scientificprotocols authored about 6 years ago
Authors: Rachel Graham
This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of MERS-CoV genomes.
This 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.
Reverse Transcription:
PCR (with Phusion PCR kit):
Annealing temperature is primer-dependent, but for most SARS-CoV primers in Table 1, annealing temperatures 52-55ºC will work.
Confirmation of PCR products and sequencing:
Primers 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.
Table 1: MERS-CoV Sequencing Primers
HCoV-EMC Primers
1F 3-22 GGCCGCATTAATACGACTCA
2F 103-122 AAAGCCCTGTTGTTTAGCGT
3F 305-324 CATGTCTTTCGTGGCTGGTG
4F 785-804 TGAGCGAGACAACACCTCTT
5F 1291-1310 ACATTTACCACTCCGCATTC
6F 1787-1806 TGTTGTCCTCGCAATTCTCT
7F 2381-2400 GGTGGTCAATGGCAAAGTTT
8F 2885-2904 CTTTGGCGGTGATCAAGTAC
9F 3377-3396 TGTGCAAGAAGAAGCACAAC
10F 3837-3856 GCTAAAGGGCCGTTACAAGT
11F 4285-4304 CAGTTGACGGTGTGCAATAT
12F 4917-4936 GCAGACAATTTGACTGCTGA
13F 5307-5326 TGGAGAGAGTGGTGCAATGT
14F 5895-5914 GGCGGTGATGCTATTAGTTT
15F 6427-6446 TGCTTAGATTGCACACCGTT
16F 6923-6942 GTTTGAAGATGCCCAAGGTT
17F 7531-7550 GCGGTATTTCATTCTGTCGT
18F 8035-8054 TCACACGCGATAAGTTGGAA
19F 8393-8412 GGATGCACTTAAACGACAGA
20F 8867-8886 TACTACATTGGCTTGGGTGA
21F 9407-9426 TATAGGTTTGTGTGCGTTCC
22F 10073-10092 CAGTGGAGATGTTGAGGCTT
23F 10691-10710 ACTTAATGGTTGCGCTTGGT
24F 11123-11142 GGCCTTCGTTATGTTGTTGG
25F 11551-11570 TTGCTTACTCACCACAGCTT
26F 12121-12140 CCTTTGCTGAGTTGGAAGCT
27F 12667-12686 CTTCTGCCGTTAAGTTGCAA
28F 13219-13238 ATGGTGGAGCTTCAGTGTGT
29F 13885-13904 TGGTTGCTGTGATGTTACCT
30F 14433-14452 AGATCTTTGTTGATGGCGTG
31F 14965-14984 TGGCAAAGCTCGTGTCTATT
32F 15415-15434 TGCTCAGGTGCTAAGCGAAT
33F 15999-16018 TCATGGTAGAGCGGTTTGTG
34F 16305-16324 TTCTCTGCTGTAAATGCTGC
35F 16749-16768 CCAAACCACCACTCAATCGT
36F 17337-17356 CCGATATTCTGGTGGTTGAT
37F 17877-17896 AAACAGCAGATACGGCACAT
38F 18287-18306 ATAGGCTTCGATGTTGAGGG
39F 18887-18906 ATAGAACGTGTGGATTGGGA
40F 19255-19274 TTGTGATGGCGGTAGTTTGT
41F 19839-19858 GCTACAAGTTCGTCCTTTGG
42F 20357-20376 AAGAAGCAACAGGAAGGTCA
43F 20813-20832 CCTGCCAATATGCGTGTTAT
44F 21117-21136 TTGGTGGGTCTGTTGCTATT
45F 21629-21648 TGTTTCTAAGGCTGACGGTA
46F 22001-22020 CGATGGATGTGGCACTTTAC
47F 22327-22346 CCACCTTGCCTGTTTATGAT
48F 22863-22882 GCTGGTCCAATATCCCAGTT
49F 23363-23382 GCAGCGCTTTGTTTATGATG
50F 23995-24014 CCAATTTACGCCAGGATGAT
51F 24615-24634 GGTGCTATTTCCGCCTCTAT
52F 25065-25084 GAGCCCATTACCTCCCTTAA
53F 25521-25540 CCGCATAAGGTTCATGTTCA
54F 25951-25970 ATCCCTAAACCCACAGCTAA
55F 26609-26628 AAGGATTGGCTTCTCGTTCA
56F 27045-27064 CTTGTCGTCGCAGCATTATC
57F 27487-27506 AACGCGCGATTCAGTTCCTC
58F 27941-27960 TTGCATGGTCCCTGATCTTT
59F 28427-28446 GGCAAAGCTACGGAACTAAT
60F 29003-29022 GCGGAACCCTAACAATGATT
61F 29603-29622 TGACCCAAAGAATCCCAACT
62F 29843-29862 AAAGTAACAAGATCGCGGCA
1R 138-157 TTAATGCCACAATCCCACCA
2R 640-659 GCCAACCAGACTGCCATTTG
3R 1062-1080 CTTTACGCTCAACATGCCA
4R 1570-1589 GCCACCAAAGATAAGTGTGA
5R 2108-2127 ACACACCAGAATCCATGTCA
6R 2382-2401 GAAACTTTGCCATTGACCAC
7R 2886-2905 TGTACTTGATCACCGCCAAA
8R 3458-3477 CAGTATCAGGCACAACAGGA
9R 3994-4013 TGCTGAAACAAGAGGAGTGA
10R 4566-4585 CCCTCGACTAAATGCCAAGA
11R 5162-5181 AATCACCGCCCTTATGTTTC
12R 5550-5569 GTTTCAATGCCCTGAAAGAC
13R 6048-6067 TTGCCTTTATACATGGCACC
14R 6582-6601 TGCCGCACTACACTCTTTAT
15R 7180-7199 GTATGCCAAACCAGTCTCAA
16R 7612-7631 GAGGTCATTTGCGACTTCTT
17R 8036-8055 TTTCCAACTTATCGCGTGTG
18R 8542-8561 CGTAAAGTCACGCAACGCAT
19R 9042-9061 GGATCATGGCAGTATGGTGT
20R 9496-9515 AACAGCAGCAACAACAGCAA
21R 10076-10095 TACAAGCCTCAACATCTCCA
22R 10566-10585 AATGCTGAACCGGTATGTGT
23R 109866-11005 AACCAATGCGCAGTACCATA
24R 11226-11245 GCTGACGAAATGGGAGTAGT
25R 11926-11945 GCATTTAACACAGAAAGCCC
26R 12474-12493 TCAGGAATTACAACGCGAAG
27R 12994-13013 CAATCTAACAGTCGCAGCAA
28R 13616-13635 TGATGCCCTTGGTCATCTAA
29R 14184-14203 TGTCTCAGCGGCCAGACAAT
30R 14630-14649 CCAGTTGTAAGTGCAGCGAC
31R 15140-15159 TTCTGATGGTACTGGCGATT
32R 15532-15551 TGACATTAGCAGTTGTCGCC
33R 16010-16029 ATAGCCAAAGACACAAACCG
34R 16596-16615 TGTTGTAGTATTGGCAAGGG
35R 17170-17189 CACACAAAGCATCAACAGCT
36R 17658-17677 CGTCACATTGCCCTTATAGA
37R 18198-18217 ATCGAGTTTAAAGCCCATCC
38R 18742-18761 GAACATCGACAAAGAAAGGG
39R 19238-19257 CAACCTGGCAAATTGAACTC
40R 19838-19857 CAAAGGACGAACTTGTAGCA
41R 20358-20377 ATGACCTTCCTGTTGCTTCT
42R 20812-20831 TAACACGCATATTGGCAGGC
43R 21118-21137 TAATAGCAACAGACCCACCA
44R 21628-21647 ACCGTCAGCCTTAGAAACAT
45R 22092-22111 GGAGTGTGATAAGTGGCAAA
46R 22636-22655 GCCAGACAGAAGAGGTGAAA
47R 23084-23103 AATAATCACCGTCTTCCCAC
48R 23570-23589 TAGAATCTCGCCGTTTAAGC
49R 23994-24013 TCATCCTGGCGTAAATTGGC
50R 24616-24635 AATAGAGGCGGAAATAGCAC
51R 25066-25085 ATTAAGGGAGGTAATGGGCT
52R 25522-25541 GTGAACATGAACCTTATGCG
53R 25946-25965 TGTGGGTTTAGGGATGTACA
54R 26326-26345 GTAAATGATGACCCGAACGT
55R 26870-26889 AATAAAGACGCCGAGAAAGC
56R 27450-27469 GGAAACATTGCCGTTTAAGG
57R 27940-27959 AAGATCAGGGACCATGCAAA
58R 28506-28525 ATGCAAGTTCAATATCCGCC
59R 29004-29023 GAATCATTGTTAGGGTTCCG
60R 29590-29609 TGGGTCAAGTTTAATGGCTC
61R 30034-30053 GGCACTGTTCACTTGCAATC
Table 2: MERS-CoV Amplicon Primer Sets
Source: Protocol Exchange. Originally published online 10 July 2014.