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Aug 2020

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A Novel PCR-Based Methodology for Viral Detection Utilizing Mechanical Homogenization
一种基于聚合酶链反应的机械均匀化病毒检测新方法   

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Abstract

The impact of viral diseases on human health is becoming increasingly prevalent globally with the burden of disease being shared between resource-rich and poor areas. As seen in the global pandemic caused by SARS-CoV-2, there is a need to establish viral detection techniques applicable to resource-limited areas that provide sensitive and specific testing with a logistically conscious mindset. Herein, we describe a direct-to-PCR technology utilizing mechanical homogenization prior to viral PCR detection, which allows the user to bypass traditional RNA extraction techniques for accurate detection of human coronavirus. This methodology was validated in vitro, utilizing human coronavirus 229E (HCoV-229E), and then clinically, utilizing patient samples to test for SARS-CoV-2 infection. In this manuscript, we describe in detail the protocol utilized to determine the limit of detection for this methodology with in vitro testing of HCoV-229E.

Keywords: Coronavirus (新冠病毒), Virology (病毒学), PCR (PCR), RT-qPCR (RT-qPCR), Diagnostics (诊断), Molecular diagnostics (分子诊断), Homogenize (均质化), Infectious disease (传染病)

Background

Polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-qPCR)-based methodologies are the current mainstay of detection for human viral infections (Elnifro et al., 2000). These PCR methodologies often rely on multiple chemical extraction steps, for lysis of the viral particles from the clinical sample, followed by a series of washing steps, which are meant to purify the exposed viral genetic material while removing other macromolecules, such as carbohydrates and proteins, that may impact downstream molecular applications (Elnifro et al., 2000). Once the purified genetic material has been isolated, it is then placed into the PCR mixture for detection of a specific targeted genetic sequence, thereby verifying the presence of the virus. This traditional extraction model for PCR detection of virus is highly sensitive and specific in confirming infection (Elnifro et al., 2000). However, as seen in the global pandemic caused by SARS-CoV-2, the stressed global supply chain could not always ensure availability of the chemical reagents needed for these extractions, driving the need for novel diagnostic techniques (Bustin et al., 2020).


Herein, we describe a methodology where mechanical homogenization was used to lyse viral particles, exposing RNA for accurate detection of the virus when placed directly into PCR, without undergoing further extraction steps or chemical washes (Morehouse et al., 2020). This methodology has been reported utilizing in vitro simulation of clinical viral samples with human coronavirus 229E (HCoV-229E) (Morehouse et al., 2020), as well as in a field validation study with the use of nasopharyngeal and oropharyngeal swabs for the detection of SARS-CoV-2 from patients (Morehouse et al., 2021). This homogenization-based direct-to-PCR technique allows for accurate viral detection of human coronaviruses, without the need for time consuming and costly chemical extraction techniques traditionally used in RT-qPCR detection workflows (Morehouse et al., 2020).


In an effort to improve access to cost effective diagnostics solutions, we demonstrate how human coronavirus can be accurately detected in clinical concentrations with similar sensitivity and specificity to the current gold standard extraction-based techniques (Morehouse et al., 2020 and Morehouse et al., 2021). It is critical that innovative diagnostic solutions such as this are continually developed with concern for the logistical challenges of providing accurate viral detection in resource-limited settings. Through the utilization of mechanical homogenization, we were able to detect HCoV-229E off spiked swabs, simulating clinically relevant concentrations of virus on nasopharyngeal swabs as an initial proof of concept study for this technique (Morehouse et al., 2021). The homogenization-based direct-to-PCR methodology was then further validated with patient samples screening for SARS-CoV-2 in a later study (Morehouse et al., 2021). In this manuscript, we lay out the detailed protocol utilized in the initial proof of concept testing of our homogenization-based direct-to-PCR technology, utilizing simulated nasopharyngeal swabs with spiked HCoV-229E to determine the limit of detection for this proposed methodology.

Materials and Reagents

  1. 2 mL screw cap tubes (Omni International Inc., catalog number: 19-628) prefilled with 1 mL of viral transport media (VTM)

  2. Corning 2 mL internal threaded polypropylene cryogenic vials, self-standing with round bottom (Corning, catalog number: 431386)

  3. Cotton tipped swabs (Dynarex, catalog number: 4305)

  4. PCR tubes: 96 well plate format (Bio-Rad, catalog number: HSP9601) with plate sealing film (Bio-Rad, catalog number: MSB1001)

  5. Viral transport media, made following the United States Centers for Disease Control and Prevention (US CDC) protocol (SOP#: DSR-052-05). While we utilized the US CDC VTM made in our laboratory for this protocol, the authors are aware that many other commercially available VTMs are eligible to be substituted based on the availability of reagents in any given laboratory environment.

  6. HCoV-229E virus stock in DMEM, prepared from cell culture

    HCoV-229E (ATCC, catalog number: VR-740) cultured following manufacturers recommendations on MRC-5 cells (ATCC, catalog number: CCL-171) in standard DMEM (Gibco, catalog number: 11965092). Cell culture media was collected following manufacturer’s instructions 72 h after inoculation, and then utilized as the virus stock for these experiments.

  7. Corning 75 cm2 U-shaped canted neck cell culture flask with vent cap (Corning, catalog number: 430641U)

  8. Corning PureCoat Amine 6-well tissue culture plates (Corning, catalog number: 354721)

  9. Luna Universal One-Step RT-qPCR Kit (New England Biolabs, catalog number: E3005S)

  10. HCoV-229E N forward and reverse primer set (Integrated DNA Technologies, Custom Oligonucleotide Purchase)

    1. N gene forward primer: 5’-AGGCGCAAGAATTCAGAACCAGAG-3’

    2. N gene reverse primer: 5’-AGCAGGACTCTGATTACGAGAAAG-3’

  11. Molecular grade agarose (Bio-Rad, catalog number: 161-3101)

  12. Ethidium bromide (Bio-Rad, catalog number: 161-0433)

  13. TBE buffer (IBI Scientific, catalog number: IB70153)

  14. 100 bp molecular ruler (Bio-Rad, catalog number: 1708202)

  15. Trypan blue (Bio-Rad, catalog number: 1450021)

Equipment

  1. Bead Ruptor Elite (Omni International Inc., catalog number: 19-042E)

  2. CFX connect (Bio-Rad, catalog number: 1855201)

  3. Gel Doc EZ Imaging System (Bio-Rad, catalog number: 1708270)

  4. Rainin Classic manual pipettes (Rainin, catalog number: 17008708)

  5. ThermoFisher Fresco 17 microcentrifuge (Thermo Fisher Scientific, catalog number: 75002421)

  6. Gel electrophoresis chamber (BioRad, catalog number: 1704487EDU)

  7. Thermo Scientific TSX ultra-low freezer (Thermo Fischer Scientific, catalog number: 09313868)

Software

  1. CFX Software Bio-Rad CFX Maestro 1.1 Version 4.1.2433.1219

  2. EZ Imager Software Image LabTM Version 6.0.0 build 25

Procedure

  1. HCoV-229E Viral Stock Preparation and Dilutions

    1. Grow a viral stock of HCoV-229E on MRC-5 cells and harvest the virus from the cell supernatant, following the manufacturer’s instructions. Use the virus to infect 80% confluent MRC-5 cells plated in T-75 flasks with a multiplicity of infection (MOI) of 1.6. Culture the infected cells for another 72 h post-infection, until greater than 70% cytopathic effect (CPE) is observed. Harvest the cell culture supernatant to create the HCoV-229E virus stock for utilization in these experiments. Centrifuge the harvested supernatant at 3,000 × g for 10 min, to pellet any cellular debris within the solution. After centrifugation, transfer the supernatant containing the virus stock to a clean 15 mL conical tube for storage and discard the pelleted debris.

    2. Following virus stock purification, complete plaque assays to determine a starting viral load. In this protocol, our viral stock had a concentration of 1.2 × 106 PFU/mL of HCoV-229E. Perform plaque assays via serial dilutions of viral stock onto MRC-5 cells plated to 80% confluence in 6-well plates. The starting cell count for this procedure was 200,000 cells/well in 2 mL of media. Following virus addition, allow the cells to grow for 5 days before evaluation of plaque formation.

    3. With 10 mL of HCoV-229E viral stock at 1.2 × 106 PFU/mL, dilute the HCoV-229E in a stepwise fashion by pipetting 1 mL of stock solution into 9 mL of viral transport media, inverting the new dilution three times, and then pipetting 1 mL into the next tube of 9 mL of viral transport media. Conduct this serial dilution until obtaining a stock of 1.2 × 101 PFU/mL. On the final dilution step, following the three inversions of the tube, pipette 1 mL of viral stock and discard it to maintain accurate concentrations. This step was conducted with all reagents thawed to room temperature.

    4. Store HCoV-229E stock dilutions in 2 mL cryogenic storage vials at -80°C until needed, if not being utilized within 2 h of stock dilution production.


  2. Sample Swab Preparation

    1. Thaw HCoV-229E stock dilutions to 4°C, if not currently available for use. Conduct the remainder of the swab preparation procedure at room temperature.

    2. Pipette 1 mL of sterile viral transport media (VTM) into clean 2 mL screw cap tubes. Do not lose the caps for these tubes, as they will be needed in the homogenization step.

    3. Once HCoV-229E stocks are thawed, prepare swab samples from each dilution for PCR detection by submerging a cotton tipped swab in the HCoV-229E dilution for 5 s, to allow for complete saturation, and then placing the swab directly into one of the prefilled 2 mL screw capped tubes.

    4. Break off the stalk of the swab while maintaining the cotton tip within the VTM and replace the screw cap on the 2 mL tube.

    5. Repeat steps B2–B4 until the desired number of swabs for each dilution have been created in preparation for PCR detection.

      Note: We recommend a minimum of 5 swabs at each concentration for preliminary limit of detection work, with a minimum of 30 additional replicates needed for confirmation once a targeted concentration has been determined.


  3. Shaker Mill Homogenization

    1. Ensure the screw capped tubes containing 1 mL of VTM and swabs are tightly sealed. Place 2 mL tube carriage on the Bead Ruptor Elite. Load the screw capped tubes onto the 2 mL carriage, then tighten the fingerplate, and close the lid.

    2. Homogenize at 4.2 m/s and room temperature for 30 s.

    3. Some froth may be generated within the tubes because of the homogenization; this is normal. To alleviate any frothing in the tubes, remove them from the Bead Ruptor Elite and spin the tubes in a microcentrifuge up to 10,000 × g for no more than 15 s.


  4. RT-qPCR

    1. Prepare the RT-qPCR HCoV-229E N Primer Mastermix, as described in the recipes section.

    2. Prepare the RT-qPCR mixture using the Luna Universal One-Step RT-qPCR kit, as described in the table below (Table 1).


      Table 1. Luna Universal One-Step RT-qPCR mixture for RT-qPCR

      Reagent Volume per reaction
      Luna Universal One-Step Reaction Mix (2×) 10 µL
      Luna WarmStart® RT Enzyme Mix (20×) 1 µL
      Forward Primer (5 µM) 1.25 µL
      Reverse Primer (5 µM) 1.25 µL
      Template RNA 1 µL
      Nuclease-Free Water 5.5 µL


    3. Prepare the PCR plate with identified spots for positive controls of purified HCoV-229E RNA, the unknown samples of homogenized HCoV-229E, and negative controls using DPEC and no HCoV-229E RNA.

    4. Load 1 µL of sample (positive, unknown, or negative) into each well, as shown in the table above.

    5. Seal the PCR plate with the plate sealing film.

    6. Place plate into the CFX Connect and run the RT-qPCR with the following cycle parameters:

      1. 55°C for 30 min

      2. 95°C for 1 min

      3. 44 cycles of 95°C for 15 s and 56°C for 30 s

      4. Hold at 4°C for further processing


  5. Amplicon Confirmation

    1. Prepare a 2% agarose gel using TBE buffer and 5 µL of 10 mg/mL ethidium bromide stock for amplicon visualization. Both 15 well and 8 well combs can be utilized, to create wells large enough to accommodate the 20 µL volume of sample. Herein, we chose to use 15 well combs in our gel due to the large number of samples we were trying to visualize.

    2. Load 20 µL of trypan blue into the 20 µL of suspected amplicon following RT-qPCR (1:1 mixture).

    3. Load 20 µL of the amplicon/trypan mixture into each well of the 2% agarose gel, with 3 µL of 100 bp molecular ruler in 12 µL of trypan blue into one of the wells.

    4. Run the gel at 125 V for approximately 1 h, or until the bands have sufficiently separated.

    5. Visualize the gel utilizing the Gel Doc EZ Imager System, to observe the size of amplicon product in comparison to the ladder. The EZ Imager Software is set to standard ethidium bromide exposure settings with targeting of band intensities for imaging of the gels. This allows for identification of the target amplicon size for the N gene mixture at 308 bp.

Data analysis

In accordance with US CDC and World Health Organization (WHO) recommendations, detection of coronavirus associated disease should be deemed as positive in PCR detection for Ct values less than 40 (Bustin et al., 2020 and Cheng et al., 2020). While these recommendations were put forward for SARS-CoV-2 detection, we have adopted them to serve as an appropriate cut off for positive detection of HCoV-229E in our protocol as well. When utilizing the Ct < 40 as the cut off for RT-qPCR, we still wanted to confirm the presence of an appropriate amplicon size (308 bp) via gel visualization, given the use of standard nucleotide primers that have the potential to dimerize giving a false positive. We were able to confidently confirm the presence of HCoV-229E in our samples, as detected by RT-qPCR.

The data supporting the in vitro limit of detection studies described herein is available in open access from the Virology Journal (Morehouse et al., 2020). Additionally, the manuscript presenting the clinical validation of this method for SARS-CoV-2 detection is also available as open access through PLOS ONE (Morehouse et al., 2021).

Notes

While this protocol was completed using traditional nucleotide primer sets targeting the N gene of HCoV-229E, a substitution of traditional primers for fluorescent probes can be made. If probes are utilized in place of primers, a change in the RT-qPCR kit will also be required, to one compatible with fluorescent probes. Additionally, if probes are used, there is no need for amplicon visualization on agarose gel following RT-qPCR, as there is minimal risk of primer self-binding or dimerization when utilizing a fluorescent quenching system.

Acknowledgments

The authors would like to thank Karl Jahn and Pete Tortorelli of Omni International Inc for the organizational and financial support of our work in developing this methodology. Additionally, we would like to thank Rachel Nash and Leah Proctor for their continued encouragement of our research efforts, with a particular acknowledgment for their sacrifices during the early days of the COVID-19 pandemic to support our work on this project.

The authors would like to acknowledge that this protocol has been adapted from previous work by Morehouse et al. (2020) entitled “A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E” and published in Virology Journal on 25 August 2020 (doi: 10.1186/s12985-020-01405-y).

Competing interests

ZPM, CMP, and RJN are named inventors on the patent describing this method held by Omni International, A PerkinElmer Company, but have no personal financial interests to disclose in the direct commercialization of this technology. CMP, GLR, and RJN are all full-time employees of Omni International Inc, A PerkinElmer Company, and GLR and RJN have no personal financial interests in the performance of the company. CMP is a shareholder of PerkinElmer, holding a personal financial interest in the performance of the company. ZPM is in a consulting relationship with Omni International, A PerkinElmer Company and holds no personal financial interests in the performance of the company. ZPM and RJN are also associated with Jeevan Biosciences LLC, a company which is not involved in the work described within this manuscript.

Ethics

No human subjects, patient samples, or animal subjects were utilized in the protocol described herein, thus no IUCAUC or IRB approval was required.

References

  1. Elnifro, E. M., Ashshi, A. M., Cooper, R. J. and Klapper, P. E. (2000). Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 13(4): 559-570.
  2. Bustin, S. A. and Nolan, T. (2020). RT-qPCR Testing of SARS-CoV-2: A Primer. Int J Mol Sci 21(8).
  3. Morehouse, Z. P., Proctor, C. M., Ryan, G. L. and Nash, R. J. (2020). A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E. Virol J 17(1): 129.
  4. Morehouse, Z. P., Samikwa, L., Proctor, C. M., Meleke, H., Kamdolozi, M., Ryan, G. L., Chaima, D., Ho, A., Nash, R. J. and Nyirenda, T. S. (2021). Validation of a direct-to-PCR COVID-19 detection protocol utilizing mechanical homogenization: A model for reducing resources needed for accurate testing. PLoS One 16(8): e0256316.
  5. Cheng, M. P., Papenburg, J., Desjardins, M., Kanjilal, S., Quach, C., Libman, M., Dittrich, S. and Yansouni, C. P. (2020). Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus 2: A Narrative Review. Ann Intern Med 172(11): 726-734.

简介

[摘要]病毒性疾病对人类健康的影响在全球范围内日益普遍,疾病负担在资源丰富地区和贫困地区之间分担。从 SARS-CoV-2 引起的全球大流行中可以看出,需要建立适用于资源有限地区的病毒检测技术,以具有后勤意识的思维方式提供敏感和特定的检测。在此,我们描述了一种在病毒 PCR 检测之前利用机械均质化的直接 PCR 技术,该技术允许用户绕过传统的 RNA 提取技术来准确检测人类冠状病毒。该方法在体外使用人类冠状病毒 229E (HCoV-229E) 进行了验证,然后在临床上使用患者样本测试 SARS-CoV-2 感染。在这份手稿中,我们详细描述了用于确定该方法的检测限的协议,并通过 HCoV-229E的体外测试。


[背景] 基于聚合酶链式反应 (PCR) 和逆转录酶 PCR (RT-qPCR) 的方法是目前检测人类病毒感染的主要方法 ( Elnifro 等人,2000)。这些 PCR 方法通常依赖于多个化学提取步骤,用于从临床样本中裂解病毒颗粒,然后进行一系列洗涤步骤,这些步骤旨在纯化暴露的病毒遗传物质,同时去除其他大分子,例如碳水化合物和蛋白质,这可能会影响下游分子应用( Elnifro 等人,2000)。分离纯化的遗传物质后,将其放入 PCR 混合物中以检测特定的目标基因序列,从而验证病毒的存在。这种用于病毒PCR检测的传统提取模型在确认感染方面具有高度敏感性和特异性( Elnifro 等人,2000)。然而,正如在 SARS-CoV-2 引起的全球大流行中所看到的那样,压力重重的全球供应链无法始终确保这些提取所需的化学试剂的可用性,从而推动了对新型诊断技术的需求( Bustin 等人,2020)。
在这里,我们描述了一种方法,其中机械均质化用于裂解病毒颗粒,当直接放入 PCR 时暴露 RNA 以准确检测病毒,无需进行进一步的提取步骤或化学洗涤(Morehouse等人,2020)。据报道,该方法利用人类冠状病毒 229E (HCoV-229E) 对临床病毒样本进行体外模拟(Morehouse等人,2020 年),以及使用鼻咽和口咽拭子进行检测的现场验证研究来自患者的 SARS-CoV-2(Morehouse等人,2021 年)。这种基于均质化的直接 PCR 技术允许对人类冠状病毒进行准确的病毒检测,而不需要传统上用于 RT-qPCR 检测工作流程的耗时且昂贵的化学提取技术(Morehouse等人,2020 年)。
为了改善获得具有成本效益的诊断解决方案的机会,我们展示了如何以与当前基于金标准提取的技术相似的敏感性和特异性在临床浓度下准确检测人类冠状病毒(Morehouse等人,2020 和 Morehouse等人。 , 2021)。至关重要的是,不断开发诸如此类的创新诊断解决方案,以应对在资源有限的环境中提供准确病毒检测的后勤挑战。通过利用机械均质化,我们能够从尖刺拭子中检测 HCoV-229E,模拟鼻咽拭子上临床相关的病毒浓度,作为该技术的初步概念验证研究(Morehouse等人,2021 年)。在后来的研究中,基于均质化的直接 PCR 方法通过患者样本筛查 SARS-CoV-2 得到了进一步验证(Morehouse等人,2021 年)。在这份手稿中,我们列出了在我们基于均质化的直接 PCR 技术的初始概念验证测试中使用的详细协议,利用带有尖刺 HCoV-229E 的模拟鼻咽拭子来确定该方法的检测限。

关键字:新冠病毒, 病毒学, PCR, RT-qPCR, 诊断, 分子诊断, 均质化, 传染病

材料和试剂
1. 2 mL 螺旋盖管(Omni International Inc.,目录号:19-628)预装 1 mL 病毒转运培养基(VTM)
2. Corning 2 mL内螺纹聚丙烯低温小瓶,自立式圆底(Corning,目录号:431386)
3. 棉签( Dynarex ,目录号:4305)
4. PCR管:96孔板格式(Bio-Rad,目录号:HSP9601),带有板密封膜(Bio-Rad,目录号:MSB1001)
5. 病毒运输培养基,按照美国疾病控制和预防中心 (US CDC) 协议 (SOP#: DSR-052-05) 制作。虽然我们使用我们实验室制造的美国 CDC VTM 进行该协议,但作者知道,根据任何给定实验室环境中试剂的可用性,许多其他市售 VTM 有资格被替代。
6. DMEM 中的 HCoV-229E 病毒原液,由细胞培养物制备
HCoV-229E(ATCC,目录号:VR-740)按照制造商对标准 DMEM(Gibco,目录号:11965092)中 MRC-5 细胞(ATCC,目录号:CCL-171)的建议进行培养。接种后 72 小时按照制造商的说明收集细胞培养基,然后用作这些实验的病毒原液。
7. Corning 75 cm 2 U形斜颈细胞培养瓶,带通气盖(Corning,目录号:430641U)
8. Corning PureCoat Amine 6孔组织培养板(Corning,目录号:354721)
9. Luna Universal One-Step RT-qPCR Kit(New England Biolabs,目录号:E3005S)
10. HCoV-229E N 正向和反向引物组(Integrated DNA Technologies,定制寡核苷酸采购)
a. N基因正向引物:5'-AGGCGCAAGAATTCAGAACCAGAG-3'
b. N基因反向引物:5'-AGCAGGACTCTGATTACGAGAAAG-3'
11. 分子级琼脂糖(Bio-Rad,目录号:161-3101)
12. 溴化乙锭(Bio-Rad,目录号:161-0433)
13. TBE 缓冲液(IBI Scientific,目录号:IB70153)
14. 100 bp分子标尺(Bio-Rad,目录号:1708202)
15. 台盼蓝(Bio-Rad,目录号:1450021)


设备


1. Bead Ruptor Elite(Omni International Inc.,目录号:19-042E)
2. CFX 连接(Bio-Rad,目录号:1855201)
3. Gel Doc EZ 成像系统(Bio-Rad,目录号:1708270)
4. Rainin Classic 手动移液器( Rainin ,目录号:17008708)
5. ThermoFisher Fresco 17 微量离心机(Thermo Fisher Scientific,目录号:75002421)
6. 凝胶电泳室( BioRad ,目录号:1704487EDU)
7. Thermo Scientific TSX 超低温冰箱(Thermo Fischer Scientific,目录号:09313868)


软件


1. CFX 软件 Bio-Rad CFX Maestro 1.1 版本 4.1.2433.1219
2. EZ Imager Software Image Lab TM版本 6.0.0 build 25


程序


A. HCoV-229E 病毒原液制备和稀释
1. 按照制造商的说明,在 MRC-5 细胞上培养 HCoV-229E 病毒储备,并从细胞上清液中收获病毒。使用该病毒感染 80% 融合的 MRC-5 细胞,这些细胞被镀在 T-75 烧瓶中,感染复数 (MOI) 为 1.6。感染后将受感染的细胞再培养 72 小时,直到观察到大于 70% 的细胞病变效应 (CPE)。收获细胞培养上清液以制造 HCoV-229E 病毒原液,用于这些实验。将收获的上清液以 3,000 × g离心10 分钟,以沉淀溶液中的任何细胞碎片。离心后,将含有病毒的上清液转移到干净的 15 mL 锥形管中进行储存,并丢弃颗粒碎片。
2. 病毒原液纯化后,完成噬菌斑测定以确定起始病毒载量。在该协议中,我们的病毒储备的 HCoV-229E 浓度为 1.2 × 10 6 PFU/mL。通过将病毒原液连续稀释到 MRC-5 细胞上进行斑块测定,这些细胞在 6 孔板中镀到 80% 的汇合度。此过程的起始细胞计数为 2 mL 培养基中的 200,000 个细胞/孔。添加病毒后,在评估斑块形成之前让细胞生长 5 天。
3. 用 10 mL 1.2 × 10 6 PFU/mL 的 HCoV-229E 病毒原液,通过移液器将 1 mL 原液移入 9 mL 病毒转运介质中,逐步稀释 HCoV-229E,将新稀释液倒置 3 次,然后然后将 1 mL 移液到下一管 9 mL 病毒运输介质中。进行此连续稀释,直到获得 1.2 × 10 1 PFU/ mL 的库存。在最后的稀释步骤中,在管的三个倒置之后,移液器 1 mL 的病毒原液并丢弃以保持准确的浓度。该步骤在所有试剂解冻至室温的情况下进行。
4. 将 HCoV-229E 库存稀释液储存在 -80°C 的 2 mL 低温储存瓶中,直到需要,如果在库存稀释生产后 2 小时内未使用。


B. 样本拭子制备
1. 如果当前无法使用,将 HCoV-229E 原液稀释液解冻至 4°C。在室温下进行剩余的拭子制备程序。
2. 将 1 mL 的无菌病毒运输介质 (VTM) 移液器放入干净的 2 mL 螺帽管中。不要丢失这些管子的盖子,因为它们在均质化步骤中是必需的。
3. 一旦 HCoV-229E 原液解冻,通过将棉签浸入 HCoV-229E 稀释液中 5 秒,从每个稀释液中制备用于 PCR 检测的拭子样本,以使其完全饱和,然后将拭子直接放入其中一个预填充液中2 毫升螺旋盖管。
4. 在将棉签保持在 VTM 内的同时折断棉签的柄,并更换 2 mL 管上的螺帽。
5. 重复步骤 B2-B4,直到为每个稀释度创建所需数量的拭子,为 PCR 检测做准备。
注意:我们建议每个浓度至少 5 个拭子用于初步检测限制,一旦确定目标浓度,至少需要额外重复 30 次以确认。


C. 摇床均质化
1. 确保含有 1 mL VTM 和拭子的螺旋盖管密封。将 2 mL 管架放在 Bead Ruptor Elite 上。将螺旋盖管装入 2 mL 托架上,然后拧紧指板,并盖上盖子。
2. 在 4.2 m/s 和室温下均质化 30 s。
3. 由于均质化,管内可能会产生一些泡沫;这个是正常的。为减轻试管中的任何泡沫,请将它们从 Bead Ruptor Elite 中取出,并在微量离心机中以高达 10,000 × g的速度旋转试管,时间不超过 15 秒。


D. 逆转录定量PCR
1. 准备 RT-qPCR HCoV-229E N Primer Mastermix ,如配方部分所述。
2. 使用 Luna 通用一步 RT-qPCR 试剂盒制备 RT-qPCR 混合物,如下表(表 1)中所述。


表 1. 用于 RT-qPCR 的 Luna 通用一步法 RT-qPCR 混合物
试剂 每次反应的体积
Luna 通用一步反应混合物 (2 × ) 10 µL
Luna WarmStart ® RT 酶混合物 (20 × ) 1 µL
正向引物 (5 µM) 1.25 µL
反向引物 (5 µM) 1.25 µL
模板 RNA 1 µL
无核酸酶水 5.5 µL


3. 为纯化的 HCoV-229E RNA 的阳性对照、均质化的 HCoV-229E 的未知样品和使用 DPEC 和无 HCoV-229E RNA 的阴性对照准备 PCR 板。
4. 将 1 μL 样品(阳性、未知或阴性)装入每个孔中,如上表所示。
5. 用封板膜封住 PCR 板。
6. 将板放入 CFX Connect 并使用以下循环参数运行 RT-qPCR:
a. 55°C 30 分钟
b. 95°C 1 分钟
c. 95°C 15 秒和 56°C 30 秒的 44 个循环
d. 保持在 4°C 用于进一步处理


E. 扩增子确认
1. 使用 TBE 缓冲液和 5 μL 的 10 mg/mL 溴化乙锭库存制备 2% 琼脂糖凝胶,用于扩增子可视化。可以使用 15 孔和 8 孔梳,以创建足够大的孔以容纳 20 µL 体积的样品。在这里,我们选择在凝胶中使用 15 孔梳,因为我们试图可视化大量样品。
2. 在 RT-qPCR(1:1 混合物)之后,将 20 μL 的台盼蓝装入 20 μL 的可疑扩增子中。
3. 将 20 μL 的扩增子/台盼混合物装入 2% 琼脂糖凝胶的每个井中,将 3 μL 的 100 bp 分子标尺在 12 μL 的台盼蓝中装入其中一个井中。
4. 在 125 V 下运行凝胶约 1 小时,或直到条带充分分离。
5. 使用 Gel Doc EZ Imager System 可视化凝胶,以观察扩增子产物与梯子相比的大小。 EZ Imager 软件设置为标准溴化乙锭曝光设置,并针对凝胶成像的条带强度进行设置。这允许在 308 bp 处识别 N 基因混合物的目标扩增子大小。


数据分析


根据美国疾控中心和世界卫生组织 (WHO) 的建议,在 PCR 检测中 Ct 值小于 40 ( Bustin ) 的冠状病毒相关疾病的检测应视为阳性。 等。 , 2020 和 Cheng等人, 2020)。虽然这些建议是针对 SARS-CoV-2 检测提出的,但我们也采用它们作为我们方案中 HCoV-229E 阳性检测的适当截止值。当使用 Ct < 40 作为 RT-qPCR 的截止值时,我们仍然想通过凝胶可视化确认是否存在适当的扩增子大小 (308 bp),因为使用了具有二聚体潜力的标准核苷酸引物假阳性。通过 RT-qPCR 检测,我们能够自信地确认样本中存在 HCoV-229E。
支持本文所述检测研究的体外限制的数据可从《病毒学杂志》(Morehouse等人,2020 年)的开放获取中获得。此外,介绍该方法对 SARS-CoV-2 检测的临床验证的手稿也可通过 PLOS ONE 开放获取(Morehouse等人,2021 年)。


笔记


虽然该协议是使用针对 HCoV-229E 的 N 基因的传统核苷酸引物组完成的,但可以将传统引物替换为荧光探针。如果使用探针代替引物,则还需要将 RT-qPCR 试剂盒更改为与荧光探针兼容的试剂盒。此外,如果使用探针,则不需要在 RT-qPCR 后在琼脂糖凝胶上进行扩增子可视化,因为使用荧光淬灭系统时引物自结合或二聚化的风险最小。


致谢


作者要感谢 Omni International Inc 的 Karl Jahn 和 Pete Tortorelli为我们开发此方法的工作提供的组织和财务支持。此外,我们要感谢 Rachel Nash 和 Leah Proctor 对我们研究工作的持续鼓励,特别感谢他们在 COVID-19 大流行初期为支持我们在这个项目上的工作所做的牺牲。
作者要承认,该协议已改编自 Morehouse等人以前的工作。 (2020) 题为“A new two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E” ,于 2020 年 8 月 25 日发表在Virology Journal上 ( doi : 10.1186/s12985-020-01405-y)。


利益争夺


ZPM、CMP 和 RJN 是 Omni International, A PerkinElmer Company 持有的描述该方法的专利的发明人,但在该技术的直接商业化过程中没有个人经济利益可披露。 CMP、GLR 和 RJN 都是 Omni International Inc, A PerkinElmer Company 的全职员工,GLR 和 RJN 在公司业绩中没有个人经济利益。 CMP 是 PerkinElmer 的股东,在公司业绩中持有个人经济利益。 ZPM 与 PerkinElmer 旗下公司 Omni International 有咨询关系,并且在公司业绩中不持有个人经济利益。 ZPM 和 RJN 还与 Jeevan Biosciences LLC 有关联,该公司不参与本手稿中描述的工作。


伦理


此处描述的方案中没有使用人类受试者、患者样本或动物受试者,因此不需要 IUCAUC 或 IRB 批准。


参考


1. Elnifro ,EM, Ashshi ,AM,Cooper,RJ 和Klapper ,PE(2000 年)。多重 PCR:在诊断病毒学中的优化和应用。 临床微生物学第 13(4) 版:559-570。
2. Bustin , SA 和 Nolan, T. (2020)。 SARS-CoV-2 的 RT-qPCR 测试:引物。 诠释 J Mol Sci 21(8)。
3. Morehouse, ZP, Proctor, CM, Ryan, GL 和 Nash, RJ (2020)。一种新的两步直接 PCR 方法,用于使用人类冠状病毒 229E 检测拭子上的病毒。 病毒学杂志17 (1):129。
4. Morehouse, ZP, Samikwa , L., Proctor, CM, Meleke , H., Kamdolozi , M., Ryan, GL, Chaima, D., Ho, A., Nash, RJ 和 Nyirenda, TS (2021)。利用机械均质化验证直接 PCR COVID-19 检测方案:减少准确检测所需资源的模型。 公共科学图书馆一号16(8):e0256316。
5. Cheng, MP, Papenburg , J., Desjardins, M., Kanjilal , S., Quach, C., Libman , M., Dittrich, S. 和Yansouni , CP (2020)。严重急性呼吸综合征相关冠状病毒 2 的诊断测试:叙述性评论。 Ann Intern Med 172(11):726-734。


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引用:Morehouse, Z. P., Proctor, C. M., Ryan, G. L. and Nash, R. J. (2022). A Novel PCR-Based Methodology for Viral Detection Utilizing Mechanical Homogenization. Bio-protocol 12(5): e4349. DOI: 10.21769/BioProtoc.4349.
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