell Culture and Virus Growth

Human coronavirus 229e (HCoV-229e) was added at a MOI of 1.4 to 60% confluent MRC-5 (lung fibroblast) cells, 48 hours after plating. The flask was maintained with DMEM infused with 5% heat inactivated FBS and 1% L-Glutamine, incubated at 37 C with 5% CO2. The cell culture supernatant was harvested at 72 hours post infection when 70% CPE was observed.


Viral RNA Extraction from Supernatant

300 µL of supernatant was added to 300 µL of RLB buffer (from Omni Universal RNA Purification Kit, PN 26-010V) in either an empty 2 mL homogenization tube (PN 19-649) or a prefilled Universal Microbial Homogenizing Mix tube (PN 19-632). Sample tubes were then homogenized by one of two methods: 1, using the Bead Ruptor Elite, 1 x 30s cycle at 4.2 m/s; or 2, vortexing for 60s using a vortex mixer similar to the Vortex Mixer 24 (PN 28-101). After the initial homogenization step was completed, the remainder of the extraction was carried out per the manufacturer’s instructions for the OMNI Universal RNA Purification Kit with one exception: 100% EtOH was substituted for the 70% EtOH called for in step 7 of the kit’s protocol. RNA was eluted from the spin column using DPEC water, allowing an on-column reaction/dissolution time of 5 mins prior to centrifugation.

HCoV-229e RT-qPCR

HCoV-229e nucleocapsid gene (N gene) was selected as a target for RT-PCR from peer reviewed publications. The N gene was targeted with forward primer 5’-AGGCGCAAGAATTCAGAACCAGAG-3’ and reverse primer 5’-AGCAGGACTCTGATTACGAGAAAG-3’. 1 µL of extracted RNA was added, for a total reaction volume of 20 µL using the proportions of primers, RNA, SYBER, RT, and DPEC laid out in the New England Biologics Luna RT-qPCR Kit. The reaction was run for 44 cycles and the resulting amplicons were loaded into a 2% agarose gel for product visualization.

Results

RT-qPCR was completed on the supernatant of MRC-5 tissue culture flasks, 72 h.p.i. once 70% CPE was observed. The efficacy of the 4 homogenization methods for processing these samples was evaluated using Cq values (Figure 1) and confirmed with a 2% agarose gel (Figure 2). The data demonstrates successful extraction of viral RNA from cell culture supernatant using the OMNI Universal RNA Purification Kit. Bead Ruptor Elite homogenization increased extracted RNA yield in comparison to traditional vortexer, as demonstrated by lower Cq values in samples processed using bead milling homogenization versus vortexing. Increased band intensity during amplicon visualization is seen from homogenization using the Bead Ruptor Elite, both with and without 0.1 mm ceramic bead media, in comparison to the bands representing a vortexer, both with and without 0.1 mm ceramic bead media. These results were confirmed via 2% agarose visualization of amplicons as shown in Figure 2 and with quantified Cq values. In negative control replicates, N gene amplification on non-infected MRC-5 culture supernatant, the late rise was attributed to primer dimer formation in these samples.

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