Molecular Diagnostic Tests for Microsporidia

Table 1

Diagnostic Primers for the Microsporidia.

Species amplified	Sequence (5^′ to 3^′)	Primer /probe*name	Annealing  temp. in  PCR/melting temp.# (°C)	Amplicon  size,   base  pairs	Reference(s); notes
Primers used in PCR
Anncaliia (Brachiola) algerae	ACTCCGGTAACGTGTGATGTG	NALGf2	55	180	[37]
	TACAAAGCATGATCCCAGTCT	NALGR1
Anncaliia (Brachiola) algerae	GCCGTTTCCGAAGTTGG	NAGf	50	192	[38]
	ATATCGACGGGACTCTCACC	NAG178r
Encephalitozoonidae and Ent. bieneusi	CACCAGGTTGATTCTGCCTGAC	PMP1 (V1)	60	Eb 250	[17]
	CCTCTCCGGAACCAAACCTG	PMP2		Ec 268
				Ei 270
				Eh 279^**
Encephalitozoonidae and Ent. bieneusi	TGAATG(G/T)GTCCCTGT	MSP1	58	Eb 508	[22]
	TCACTCGCCGCTACT	MSP2A		Ec 289
	GTTCATCGCACTACT	MSP2B		Ei 305
	GGAATTCACACCGCCCGTC(A/G)(C/T)TAT	MSP3
	CCAAGCTTATGCTTAAGT(C/T)(A/C)AA(A/G)GGGT	MSP4A
	CCAAGCTTATGCTTAAGTCCAGGGAG	MSP4B
Encephalitozoonidae and Ent. bieneusi	CCAGGUTGATUCTGCCUGACG	Mic3U	65/62	Eb 132	[20]
	TUACCGCGGCUGCUGGCAC	Mic421U		Ec 113
	AAGGAGCCTGAGAGATGGCT	Mic266		Eh 134
	CAATTGCTTCACCCTAAGGTC	Eb379		Ei 128
	GACCCCTTTGCACTCGCACAC	Ec378
	TGCCCTCCAGTAAATCACAAC	Eh410
	CCTCCAATCAATCTCGACTC	Ei395
Encephalitozoonidae and Ent. bieneusi	CACCAGGTTGATTCTGCC	C1 (V1)	56	Eb 1170	[18]
	GTGACGGGCGGTGTCTAC	C2		Ec 1190
				Eh 1205
				Ei 1186^***
Encephalitozoonidae	TGCAGTTAAAATGTCCGTAGT	int530f	40	1000	Didier et al., [39]
	TTTCACTCGCCGCTACTCAG	int580r
Enc. intestinalis	CACCAGGTTGATTCTGCCTGAC	V1	58	375	[40]
	CTCGCTCCTTTACACTCGAA	Si500
Enc. intestinalis	GGGGGTAGGAGTGTTTTTG	3	65	930	Schuitema et al., 1993, [41]
	CAGCAGGCTCCCTCGCCATC	3
Enc. intestinalis	TTTCGAGTGTAAGGAGTCGA	SINTF1	55	520	[42, 43]
	CCGTCCTCGTTCTCCTGCCCG	SINTR
Enc. cuniculi	ATGAGAAGTGATGTGTGTGCG	ECUNF	55	549	[43, 44]
	TGCCATGCACTCACAGGCATC	ECUNR
Enc. hellem	TGAGAAGTAAGATGTTTAGCA	EHELF	55	547	[44]
	GTAAAAAGACTCTCACACTCA	EHELR
Ent. bieneusi	GAAACTTGTCCACTCCTTACG	EBIEF1	55	607	[42]
	CCATGCACCACTCCTGCCATT	EBIER1
Ent. bieneusi	CACCAGGTTGATTCTGCCTGAC	V1	48	353	[45, 46]
	ACTCAGGTGTTATACTCACGTC	EB450
Ent. bieneusi	CACCAGGTTGATTCTGCCTGAC	V1	54	446	[24, 47]
	CAGCATCCACCATAGACAC	Mic3
Ent. bieneusi	TCAGTTTTGGGTGTGGTATCGG	Eb.gc	49	210	[48]
	GCTACCCATACACACATCATTC	Eb.gt
Ent. bieneusi	GCCTGACGTAGATGCTAGTC	2	55	1265	[49]
	ATGGTTCTCCAACTGAAACC	2
Vittaforma corneae	TGAGACGTGAAGATGAGTATC	NCORF1	55	375	Pieniazek NJ and Visvesvara GS personal communication
	TCCCTGCCCACTGTCTCCAAT	NCORR1
Primers and probes used in hybridization
Encephalitozoon spp.	CAGGTTGATTCTGCCTGACG	FP	63		Notermans et al., [50]
	ATCTCTCAGGCTCCCTCTCC	RP
Enc. hellem	ACT CTCACA CTC ACT TCA G	HEL878F	54		[51]
Ent. bieneusi	CGGTGGTGTGTGTAGGCGTGAGAGTGTATC	probe	55		[52]
			(hybridization)
Primers and probes used in RT-PCR or fluorogenic PCR
EncephP1	CCC TGT CCT TTG TAC ACA CCG CCC	EncephP1	68		[53]
EcunF1	TCC TAG TAA TAG CGG CTG ACG AA	EcunF1	59
EcunR2	ACT CAG GAC TCA GAC CTT CCG A	EcunR2	59
EhelF1	GAA TGA TTG AAC AAG TTA TTT TGA ATG TG	EhelF1	59
EhelR2	AAC ACG AAA GAC TCA GAC CTC TCA	EhelR2	58
EintF1	AAT TCC TAG TAA TAA CGA TTG AAC AAG TTG	EintF1	59
EintR2	ACG AAG GAC TCA GAC CTT CCA A	EintR2	59
Ent. bieneusi	CGCTGTAGTTCCTGCAGTAAACTATGCC	FEB1	65	102	[33, 34]
	CTTGCGAGCGTACTATCCCCAGAG	REB1
	ACGTGGGCGGGAGAAATCTTTAGTGTTCGGG	probe
Enc. intestinalis	GCAAGGGAGGAATGGAACAGAACAG	FEI1	65	127	[33]
	TTCAGAAGCCCATTACACAGC	REI1
	CGGGCGGCACGCGCACTACGATA	probe
Ent. bieneusi	TGTGTAGGCGTGAGAGTGTATCTG	EbITS-89F	60	103	[54]
	CATCCAACCATCACGTACCAATC	EbITS-191R
	CACTGCACCCACATCCCTCACCCTT	EbITS-114revT
Encephalitozoon spp.	CACCAGGTTGATTCTGCCTGAC	MSP1F	60
	CTAGTTAGGCCATTACCCTAACTACCA	Eint227R
	CTATCACTGAGCCGTCC	Eint82Trev
Encephalitozoon	GTCCGT TAT GCC CTG AGA T		60	268
	ACA GCA GCC ATG TTA CGACT
	GCC CGT CGC TATCTA AGA TGA CGC A	probe 1
	TGG ACG AAG ATT GGA AGGTCT GAG TC	probe 2
Primers and probes used in oligonucleotide microarray
microsporidia-generic	GATTCTGCCTGACGTGGATGCTATT	Msp1	58		[55]
	ATTCCGGAGAGGGAGCCTGAGAGAT	Msp2	61
	ATTGACGGAAGGACACTACCAGGA	Msp3	57
	GTGCGGCTTAATTTGACTCAACGCG	Msp4	58
Ent. bieneusi	ACGGCTCAGTAATGTTGCGGTAATT	Eb1	56
	CCTATCAGCTTGTTGGTAGTGTAAA	Eb2	54
	TCATGAGACGTGAGTATAAGACCTG	Eb3	56
	ATCGAATACGTGAGAATGGCAGGAGT	Eb4	58
	CTAAAAGCGGAGAATAAGGCGCAAC	Eb5	58
	CGTTGTTCAATAGCGATGAGTTTGC	Eb6	56
	GGTGAAACTTAAAGCGAAATTGACGG	Eb7	56
	AGCCTGTGTGTGAGAATACGTGG	Eb8	56
Encephalitozoon	ACGGCTCAGTGATAGTACGATGATT	Ence1	56
	TATCAGCTGGTAGTTAGGGTAATGG	Ence2	56
	GAGTGAAACTTGAAGAGATTGACGG	Ence3	56
Enc. cuniculi	TGTGGGGTTGGCAAGTAAGTTGTGG	Ec1	59
	ATGAGAAGTGATGTGTGTGCGAGTG	Ec2	58
	GCCTGTGAGTGCATGGCATGAG	Ec3	59
	GGGAAACTGCAGATAGTGGTCTGC	Ec4	59
	GGATGTAGTGATGTGTGTGGCAGAG	Ec5	59
	CTGGACGGGACAGTGTGTGTTGT	Ec6	59
Enc. hellem	TAAGTTCTGGGGGTGGTAGTTTGTA	Eh1	56
	GCGGTTATGAGAAGTAAGATGTTTAGCA	Eh2	57
	CTGAAGTGAGTGTGAGAGTGTTTTTAC	Eh3	57
	ATTGGGAGCCTGGATGTAACTGTGG	Eh4	59
	GGATGTAGTTTTATTGTAGCAGAGG	Eh5	54
	TGGACGGGACTGTTTTAGTGTTGTC	Eh6	58
Enc. intestinalis	TTGACACGAGCCAAGTAAGTTGTAG	Ei1	56
	TTTCGAGTGTAAAGGAGTCGAGATTGA	Ei2	57
	GGCAGGAGAACGAGGACGGGAT	Ei3	60
	CAGGTAGGGGGCTAGGAGTGTTTTT	Ei4	59
	TATGTCCTGATGTGGATGTAAGAGG	Ei5	56
	TGGACGGGACTATATAGTGTTGTG	Ei6	56
microsporidia-generic	TTTMMAACGGCCATGCACCAC	MspR3	52	var.	Used to label initial PCR products

* Italics indicate hybridization probe. ^** PstI and HaeIII restriction analysis differentiates these amplicons. ^*** HindIII and HinfI restriction analysis differentiates these amplicons. ^# Italics indicate melting temperature.