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

Table 4

List of primers designed to detect mcr genes by PCR.

Targeted genes	Analyze	Method	Primer sequences	Cycle (nb: steps)	Product (bp)	Study
mcr-1	Original study	Std	CLR F: 5′-CGGTCAGTCCGTTTGTTC-3′	25: 94°C, 30 s; 58°C, 90 s; 72°C, 60 s	309	[6]
			CLR R: 5′-CTTGGTCGGTCTGTAGGG-3′
	105 colistin-resistant strains	Std	mcr-1_F: 5′-TGTGGTACCGACGCTCGGTCAG-3′			[152]
			mcr-1_R: 5′-TCAGCGGATGAATGCGGTGC-3′
	45 colistin-resistant strains in 2 spiked stools	HotStarTaqMasterMix	mcr-1_s: 5′-ATGGCACGGTCTATGATA-3′	45: 95°C, 30 s; 55°C, 30 s; 72°C, 30 s	155	[160]
		Mcr-1_FAM-BHQ: 5′-CTACAGACCGACCAAGCCGA-3′	mcr-1_as: 5′-CGGATAATCCACCTTAACA-3′
	In silico study	Pr 5 = -HEX-C	mcr-1-286F: 5′-ACTTATGGCACGGTCTATGA-3′	40: 95°C, 10 s; −56°C, 40 s		[162]
		CAAGCCGA-ZEN-GACCAAGGATC-3IABkFQ-3	mcr-1-401R: 5′-ACACCCAAACCAATGATACG-3′
	20 strains in 3 spiked stools	SYBR Green	mcr-1-qF1: 5′-ACACTTATGGCACGGTCTATG-3′	40: 95°C, 3 s; 60°C, 20 s; 72°C, 7 s	120	[148]
			mcr-1-qR1: 5′-GCACACCCAAACCAATGATAC-3′
			mcr-1-qF2: 5′-TGGCGTTCAGCAGTCATTAT-3′			[165]
			mcr-1-qR2: 5′-AGCTTACCCACCGAGTAGAT-3′
	2046 strains	Std	mcr-1-F: 5′-ATGATGCAGCATACTTCTGTGTG-3′		1646	[148]
			mcr-1-R: 5′-TCAGCGGATGAATGCGGTGC-3′
	Wastewater samples	SYBR Green	mcr-1-F1: 5′-TGTTCTTGTGGCGAGTGTTG-3′	40: 95°C, 15 s; 60°C, 30 s		[158]
			mcr-1-R1: 5′-CGCGCCCATGATTAATAGCA-3′
	78 stool	SYBR Green	mcr-1-FW: 5′-ACGCCATCTGCAACACCAA-3′	30/40: 95°C, 15 s; −63°C, 10 s; −72°C, 10 s	59	[61]
			mcr-1-RV: 5′-GCCAACGAGCATACCGACAT-3′
	100 strains: 18 colistin-resistant strains in 833 faecal samples	TaqMan probe: 6 FAM–GACCGCGACCGCCAATCTTACC-TAMRA	F1: GCAGCATACTTCTGTGTGGTAC	35: 95°C, 30 s; −60°C, 1 min	145	[149]
			R1: ACAAAGCCGAGATTGTCCGCG
		Std	F1: GCAGCATACTTCTGTGTGGTAC		554
			R3: TATGCACGCGAAAGAAACTGGC
	1495 E. coli strains and 571 KP strains	Std	Mcr-1-forward: 5ʹ-GCTCGGTCAGTCCGTTTG-3ʹ			[150]
			Mcr-1-reverse: 5ʹ-GAATGCGGTGCGGTCTTT-3′
	51 strains	FastStart Universal Probe Master kit	M-F: CATCGCGGACAATCTCGG	40: 95°C, 15 s; −60°C, 1 min	116	[161]
	18 samples	FAM-AACAGCGTGGTGATCAGTAGCAT-BHQ	M-R: AAATCAACACAGGCTTTAGCAC
	241 isolates	Std	MCR-1-F2: 5′-CTCATGATGCAGCATACTTC-3′		Entire gene	[151]
			MCR-1-R2: 5′-CGAATGGAGTGTGCGGTG-3′
	Clinical E. coli isolates	TaqMan Fast Advanced Master Mix	MCR-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_Probe	RT-mcr-1_F: TGGCGTTCAGCAGTCATTAT	30°C–95°C, 15 s; −60°C, 1 min		[159]
		Cy5-AGTTTCTTTCGCGTGCATAAGCCG-BBQ-650	RT-mcr-1_R: AGCTTACCCACCGAGTAGAT
	62 isolates	MCR1_22,763_Pb1 FAM-TGGTCTCGG/ZEN/CTTGGTCGGTCTGTAGGGC-3IABkFQ	MCR1_22,697_F1: 5′-CACTTATGGCACGGTCTATGA-3′			[68]
			MCR1_22,810_R1: 5′-CCCAAACCAATGATACGCAT-3′
	31 colistin-resistant isolates	Std	mcr-1_F: 5′-ATGATGCAGCATACTTCTGTGTGG-3′			[157]
			mcr- 1_R: 5′-GTGCGGTCTTTGACTTTGTCC-3′
	122 faecal samples	TaqMan probe: 5′-TTGACCGCGACCGCCAATCTTA-3′ FAM	[6]	45: 15 s, 95°C; −1 min, −60°C	309	[63]
	48 E. coli and 27 KP strains		mcr-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-2	136 colistin-resistant isolates	Std	MCR2-IF: 5′-TGTTGCTTGTGCCGATTGGA-3′	33: 95°C, 3 min; 65°C, 30 s; 72°C, 1 min	567	[7]
	31 coli-resistant isolates		MCR2-IR: 5′-AGATGGTATTGTTGGTTGCTG-3′			[157]
	1200 isolates					[163]
	6 isolates	Std	MCR-2-F(EcoRI): 5′-AACCGAATTCATGACATCACATCACTCTTG-3′			[164]
			MCR-2-R (SalI): 5′-CCGGTCGACTTACTGGATAAATGCCGCGC-3′
	2396 strains	Std	Mcr-2 full Fw: 5′-ATGACATCACATCACTCTTGG-3′	34: 95°C, 1 min; 52°C, 30 s; 72°C, 1 min		[165]
	1144 samples		Mcr-2 full Rv: 5′-TTACTGGATAAATGCCGCGC-3′			[65, 166]
	436 cultures	TaqMan mcr-2_Probe	Mcr-2_fwd: TTGTCGTGCTGTTATCCTATCG	30: 95°C, 15 s; −60°C, 1 min		[167]
		ROX-ACTGATTATGGGTGCGGTGACGAG-BHQ-2	Mcr-2_rev: CCGTGCCATAAGTATCGGTAAA
mcr-1 and mcr-2	1200 isolates	Std	mcr1-2 universal F: ACTTATGGCACGGTCTATGATAC	30: 94°C, 30 s; 58°C, 30 s; 72°C, 2 min	1311	[163]
			mcr1-2 universal R: CCGCGGTGACATCAAACA
		Std	MCR-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 samples	Mcr-generic probe TATCACGCCACAAGATAC	Mcr-generic fw: GCCAAATACCAAGAAAATG		98 bp	[71]
			Mcr-generic rev: TTATCCATCACGCCTTTT
mcr-3	580 E. coli strains	Std	MCR3-F: 5′-TTGGCACTGTATTTTGCATTT-3′	30: 95°C, 30 s; 50°C, 30 s; 72°C, 45 s	542	[10]
			MCR3-R: 5′-TTAACGAAATTGGCTGGAACA-3′
mcr-1, mcr-2, and mcr-3	25 isolates: 17 mcr-1 and 8 mcr-3 20 samples	SYBR Green	mcr1-qf: AAAGACGCGGTACAAGCAAC MCR-1	40: 95°C, 30 s; 60°C, 30 s; 72°C, 30 s	213	[169]
			mcr1-qr: GCTGAACATACACGGCACAG		92
			mcr2-qf: CGACCAAGCCGAGTCTAAGG MCR-2		169
			mcr2-qr: CAACTGCGACCAACACACTT
			mcr3-qf: ACCTCCAGCGTGAGATTGTTCCA MCR-3
			mcr3-qr: GCGGTTTCACCAACGACCAGAA
mcr-4	125 isolates	Std	Mcr-4 FW: ATTGGGATAGTCGCCTTTTT		487	[11]
			Mcr-4 RV: TTACAGCCAGAATCATTATCA
mcr-5	12 Salmonella paratyphi B isolates	Std	MCR5_fw: 5′-ATGCGGTTGTCTGCATTTATC-30′	30: 95°C, 30 s; 50°C, 95 s; 72°C, 95 s	1644	[12]
			MCR5_rev: 5′-TCATTGTGGTTGTCCTTTTCTG-3′
mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5	49 E. coli and Salmonella isolates	Std	mcr1_fw: AGTCCGTTTGTTCTTGTGGC	25: 94°C, 30s; 58°C, 90s; 72°C, 60s	320	[170]
			mcr1_rev: AGATCCTTGGTCTCGGCTTG
			mcr2_fw: CAAGTGTGTTGGTCGCAGTT		715
			mcr2_rev: TCTAGCCCGACAAGCATACC
			mcr3_fw: AAATAAAAATTGTTCCGCTTATG		929
			mcr3_rev: AATGGAGATCCCCGTTTTT
			mcr4_fw: TCACTTTCATCACTGCGTTG		1116
			mcr4_rev: TTGGTCCATGACTACCAATG
			mcr5_fw: ATGCGGTTGTCTGCATTTATC		1644
			mcr5_rev: TCATTGTGGTTGTCCTTTTCTG

Std: standard; KP: K. pneumoniae.