Review Article

Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains

Table 4

List of primers designed to detect mcr genes by PCR.

Targeted genesAnalyzeMethodPrimer sequencesCycle (nb: steps)Product (bp)Study

mcr-1Original studyStdCLR F: 5′-CGGTCAGTCCGTTTGTTC-3′25: 94°C, 30 s; 58°C, 90 s; 72°C, 60 s309[6]
CLR R: 5′-CTTGGTCGGTCTGTAGGG-3′
105 colistin-resistant strainsStdmcr-1_F: 5′-TGTGGTACCGACGCTCGGTCAG-3′[152]
mcr-1_R: 5′-TCAGCGGATGAATGCGGTGC-3′
45 colistin-resistant strains in 2 spiked stoolsHotStarTaqMasterMixmcr-1_s: 5′-ATGGCACGGTCTATGATA-3′45: 95°C, 30 s; 55°C, 30 s; 72°C, 30 s155[160]
Mcr-1_FAM-BHQ: 5′-CTACAGACCGACCAAGCCGA-3′mcr-1_as: 5′-CGGATAATCCACCTTAACA-3′
In silico studyPr 5 = -HEX-Cmcr-1-286F: 5′-ACTTATGGCACGGTCTATGA-3′40: 95°C, 10 s; −56°C, 40 s[162]
CAAGCCGA-ZEN-GACCAAGGATC-3IABkFQ-3mcr-1-401R: 5′-ACACCCAAACCAATGATACG-3′
20 strains in 3 spiked stoolsSYBR Greenmcr-1-qF1: 5′-ACACTTATGGCACGGTCTATG-3′40: 95°C, 3 s; 60°C, 20 s; 72°C, 7 s120[148]
mcr-1-qR1: 5′-GCACACCCAAACCAATGATAC-3′
mcr-1-qF2: 5′-TGGCGTTCAGCAGTCATTAT-3′[165]
mcr-1-qR2: 5′-AGCTTACCCACCGAGTAGAT-3′
2046 strainsStdmcr-1-F: 5′-ATGATGCAGCATACTTCTGTGTG-3′1646[148]
mcr-1-R: 5′-TCAGCGGATGAATGCGGTGC-3′
Wastewater samplesSYBR Greenmcr-1-F1: 5′-TGTTCTTGTGGCGAGTGTTG-3′40: 95°C, 15 s; 60°C, 30 s[158]
mcr-1-R1: 5′-CGCGCCCATGATTAATAGCA-3′
78 stoolSYBR Greenmcr-1-FW: 5′-ACGCCATCTGCAACACCAA-3′30/40: 95°C, 15 s; −63°C, 10 s; −72°C, 10 s59[61]
mcr-1-RV: 5′-GCCAACGAGCATACCGACAT-3′
100 strains: 18 colistin-resistant strains in 833 faecal samplesTaqMan probe: 6 FAM–GACCGCGACCGCCAATCTTACC-TAMRAF1: GCAGCATACTTCTGTGTGGTAC35: 95°C, 30 s; −60°C, 1 min145[149]
R1: ACAAAGCCGAGATTGTCCGCG
StdF1: GCAGCATACTTCTGTGTGGTAC554
R3: TATGCACGCGAAAGAAACTGGC
1495 E. coli strains and 571 KP strainsStdMcr-1-forward: 5ʹ-GCTCGGTCAGTCCGTTTG-3ʹ[150]
Mcr-1-reverse: 5ʹ-GAATGCGGTGCGGTCTTT-3′
51 strainsFastStart Universal Probe Master kitM-F: CATCGCGGACAATCTCGG40: 95°C, 15 s; −60°C, 1 min116[161]
18 samplesFAM-AACAGCGTGGTGATCAGTAGCAT-BHQM-R: AAATCAACACAGGCTTTAGCAC
241 isolatesStdMCR-1-F2: 5′-CTCATGATGCAGCATACTTC-3′Entire gene[151]
MCR-1-R2: 5′-CGAATGGAGTGTGCGGTG-3′
Clinical E. coli isolatesTaqMan Fast Advanced Master MixMCR-1F: 5′-CATCGCTCAAAGTATCCAGTGG-3′[69]
5′-Cy5-TGCAGACGCACAGCAATGCCTATGAT-TAO-3′MCR-1R: 5′-CCATGTAGATAGACACCGTTCTCAC-3′
10,609 E. coli isolates (505R)TaqMan RT-mcr-1_ProbeRT-mcr-1_F: TGGCGTTCAGCAGTCATTAT30°C–95°C, 15 s; −60°C, 1 min[159]
Cy5-AGTTTCTTTCGCGTGCATAAGCCG-BBQ-650RT-mcr-1_R: AGCTTACCCACCGAGTAGAT
62 isolatesMCR1_22,763_Pb1 FAM-TGGTCTCGG/ZEN/CTTGGTCGGTCTGTAGGGC-3IABkFQMCR1_22,697_F1: 5′-CACTTATGGCACGGTCTATGA-3′[68]
MCR1_22,810_R1: 5′-CCCAAACCAATGATACGCAT-3′
31 colistin-resistant isolatesStdmcr-1_F: 5′-ATGATGCAGCATACTTCTGTGTGG-3′[157]
mcr- 1_R: 5′-GTGCGGTCTTTGACTTTGTCC-3′
122 faecal samplesTaqMan probe: 5′-TTGACCGCGACCGCCAATCTTA-3′ FAM[6]45: 15 s, 95°C; −1 min, −60°C309[63]
48 E. coli and 27 KP strainsmcr-1-F1: 5′-ATGATGCAGCATACTTCTGTG-3′[153, 155]
mcr-1-R1: 5′-TCAGCGGATGAATGCGGTG-3′
CLR5-F1: 5′-ATGATGCAGCATACTTCTGTGTGG-3′[156]
CLR5-R1: 5′-TCAGCGGATGAATGCGGTGC-3′
CLR5-F: 5′-CGGTCAGTCCGTTTGTTC-3′[176]
Mcr1-Rv2: 5′-CCAGCGTATCCAGCACATTT-3′

mcr-2136 colistin-resistant isolatesStdMCR2-IF: 5′-TGTTGCTTGTGCCGATTGGA-3′33: 95°C, 3 min; 65°C, 30 s; 72°C, 1 min567[7]
31 coli-resistant isolatesMCR2-IR: 5′-AGATGGTATTGTTGGTTGCTG-3′[157]
1200 isolates[163]
6 isolatesStdMCR-2-F(EcoRI): 5′-AACCGAATTCATGACATCACATCACTCTTG-3′[164]
MCR-2-R (SalI): 5′-CCGGTCGACTTACTGGATAAATGCCGCGC-3′
2396 strainsStdMcr-2 full Fw: 5′-ATGACATCACATCACTCTTGG-3′34: 95°C, 1 min; 52°C, 30 s; 72°C, 1 min[165]
1144 samplesMcr-2 full Rv: 5′-TTACTGGATAAATGCCGCGC-3′[65, 166]
436 culturesTaqMan mcr-2_ProbeMcr-2_fwd: TTGTCGTGCTGTTATCCTATCG30: 95°C, 15 s; −60°C, 1 min[167]
ROX-ACTGATTATGGGTGCGGTGACGAG-BHQ-2Mcr-2_rev: CCGTGCCATAAGTATCGGTAAA

mcr-1 and mcr-21200 isolatesStdmcr1-2 universal F: ACTTATGGCACGGTCTATGATAC30: 94°C, 30 s; 58°C, 30 s; 72°C, 2 min1311[163]
mcr1-2 universal R: CCGCGGTGACATCAAACA
StdMCR-1/2-Fw: 5′-TAT CGC TAT GTG CTA AAG CC-3′715 bp[168]
MCR-1/2-Rv: 5′-TCT TGG TAT TTG GCG GTA TC-3′
621 faecal samplesMcr-generic probe TATCACGCCACAAGATACMcr-generic fw: GCCAAATACCAAGAAAATG98 bp[71]
Mcr-generic rev: TTATCCATCACGCCTTTT

mcr-3580 E. coli strainsStdMCR3-F: 5′-TTGGCACTGTATTTTGCATTT-3′30: 95°C, 30 s; 50°C, 30 s; 72°C, 45 s542[10]
MCR3-R: 5′-TTAACGAAATTGGCTGGAACA-3′

mcr-1, mcr-2, and mcr-325 isolates: 17 mcr-1 and 8 mcr-3 20 samplesSYBR Greenmcr1-qf: AAAGACGCGGTACAAGCAAC MCR-140: 95°C, 30 s; 60°C, 30 s; 72°C, 30 s213[169]
mcr1-qr: GCTGAACATACACGGCACAG92
mcr2-qf: CGACCAAGCCGAGTCTAAGG MCR-2169
mcr2-qr: CAACTGCGACCAACACACTT
mcr3-qf: ACCTCCAGCGTGAGATTGTTCCA MCR-3
mcr3-qr: GCGGTTTCACCAACGACCAGAA

mcr-4125 isolatesStdMcr-4 FW: ATTGGGATAGTCGCCTTTTT487[11]
Mcr-4 RV: TTACAGCCAGAATCATTATCA

mcr-512 Salmonella paratyphi B isolatesStdMCR5_fw: 5′-ATGCGGTTGTCTGCATTTATC-30′30: 95°C, 30 s; 50°C, 95 s; 72°C, 95 s1644[12]
MCR5_rev: 5′-TCATTGTGGTTGTCCTTTTCTG-3′

mcr-1, mcr-2, mcr-3, mcr-4 and mcr-549 E. coli and Salmonella isolatesStdmcr1_fw: AGTCCGTTTGTTCTTGTGGC25: 94°C, 30s; 58°C, 90s; 72°C, 60s320[170]
mcr1_rev: AGATCCTTGGTCTCGGCTTG
mcr2_fw: CAAGTGTGTTGGTCGCAGTT715
mcr2_rev: TCTAGCCCGACAAGCATACC
mcr3_fw: AAATAAAAATTGTTCCGCTTATG929
mcr3_rev: AATGGAGATCCCCGTTTTT
mcr4_fw: TCACTTTCATCACTGCGTTG1116
mcr4_rev: TTGGTCCATGACTACCAATG
mcr5_fw: ATGCGGTTGTCTGCATTTATC1644
mcr5_rev: TCATTGTGGTTGTCCTTTTCTG

Std: standard; KP: K. pneumoniae.