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Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated with the development and exacerbation of chronic lung conditions including asthma, and it is a major cause of hospitalizations in infants. Because of its clinical relevance, experimental animal models to study RSV in vivo are needed. The most common and accessible animal model in research laboratories is the mouse. However, commonly use RSV strains poorly establish infection in mice and thus titration of the virus from mouse lungs to confirm infection is not sensitive enough to detect early viral infection. Here we discuss in detail how to infect BALB/c mice with RSV and how to detect RSV genomes in the lung using reverse transcription quantitative PCR (RT-qPCR). This method allows detection of viral genomes as early as day 1 post-infection (shown in Figure 2), whereas traditional TCID50 fails to detect significant virus until after day 2 post-infection. Of note, despite of higher sensitivity, genome RT-qPCR only shows the production of viral genomes and thus positive results for this assay are not proof of production of infectious viral particles.

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Respiratory Syncytial Virus Infection in Mice and Detection of Viral Genomes in the Lung Using RT-qPCR
小鼠呼吸道合胞体病毒感染和采用RT-qPCT检测肺部的病毒基因组

微生物学 > 体内实验模型 > 病毒
作者: Yan Sun
Yan SunAffiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a2319
 and Carolina B. López
Carolina B. LópezAffiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, USA
For correspondence: lopezca@vet.upenn.edu
Bio-protocol author page: a3156
Vol 6, Iss 10, 5/20/2016, 1620 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1819

[Abstract] Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated with the development and exacerbation of chronic lung conditions including asthma, and it is a major cause of hospitalizations in infants. Because of its clinical relevance, experimental animal models to study RSV in vivo are needed. The most common and accessible animal model in research laboratories is the mouse. However, commonly use RSV strains poorly establish infection in mice and thus titration of the virus from mouse lungs to confirm infection is not sensitive enough to detect early viral infection. Here we discuss in detail how to infect BALB/c mice with RSV and how to detect RSV genomes in the lung using reverse transcription quantitative PCR (RT-qPCR). This method allows detection of viral genomes as early as day 1 post-infection (shown in Figure 2), whereas traditional TCID50 fails to detect significant virus until after day 2 post-infection. Of note, despite of higher sensitivity, genome RT-qPCR only shows the production of viral genomes and thus positive results for this assay are not proof of production of infectious viral particles.
Keywords: Respiratory syncytial virus(呼吸道合胞病毒), Respiratory infection(呼吸道感染), Virus detection(病毒检测)

[Abstract]

Materials and Reagents

  1. Vacuum-driven Sterilcup® 500 ml Millipore ExpressPLUS 0.22 μm PES (Merck Millipore Corporation, catalog number: SCGPU05RE )
  2. FastPrep® tubes (MP Biomedicals, catalog number: 5076-400 )
  3. ¼” ceramic sphere (MP Biomedicals LLC, catalog number: 116540412 )
  4. CryoTubesTM vials for freezing viruses (Thermo Fisher Scientific, catalog number: 377267 )
  5. Surgical scissors (Roboz Surgical Instrument Co)
  6. Tweezers (Roboz Surgical Instrument Co)
  7. 384-well PCR plate (VWR International, catalog number: 82051-470 )
  8. PCR strip tubes, 0.2 ml (Bioexpress, catalog number: T-3035-2 )
  9. 0.22 μm filter used in the pipette aid (VWR International, catalog number: 28145-481)
  10. BSL2 personal protective equipment (PPE)
  11. Mice (Balb/c, 6-8 weeks old, gender-mixed) (Taconic)
  12. Human RSV strain A2 (ATCC, catalog number: VR-1540 )
  13. SYBR green master mix (Applied Biosystems, catalog number: 4368708 )
  14. qPCR primer sets

    Genes
    Forward primer
    Reverse primer
    RSV g
    5’AACATACCTGCCCAGAATC3’
    5’GGTCTTGACTGTTGTAGATTGCA3’
    Rsp11
    5’CGTGACGAAGATGAAGATGC3’
    5’ GCACATTGAATCGCACAGTC3’
    α-tubulin
    5’ TGCCTTTGTGCACTGGTATG3’
    5’ CTGGAGCAGTTTGACGACAC3’

  15. RSV genome RT primer: 5’GATAAATATAGGCATGGGGAAAGTG3’
  16. TRIzol reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596018 )
  17. Chloroform (Sigma-Aldrich, catalog number: C2432-25 ml )
  18. Isopropanol (Sigma-Aldrich, catalog number: 437522 )
    Note: It is also named “2-propanol” on Sigma-Aldrich website.
  19. Ultra-Pure nuclease-free water
  20. SuperScript® III First-Strand Synthesis System (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-051 )
  21. High capacity RNA-to-cDNATM kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4387406 )
  22. Ketamine (Henry Schein)
  23. Xylazine (U.S. Food and Drug Administration, AnaSed, catalog number: 139-236 )
  24. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  25. Ketamine/Xylazine solution (see Recipes)
  26. Primer preparation (see Recipes)

Equipment

  1. Biosafety hood in biosafety level 2 facility
  2. Applied Biosystem ViiATM 7 lightcycler (Life Technologies, catalog number: 4453536 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: 4453536”.
  3. Eppendorf centrifuge 5415R (Eppendorf AG, catalog number: 22636570 )
    Note: This product has been discontinued by Eppendorf AG.
  4. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, catalog number: ND-1000 )
  5. BioRad C1000 thermal cycler (Bio-Rad Laboratories, catalog number: 1851148EDU )
  6. MP Fastprep-24 homogenizer (MP Biomedicals, catalog number: 116004500 )

Procedure

  1. Infection (Figure 1)
    Note: This section describes the infection of Balb/C mice with RSV using the intranasal route.
    1. Dilute RSV stock [for preparation of RSV stocks see Sun and López (2016)] in sterile PBS to a concentration of 5 x 106 TCID50 / 35 μl. Keep the diluted virus on ice.
    2. Anesthetize the mice with 40 μl/20 g of weight of Ketamine/Xylazine solution. Make sure each mouse is completely asleep and unresponsive to a toe pinch stimulus.
    3. Hold the mouse vertically with the head tilted back and slowly deliver 35 μl of virus dilution into its nostrils using a 100 μl pipette. The solution should be completely aspirated into the lung and not be swallowed through the mouth.
    4. Place the mouse back into its cage and monitor it until it wakes from anesthesia.

      Figure 1. Procedure for intranasal immunization of mice. A. Use a P100 pipette to administer 35 μl of virus solution per mouse. B. Hand-hold a fully-anesthetized mouse vertically and place the virus solution into its nostril drop by drop. C. Place the mouse back into its cage and monitor it until it awakens.

  2. Lung tissue collection
    Note: This section describes the collection of infected tissue from mice to later examine viral load.
    1. Prepare collection tubes: For each sample, add 1 ml TRIzol into one MP Fastprep tube containing one ceramic sphere. Other similar homogenizing techniques can be used.
    2. Euthanize the mice at the desired time point post infection (usually 6 h, 10 h, 24 h, and daily for up to one week) using CO2 or other approved method.
    3. Place a dead mouse on polystyrene foam and pin each limb with needles, belly up.
    4. Spray the fur of the mouse using 70% ethanol.
    5. Use scissors to cut open the abdominal and thoracic cavities. Opening the abdominal cavity exposing the underside of the diaphragm. Use scissors to pierce through the diaphragm. Cut away the ribcage of the mouse and fully expose the heart and lungs. Collect two of the left lobes and place into a Fastprep tube containing TRIzol. Always take the same lobes of the lung to maintain consistency. Other lobes can be used for other purposes.
    6. Homogenize the lung on the FastPrep-24 homogenizer or similar equipment. Homogenize for 20 sec. Repeat three times.
    7. Aliquot the homogenized samples into 500 μl aliquots. Place them at -80 °C for storage.

  3. Total RNA extraction
    Note: This section describes the extraction of RNA from infected tissue, which will be later used for detection of viral genomes.
    1. Thaw one aliquot and add 500 μl of fresh TRIzol for a 1 ml total volume.
    2. Add 200 μl of chloroform and shake vigorously for 15 sec.
    3. Leave for 3 min at room temperature to allow for phase separation. Centrifuge for 15 min at 12,800 x g in a microcentrifuge at 4 °C.
    4. Carefully collect 3/4 of the upper aqueous phase without disturbing or touching the interface or organic layer. Combine the aqueous phase with an equal volume of isopropanol. Gently invert to mix.
    5. Leave for 10-20 min at -20 °C for precipitation.
    6. Centrifuge for 15 min at 12,800 x g in a microcentrifuge at 4 °C.
    7. Discard the supernatant with care not to lose the pellet.
    8. Wash the pellet with 1 ml of 75% ethanol. Vortex briefly or pipette up and down the sample multiple times.
    9. Centrifuge for 10 min at 12,800 x g in a microcentrifuge at 4 °C.
    10. Remove the supernatant and invert the tubes. Allow the pellet to air-dry until clear.
    11. Resuspend the pellet in 30-40 μl of ultra-pure nuclease-free dH2O.
    12. Incubate at 65 °C for 5 min.
    13. Measure RNA concentration in a Nanodrop 1000 or equivalent.

  4. Reverse transcription (RT)
    Note: This section describes the conversion of total RNA prepared in section C into cDNA. In brief, mRNAs are converted into total cDNA (T-cDNA) using oilgo-dT. This cDNA will be then used to amplify housekeeping genes as controls. RSV genomes are converted into viral genome cDNA (V-cDNA) using a RSV genome RT primer that specifically binds to the 5’ end of the genome. V-cDNA will be used for the amplification of RSV viral genomes.

    D1. RT of total mRNA (T-cDNA)
    1. Use 2 μg of RNA for reverse transcription.
    2. Prepare the RT reaction mix in PCR tubes on ice using the High Capacity RNA-cDNA kit as follows (per sample): 2x RT buffer 5 μl, 20x RT Enzyme mix 0.5 μl, RNA 2 μg, nuclease-free H2O quantity sufficient to 10 μl.
      Note: The oligo-dT primers are included in the RT buffer.
    3. Gently mix and briefly centrifuge to collect all the components at the bottom of the tube.
    4. Place into a BioRad C1000 Thermal Cycler and run program to synthesize cDNA as follow: 37 °C for 60 min, 95 °C for 5 min, 4 °C hold.
    5. Store total cDNA (T-cDNA) at -20 °C until ready for qPCR analysis.

    D2. RT of viral genomes (V-cDNA)
    1. Use 2 μg of RNA for reverse transcription.
    2. Mix the following ingredients per sample in one tube: 1 μl of the genome RT primer (50 μM), 1 μl of dNTPs (10 mM), 2 μg of RNA, nuclease-free H2O (add up to 10 μl).
    3. Incubate 10 min at 65 °C.
    4. Prepare the RT reaction mix in the PCR tubes on ice using SuperScript® III First-Strand Synthesis System as follows (per sample): 5x RT buffer 2 μl, 25 mM MgCl2 4 μl, 0.1 M DTT 2 μl, RNase OUT 1 μl, SS III 1 μl.
    5. After incubation, add 10 μl of RT mix to each sample. Mix well.
    6. Incubate at 50 °C for 50 min.
    7. Heat at 85 °C for 5 min.
    8. Add 1 μl of RNase H per sample (from SuperScript III reverse transcriptase kit) and incubate at 37 °C for 20 min.
    9. Keep the viral genome cDNA (V-cDNA) at -20 °C until ready for the qPCR analysis.

  5. Quantitative PCR (qPCR)
    Note: This section describes the quantification of viral genomes presented in the infected tissue. T-cDNA and V-cDNA generated from section D are used as templates for housekeeping genes and RSV genomes, respectively. The copy numbers of two housekeeping genes are used to calculate the housekeeping index (hki) that is then used to calculate the relative copy number of viral genomes. An example of the results from viral genome qPCR at day 1 post-infection in vivo is shown in Figure 2.
    1. qPCR reactions are performed in triplicate using specific primers and the Power SYBR® Green PCR Master Mixture in a Viia7 Applied Biosystem Lightcycler or equivalent.
    2. Thaw both T- and V-cDNA and spin down before use.
    3. Dilute cDNA 1:40 using dH2O.
    4. Load the plate with 4 μl of the diluted cDNA/well in triplicate.
    5. Prepare Master Mix: SYBR green (5 μl) with 1 μl of qPCR primer mix. Load the plate with Master Mix 6 μl/well. For housekeeping genes, Rsp11 and α-tubulin are examined as reference using T-cDNAs as template. For RSV genome, RSV g primer pair is used and V-cDNAs serve as template.
    6. Run the qPCR using the following program (Table 1):

      Table 1. DI-PCR program


    7. Viral genome copy numbers are normalized based on levels of Rsp11 and α-tubulin for mouse samples using the formula: relative copy number=2,500*1.93^ (Ctgenome - hki), hki=median of CtRsp11 and Ctα-tubulin. Sequences of primers used for qPCR can be found in the materials under qPCR primers. Results see Figure 2.


      Figure 2. RSV viral genome qPCR. Mice were infected with PBS (mock) or RSV. Lungs were harvest on day 1 post-infection and analyzed using a RSV genome RT-qPCR assay. Each dot represents RSV genome quantification per mouse.

Recipes

  1. Ketamine/Xylazine solution (2 ml)
    1 ml ketamine
    0.14 ml xylazine
    1 ml PBS
    Mixed in BSL2 hood and store at room temperature
  2. Primer preparation
    Primers are diluted into 100 μM as stock solution.
    For reverse transcription (RT primer), dilute the stock solution 1:2 using nuclease-free H2O for a working concentration of 50 μM.
    For qPCR primers, reverse and forward primers are used as a pair. Prepare 750 μl of primer mix as a working stock as described below:
    37.5 μl stock solution of forward primer
    37.5 μl stock solution of reverse primer
    675 μl nuclease-free H2O

Acknowledgments

This work was supported by grant AI083284 from the National Institute of Health (CBL). The protocol described herein was based on the following paper: Sun et al. (2015); Sun and López (2016).

References

  1. Sun, Y., and López, C. B. (2016). Preparation of respiratory syncytial virus with high or low content of defective viral particles and their purification from viral stocks. Bio-protocol 6(9): e1820.
  2. Sun, Y., Jain, D., Koziol-White, C. J., Genoyer, E., Gilbert, M., Tapia, K., Panettieri, R. A., Jr., Hodinka, R. L. and Lopez, C. B. (2015). Immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans. PLoS Pathog 11(9): e1005122.
  3. Tapia, K., Kim, W. K., Sun, Y., Mercado-Lopez, X., Dunay, E., Wise, M., Adu, M. and Lopez, C. B. (2013). Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity. PLoS Pathog 9(10): e1003703.

材料和试剂

  1. 真空驱动的Sterilcup 500ml Millipore ExpressPLUS0.22μmPES(Merck Millipore Corporation,目录号:SCGPU05RE)
  2. FastPrep 管(MP Biomedicals,目录号:5076-400)
  3. ¼"陶瓷球(MP Biomedicals LLC,目录号:116540412)
  4. 用于冷冻病毒的CryoTubes TM sup/TM小瓶(Thermo Fisher Scientific,目录号:377267)
  5. 外科剪刀(Roboz Surgical Instrument Co)
  6. 镊子(Roboz外科手术器械公司)
  7. 384孔PCR板(VWR International,目录号:82051-470)
  8. PCR条管,0.2ml(Bioexpress,目录号:T-3035-2)
  9. 用于移液管助剂(VWR International,目录号:28145-481)中的0.22μm过滤器
  10. BSL2个人防护装备(PPE)
  11. 小鼠(Balb/c,6-8周龄,性别混合)(Taconic)
  12. 人RSV株A2(ATCC,目录号:VR-1540)
  13. SYBR绿主混合物(Applied Biosystems,目录号:4368708)
  14. qPCR引物组

    基因
    正向引号
    反向引号
    RSV g
    5'AACATACCTGCCCAGAATC3'
    5'GGTCTTGACTGTTGTAGATTGCA3'
    Rsp11
    5'CGTGACGAAGATGAAGATGC3'
    5'GCACATTGAATCGCACAGTC3'
    微管蛋白
    5'TGCCTTTGTGCACTGGTATG3'
    5'CTGGAGCAGTTTGACGACAC3'

  15. RSV基因组RT引物:5'GATAAATATAGGCATGGGGAAAGTG3'
  16. TRIzol试剂(Thermo Fisher Scientific,Ambion TM ,目录号:15596018)
  17. 氯仿(Sigma-Aldrich,目录号:C2432-25ml)
  18. 异丙醇(Sigma-Aldrich,目录号:437522)
    注意:在Sigma-Aldrich网站上也称为"2-丙醇"。
  19. 超纯无核酸酶水
  20. SuperScript III第一链合成系统(Thermo Fisher Scientific,Invitrogen TM ,目录号:18080-051)
  21. 高容量RNA-to-cDNA 试剂盒(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4387406)
  22. 氯胺酮(Henry Schein)
  23. 赛拉嗪(美国食品和药物管理局,AnaSed,目录号:139-236)
  24. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023)
  25. 氯胺酮/赛拉嗪溶液(参见配方)
  26. 底漆准备(见配方)

设备

  1. 生物安全2级生物安全罩
  2. Applied Biosystem ViiA TM supra TM lightcycler(Life Technologies,目录号:4453536)
    注意:目前,它是"Thermo Fisher Scientific,目录号:4453536"。
  3. Eppendorf离心机5415R(Eppendorf AG,目录号:22636570) 注意:此产品已由Eppendorf AG停产。
  4. NanoDrop 1000分光光度计(Thermo Fisher Scientific,目录号:ND-1000)
  5. BioRad C1000热循环仪(Bio-Rad Laboratories,目录号:1851148EDU)
  6. MP Fastprep-24匀浆器(MP Biomedicals,目录号:116004500)

程序

  1. 感染(图1)
    注意:本节介绍使用鼻内途径的Balb/C小鼠感染RSV。
    1. 稀释RSV原液[用于RSV原种的制备参见Sun和López (2016)]在无菌PBS中稀释至浓度为5×10 6 TCID 50 /35μl。 将稀释的病毒置于冰上。
    2. 麻醉小鼠与40微升/20   克重量的氯胺酮/甲苯噻嗪溶液。 确保每个鼠标都是 完全睡眠和对脚趾捏力刺激无反应
    3. 保持 鼠标垂直与头部倾斜回来,并缓慢地递送35微升 使用100μl移液管将病毒稀释到其鼻孔中。 解决方案   应完全吸入肺部,不要吞咽 通过嘴。
    4. 将鼠标放回其笼子,并监视它,直到它从麻醉苏醒
      图1.小鼠鼻内免疫的程序。 A.使用P100 移液管以每只小鼠施用35μl病毒溶液。 B.手持a 完全麻醉的鼠标垂直和放置病毒溶液它的鼻孔一滴一滴。 C.将鼠标放回笼子里 监视它,直到它醒了。

  2. 肺组织收集
    注意:本节介绍从小鼠感染的组织的收集,以后检查病毒载量。
    1. 准备收集管:对于每个样品,加入1 ml TRIzol到一个MP Fastprep管包含一个陶瓷球。 其他类似均质 可以使用技术
    2. 安乐死小鼠在所需的时间 点后感染(通常6小时,10小时,24小时,每天最多一个 周)或使用CO 2或其它批准的方法。
    3. 把一只死了的老鼠放在聚苯乙烯泡沫上,用针刺住每个肢体,肚子朝上
    4. 用70%乙醇喷涂鼠标的毛皮。
    5. 使用剪刀打开腹腔和胸腔。 打开腹腔暴露隔膜的下侧。 使用剪刀刺穿隔膜。 切掉胸腔 小鼠和充分暴露心脏和肺。 收集左边的两个 叶并放入含有TRIzol的Fastprep管中。 总是带走 相同肺叶保持一致性。 可以使用其他叶 为其他目的。
    6. 在FastPrep-24匀浆器或类似设备上均化肺。 均化20秒。 重复三次。
    7. 将匀浆样品等分至500μl等分试样。 将其置于-80℃下储存。

  3. 总RNA提取
    注意:本节介绍从感染组织中提取RNA,以后用于检测病毒基因组。
    1. 解冻一个等分试样,加入500μl新鲜TRIzol,总体积为1 ml
    2. 加入200微升氯仿,并剧烈摇动15秒。
    3. 在室温下放置3分钟,以进行相分离。 在微量离心机中在4℃下以12,800×g离心15分钟。
    4. 小心地收集3/4的上层水相而不打扰 或接触界面或有机层。 合并水相 与等体积的异丙醇。 轻轻倒转混合。
    5. -20℃放置10-20分钟以沉淀。
    6. 在微量离心机中于4℃下以12,800×g离心15分钟。
    7. 弃去上清液,小心不要丢失沉淀。
    8. 用1ml的75%乙醇洗涤沉淀。 短暂涡旋或用吸移管向上和向下多次样品。
    9. 在微量离心机中于4℃下以12,800×g离心10分钟。
    10. 除去上清液并倒置试管。 让沉淀物风干至澄清。
    11. 将沉淀重悬在30-40μl超纯无核酸酶dH 2 O中。
    12. 在65℃孵育5分钟。
    13. 在Nanodrop 1000或等价物中测量RNA浓度。

  4. 逆转录(RT)
    注意:本节介绍C部分中制备的总RNA转化为cDNA。简言之,使用oil-dT将mRNA转化成总cDNA(T-cDNA)。然后将该cDNA用于扩增作为对照的持家基因。使用特异性结合基因组5'端的RSV基因组RT引物将RSV基因组转化为病毒基因组cDNA(V-cDNA)。 V-cDNA将用于扩增RSV病毒基因组。

    D1。 RT的总mRNA(T-cDNA)
    1. 使用2μgRNA进行逆转录
    2. 准备RT 反应混合物在PCR管中在冰上使用高容量RNA-cDNA试剂盒作为  (每个样品):2x RT缓冲液5μl,20x RT酶混合物0.5μl,RNA 2  μg,无核酸酶的H 2 O 2量足以达到10μl。
      注意:oligo-dT引物包含在RT缓冲液中。
    3. 轻轻混合并短暂离心以收集管底部的所有组分
    4. 放入BioRad C1000 Thermal Cycler并运行程序 合成cDNA如下:37℃60分钟,95℃5分钟,4℃保持
    5. 将总cDNA(T-cDNA)储存于-20℃,直到准备进行qPCR分析

    D2。 病毒基因组(V-cDNA)RT
    1. 使用2μgRNA进行逆转录
    2. 混合以下 每个样品的成分在一个管中:1μl的基因组RT引物(50 μM),1μl的dNTP(10mM),2μg的RNA,无核酸酶的H 2 O(总计为10   μl)。
    3. 在65℃孵育10分钟。
    4. 准备RT反应 使用SuperScript III Supers III第一链在冰上使用PCR管混合 合成系统如下(每个样品):5x RT缓冲液2μl,25mM MgCl 24μl,0.1M DTT2μl,RNase OUT1μl,SS III1μl。
    5. 孵育后,向每个样品中加入10μlRT混合物。 混合良好。
    6. 在50℃孵育50分钟。
    7. 在85℃加热5分钟
    8. 每个样品加入1μlRNase H(从SuperScript III逆转录酶试剂盒),并在37℃孵育20分钟。
    9. 保持病毒基因组cDNA(V-cDNA)在-20°C,直到准备好qPCR分析

  5. 定量PCR(qPCR)
    注意:本节描述了感染组织中存在的病毒基因组的定量。 将从D部分产生的T-cDNA和V-cDNA分别用作管家基因和RSV基因组的模板。 两个管家基因的拷贝数用于计算随后用于计算病毒基因组的相对拷贝数的持家性指数(hki)。 图2中显示了感染后第1天的病毒基因组qPCR结果的实例。
    1. qPCR反应使用特异性引物一式三份进行 在Viia7 Applied Biosystem中的Power SYBR Green PCR Master Mixture Lightcycler或同等产品。
    2. 解冻T-和V-cDNA,并在使用前旋转
    3. 使用dH 2 O稀释cDNA 1:40
    4. 加载板的4μl稀释的cDNA /孔一式三份。
    5. 准备主混合物:SYBR绿色(5微升)与1微升的qPCR引物混合。   加载板与主混合6μl/孔。 对于管家基因,使用T-cDNA作为模板检查Rsp11 和α-微管蛋白作为参考。 对于 RSV基因组,使用RSV g引物对,V-cDNA作为模板
    6. 使用以下程序运行qPCR(表1):

      表1. DI-PCR程序


    7. 基于小鼠样品的Rsp11 和α-微管蛋白的水平对病毒基因组拷贝数进行归一化,使用公式:相对拷贝 数字= 2,500 * 1.93 ^(Ct基因组-hki),hki = Ct Rsp11的中位数和 Ct α-微管蛋白 。 用于qPCR的引物序列可见于 材料在qPCR引物下。 结果见图2.


      图2. RSV 病毒基因组qPCR。用PBS(模拟)或RSV感染小鼠。 肺   在感染后第1天收获并使用RSV基因组RT-qPCR分析   测定。 每个点代表每只小鼠的RSV基因组定量。

食谱

  1. 氯胺酮/甲苯噻嗪溶液(2ml) 1 ml氯胺酮 0.14ml赛拉嗪
    1 ml PBS
    在BSL2罩中混合并在室温下贮存
  2. 底漆准备
    将引物稀释到100μM作为储备溶液 对于逆转录(RT引物),使用不含核酸酶的H 2 O以1:50的工作浓度稀释储备溶液1:2。
    对于qPCR引物,使用反向和正向引物作为一对。 准备750微升引物混合物作为工作库存,如下所述:
    37.5μl正向引物的储液 37.5μl反向引物的储液
    675μl不含核酸酶的H 2

致谢

这项工作是由国家卫生研究所(CBL)授权AI083284支持。本文所述的方案基于以下论文:Sun等人(2015); Sun和López(2016)。

参考文献

  1. Sun,Y.,andLópez,C. B.(2016)。 准备具有高或低含量的缺陷性病毒颗粒的呼吸道合胞病毒,并从病毒性原种中纯化。 生物协议 6(9):e1820
  2. Sun,Y.,Jain,D.,Koziol-White,C.J.,Genoyer,E.,Gilbert,M.,Tapia,K.,Panettieri,R.A.,Jr.,Hodinka,R.L.and Lopez, 来自呼吸道合胞病毒的免疫刺激性缺陷型病毒基因组在小鼠和人类感染期间促进强烈的先天抗病毒反应。 PLoS Pathog 11(9):e1005122。
  3. Tapia,K.,Kim,W. K.,Sun,Y.,Mercado-Lopez,X.,Dunay,E.,Wise,M.,Adu,M.and Lopez,C.B。 体内产生的有缺陷的病毒基因组 提供关键的危险信号触发 肺抗病毒免疫。 PLoS Pathog 9(10):e1003703。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sun, Y. and López, C. B. (2016). Respiratory Syncytial Virus Infection in Mice and Detection of Viral Genomes in the Lung Using RT-qPCR. Bio-protocol 6(10): e1819. DOI: 10.21769/BioProtoc.1819; Full Text
  2. Sun, Y., Jain, D., Koziol-White, C. J., Genoyer, E., Gilbert, M., Tapia, K., Panettieri, R. A., Jr., Hodinka, R. L. and Lopez, C. B. (2015). Immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans. PLoS Pathog 11(9): e1005122.




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