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MERS-CoV PCR/Sequencing Primers

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  "description": "Authors: Rachel Graham\n\n### Abstract\n\nThis protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of MERS-CoV genomes.\n\n### Introduction\n\nThis protocol details the steps, reagents, and conditions required to sequence MERS-CoV genomes in the forward and reverse directions. The protocol begins with the RT-PCR step, assuming that MERS-CoV RNA purified using a standard procedure (i.e., TRIzol extraction) has already been performed.\n\n### Materials\n\n1. Invitrogen SuperScript III kit\n- dNTPS (10 mM)\n- Random Hexamers\n- RNasin (if desired)\n- Thermo Phusion PCR enzyme\n- Primers (2 mM stock \u2013 see Tables 1 and 2)\n- Agarose\n- 1X TAE Buffer\n- Ethidium Bromide\n\n### Equipment\n\n1. 70\u00baC water bath\n- 55\u00baC water bath\n- Thermal cycler\n\n### Procedure\n\nReverse Transcription:\n\n1. In a 1.5-\u00b5L Eppendorf tube, add 1-4 \u00b5L RNA and 1 \u00b5L Random Hexamers.\n- Incubate at 70\u00baC for 5 min.\n- Place reaction briefly on ice and assemble the reverse transcription reaction using the kit-supplied reagents (a master mix can be made if multiple reactions are being run):\n  - 4 \u00b5L 5X First-Strand Buffer\n  - 2 \u00b5L DTT\n  - 1 \u00b5L SuperScript III reverse transcriptase\n  - 1 \u00b5L dNTPs\n  - 1 \u00b5L RNasin (if desired)\n  - x \u00b5L H2O to 20 \u00b5L\n- Incubate at 55\u00baC for 45 min to 1 h.\n- Inactivate the reverse transcriptase at 70\u00baC for 15 min. Place reaction on ice after inactivation.\n- Proceed with PCR setup.\n\nPCR (with Phusion PCR kit):\n\n1. Assemble PCR reactions to generate amplicons according to those detailed in Table 2 (for whole-genome sequencing) or with any combination of forward and reverse primers from Table 1.\n- PCR reaction setup:\n  - 2 \u00b5L First-strand template\n  - 1 \u00b5L Forward Primer\n  - 1 \u00b5L Reverse Primer\n  - 5 \u00b5L 10X HF Buffer\n  - 1 \u00b5L dNTPs\n  - 0.5 \u00b5L Phusion polymerase\n  - x \u00b5L H2O to 50 \u00b5L\n- PCR reactions are run under standard PCR conditions:\n  - 98\u00baC 5 min\n  - 35 cycles of:\n  - 98\u00baC 15 sec\n  - x\u00baC for 30 sec*\n  - 72\u00baC for ~45 sec/kb\n  - 72\u00baC 10 min\n  - 8\u00baC Hold\n\nAnnealing temperature is primer-dependent, but for most SARS-CoV primers in Table 1, annealing temperatures 52-55\u00baC will work.\n\nConfirmation of PCR products and sequencing:\n\n1. Run PCR products (5 \u00b5L/reaction) on a 0.8% agarose/1X TAE gel to verify PCR success.\n- Purify PCR products with PCR purification kit of choice (the Qiagen PCR Purification Kit works well).\n- PCR products can be diluted to 150-200 \u00b5L/reaction to ensure that enough product is present for assembling sequencing reactions.\n- Assemble sequencing reactions according to the primer/amplicon combinations outlined in Table 2.\n\n### Timing\n\n- Reverse transcription: 1-1.5 h\n- PCR: 2-4 h\n- Sequencing: facility-dependent\n\n### Anticipated Results\n\nPrimers have been designed to give clean, single-band PCR products. If multiple bands are detected, alternate annealing temperatures may be required. It may also be possible that alternate bands indicate multiple genome patterns at that locus.\n\n### Figures\n\n**Table 1: MERS-CoV Sequencing Primers** \n\nHCoV-EMC Primers\n\n1F 3-22\nGGCCGCATTAATACGACTCA\n\n2F 103-122\nAAAGCCCTGTTGTTTAGCGT\n\n3F 305-324\nCATGTCTTTCGTGGCTGGTG\n\n4F 785-804\nTGAGCGAGACAACACCTCTT\n\n5F 1291-1310\nACATTTACCACTCCGCATTC\n\n6F 1787-1806\nTGTTGTCCTCGCAATTCTCT\n\n7F 2381-2400\nGGTGGTCAATGGCAAAGTTT\n\n8F 2885-2904\nCTTTGGCGGTGATCAAGTAC\n\n9F 3377-3396\nTGTGCAAGAAGAAGCACAAC\n\n10F 3837-3856\nGCTAAAGGGCCGTTACAAGT\n\n11F 4285-4304\nCAGTTGACGGTGTGCAATAT\n\n12F 4917-4936\nGCAGACAATTTGACTGCTGA\n\n13F 5307-5326\nTGGAGAGAGTGGTGCAATGT\n\n14F 5895-5914\nGGCGGTGATGCTATTAGTTT\n\n15F 6427-6446\nTGCTTAGATTGCACACCGTT\n\n16F 6923-6942\nGTTTGAAGATGCCCAAGGTT\n\n17F 7531-7550\nGCGGTATTTCATTCTGTCGT\n\n18F 8035-8054\nTCACACGCGATAAGTTGGAA\n\n19F 8393-8412\nGGATGCACTTAAACGACAGA\n\n20F 8867-8886\nTACTACATTGGCTTGGGTGA\n\n21F 9407-9426\nTATAGGTTTGTGTGCGTTCC\n\n22F 10073-10092\nCAGTGGAGATGTTGAGGCTT\n\n23F 10691-10710\nACTTAATGGTTGCGCTTGGT\n\n24F 11123-11142\nGGCCTTCGTTATGTTGTTGG\n\n25F 11551-11570\nTTGCTTACTCACCACAGCTT\n\n26F 12121-12140\nCCTTTGCTGAGTTGGAAGCT\n\n27F 12667-12686\nCTTCTGCCGTTAAGTTGCAA\n\n28F 13219-13238\nATGGTGGAGCTTCAGTGTGT\n\n29F 13885-13904\nTGGTTGCTGTGATGTTACCT\n\n30F 14433-14452\nAGATCTTTGTTGATGGCGTG\n\n31F 14965-14984\nTGGCAAAGCTCGTGTCTATT\n\n32F 15415-15434\nTGCTCAGGTGCTAAGCGAAT\n\n33F 15999-16018\nTCATGGTAGAGCGGTTTGTG\n\n34F 16305-16324\nTTCTCTGCTGTAAATGCTGC\n\n35F 16749-16768\nCCAAACCACCACTCAATCGT\n\n36F 17337-17356\nCCGATATTCTGGTGGTTGAT\n\n37F 17877-17896\nAAACAGCAGATACGGCACAT\n\n38F 18287-18306\nATAGGCTTCGATGTTGAGGG\n\n39F 18887-18906\nATAGAACGTGTGGATTGGGA\n\n40F 19255-19274\nTTGTGATGGCGGTAGTTTGT\n\n41F 19839-19858\nGCTACAAGTTCGTCCTTTGG\n\n42F 20357-20376\nAAGAAGCAACAGGAAGGTCA\n\n43F 20813-20832\nCCTGCCAATATGCGTGTTAT\n\n44F 21117-21136\nTTGGTGGGTCTGTTGCTATT\n\n45F 21629-21648\nTGTTTCTAAGGCTGACGGTA\n\n46F 22001-22020\nCGATGGATGTGGCACTTTAC\n\n47F 22327-22346\nCCACCTTGCCTGTTTATGAT\n\n48F 22863-22882\nGCTGGTCCAATATCCCAGTT\n\n49F 23363-23382\nGCAGCGCTTTGTTTATGATG\n\n50F 23995-24014\nCCAATTTACGCCAGGATGAT\n\n51F 24615-24634\nGGTGCTATTTCCGCCTCTAT\n\n52F 25065-25084\nGAGCCCATTACCTCCCTTAA\n\n53F 25521-25540\nCCGCATAAGGTTCATGTTCA\n\n54F 25951-25970\nATCCCTAAACCCACAGCTAA\n\n55F 26609-26628\nAAGGATTGGCTTCTCGTTCA\n\n56F 27045-27064\nCTTGTCGTCGCAGCATTATC\n\n57F 27487-27506\nAACGCGCGATTCAGTTCCTC\n\n58F 27941-27960\nTTGCATGGTCCCTGATCTTT\n\n59F 28427-28446\nGGCAAAGCTACGGAACTAAT\n\n60F 29003-29022\nGCGGAACCCTAACAATGATT\n\n61F 29603-29622\nTGACCCAAAGAATCCCAACT\n\n62F 29843-29862\nAAAGTAACAAGATCGCGGCA\n\n1R 138-157\nTTAATGCCACAATCCCACCA\n\n2R 640-659\nGCCAACCAGACTGCCATTTG\n\n3R 1062-1080\nCTTTACGCTCAACATGCCA\n\n4R 1570-1589\nGCCACCAAAGATAAGTGTGA\n\n5R 2108-2127\nACACACCAGAATCCATGTCA\n\n6R 2382-2401\nGAAACTTTGCCATTGACCAC\n\n7R 2886-2905\nTGTACTTGATCACCGCCAAA\n\n8R 3458-3477\nCAGTATCAGGCACAACAGGA\n\n9R 3994-4013\nTGCTGAAACAAGAGGAGTGA\n\n10R 4566-4585\nCCCTCGACTAAATGCCAAGA\n\n11R 5162-5181\nAATCACCGCCCTTATGTTTC\n\n12R 5550-5569\nGTTTCAATGCCCTGAAAGAC\n\n13R 6048-6067\nTTGCCTTTATACATGGCACC\n\n14R 6582-6601\nTGCCGCACTACACTCTTTAT\n\n15R 7180-7199\nGTATGCCAAACCAGTCTCAA\n\n16R 7612-7631\nGAGGTCATTTGCGACTTCTT\n\n17R 8036-8055\nTTTCCAACTTATCGCGTGTG\n\n18R 8542-8561\nCGTAAAGTCACGCAACGCAT\n\n19R 9042-9061\nGGATCATGGCAGTATGGTGT\n\n20R 9496-9515\nAACAGCAGCAACAACAGCAA\n\n21R 10076-10095\nTACAAGCCTCAACATCTCCA\n\n22R 10566-10585\nAATGCTGAACCGGTATGTGT\n\n23R 109866-11005\nAACCAATGCGCAGTACCATA\n\n24R 11226-11245\nGCTGACGAAATGGGAGTAGT\n\n25R 11926-11945\nGCATTTAACACAGAAAGCCC\n\n26R 12474-12493\nTCAGGAATTACAACGCGAAG\n\n27R 12994-13013\nCAATCTAACAGTCGCAGCAA\n\n28R 13616-13635\nTGATGCCCTTGGTCATCTAA\n\n29R 14184-14203\nTGTCTCAGCGGCCAGACAAT\n\n30R 14630-14649\nCCAGTTGTAAGTGCAGCGAC\n\n31R 15140-15159\nTTCTGATGGTACTGGCGATT\n\n32R 15532-15551\nTGACATTAGCAGTTGTCGCC\n\n33R 16010-16029\nATAGCCAAAGACACAAACCG\n\n34R 16596-16615\nTGTTGTAGTATTGGCAAGGG\n\n35R 17170-17189\nCACACAAAGCATCAACAGCT\n\n36R 17658-17677\nCGTCACATTGCCCTTATAGA\n\n37R 18198-18217\nATCGAGTTTAAAGCCCATCC\n\n38R 18742-18761\nGAACATCGACAAAGAAAGGG\n\n39R 19238-19257\nCAACCTGGCAAATTGAACTC\n\n40R 19838-19857\nCAAAGGACGAACTTGTAGCA\n\n41R 20358-20377\nATGACCTTCCTGTTGCTTCT\n\n42R 20812-20831\nTAACACGCATATTGGCAGGC\n\n43R 21118-21137\nTAATAGCAACAGACCCACCA\n\n44R 21628-21647\nACCGTCAGCCTTAGAAACAT\n\n45R 22092-22111\nGGAGTGTGATAAGTGGCAAA\n\n46R 22636-22655\nGCCAGACAGAAGAGGTGAAA\n\n47R 23084-23103\nAATAATCACCGTCTTCCCAC\n\n48R 23570-23589\nTAGAATCTCGCCGTTTAAGC\n\n49R 23994-24013\nTCATCCTGGCGTAAATTGGC\n\n50R 24616-24635\nAATAGAGGCGGAAATAGCAC\n\n51R 25066-25085\nATTAAGGGAGGTAATGGGCT\n\n52R 25522-25541\nGTGAACATGAACCTTATGCG\n\n53R 25946-25965\nTGTGGGTTTAGGGATGTACA\n\n54R 26326-26345\nGTAAATGATGACCCGAACGT\n\n55R 26870-26889\nAATAAAGACGCCGAGAAAGC\n\n56R 27450-27469\nGGAAACATTGCCGTTTAAGG\n\n57R 27940-27959\nAAGATCAGGGACCATGCAAA\n\n58R 28506-28525\nATGCAAGTTCAATATCCGCC\n\n59R 29004-29023\nGAATCATTGTTAGGGTTCCG\n\n60R 29590-29609\nTGGGTCAAGTTTAATGGCTC\n\n61R 30034-30053\nGGCACTGTTCACTTGCAATC\n\n**Table 2: MERS-CoV Amplicon Primer Sets** \n\n![Table 2](http://i.imgur.com/CvlCatG.png "Table 2")\n\n*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/3273). Originally published online 10 July 2014*.", 
  "license": "https://creativecommons.org/publicdomain/zero/1.0/legalcode", 
  "creator": [
    {
      "affiliation": "ScientificProtocols.org", 
      "@type": "Person", 
      "name": "sprotocols"
    }
  ], 
  "headline": "MERS-CoV PCR/Sequencing Primers", 
  "image": "https://zenodo.org/static/img/logos/zenodo-gradient-round.svg", 
  "datePublished": "2014-12-31", 
  "url": "https://zenodo.org/record/13652", 
  "@context": "https://schema.org/", 
  "identifier": "https://doi.org/10.5281/zenodo.13652", 
  "@id": "https://doi.org/10.5281/zenodo.13652", 
  "@type": "ScholarlyArticle", 
  "name": "MERS-CoV PCR/Sequencing Primers"
}
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