Table 2.

Summary of PCR oligonucleotides used for the specific detection of Cyclospora cayetanensis in fresh produce.

Type of PCR	Target	Sequence	Amplicon size (bp)	Reference
Nesteda	18S rRNA	1st: CYCIFE: 5’-TACCCAATGAAAACAGTTT-3’; CYC2RB: 5’-CAGGAGAAGCCAAGGTAGG-3′	294	Jinneman et al. (1998)
		2nd: CYCF3E: 5’-CCTTCCGCGCTTCGCTGCGT-3’; CYC4RB: 5’-CGTCTTCAAACCCCCTACTG-3’
		1st: CYCIFE + CYC2RB		Dixon et al. (2013)
		2nd: CYCF3E + CYC4RB
nested		1st: F1E: 5´-TACCCAATGAAAACAGTTT-3´; R2B: 5´-CAGGAGAAGCCAAGGTAGG-3´	636	Orlandi and Lampel (2000)
		2nd.: F3E: 5´-CCTTCCGCGCTTCGCTGCGT-3´; R4B: 5´-CGTCTTCAAACCCCCTACTG-3´	294
		1st: F1E + R2B		Sim et al. (2017)
		2nd: F3E + R4B
		1st: F1E + R2B	636	Shields et al. (2013)
		2nd: CC719: 5´-GTAGCCTTCCGCGCTTCG-3´; CRP999: 5´-CGTCTTCAAACCCCCTACTGTCG-3´	298
		1st: F1E + R2B	636	Murphy et al., 2017, Murphy et al., 2018
		2nd: CC719 + CRP999
		1st: CYCIFE + CYC2RB	630	Resendiz-Nava et al. (2020)
		2nd: CC719 + CRP999	298
		1st: CYCF IE: 5’-GGAATTCCTACCCAATGAAAACAGTTT-3’; CYCR2B: 5’-CGGGATCCAGGAGAAGCCAAGGTAGG-3’	101	Steele et al. (2003)
		2nd: CYCF3E: 5’-GGAATTCCTTCCGCGCTTCGCTGCGT-3’; CYCR4B: 5’-CGGGATCCCGTCTTCAAACCCCCTACTG-3’
		1st: ExCycF: 5′-AATGTAAAACCCTTCCAGAGTAAC-3′; ExCycR: 5′-GCAATAATCTATCCCCATCACG-3′	N/A	Li et al. (2019)
		2nd: NesCycF: 5′-AATTCCAGCTCCAATAGTGTAT-3′; NesCycR: 5′-CAGGAGAAGCCAAGGTAGGCRTTT-3
		1st: ExCycF: 5′-AATGTAAAACCCTTCCAGAGTAAC-3′; ExCycR: 5′-GCAATAATCTATCCCCATCACG-3′	500	Chandra et al. (2014)
		2nd: NesCycF: 5′-AATTCCAGCTCCAATAGTGTAT-3′; NesCycR: 5′-CAGGAGAAGCCAAGGTAGGCRTTT-3′	294
		HMPr46: 5′- TCGTGATGGGGATAGATTA-3´; HMPr43: 5´- GCTCTATTTACGCAACTTTC-3´; HMPro61 FAM: 5´-CTGGTCAGTCCAATGAGTTCACA-3´	200	Shapiro et al. (2019)
nested multiplexb, c		1st: m18SeF: 5′-CGGGTAACGGGGAATTAGGG-3′; m18SeR: 5′-TCAGCCTTGCGACCATACTC-3′	751–779	Shapiro et al. (2019)
		2nd: m18ScycF: 5′-TCGTGGTCATCCGGCCTT-3′; m18ScycR: 5′-TCGTCTTCAAACCCCCTACTG-3′	359
qPCR MCA		Cyclo: 5′-AGTGACGAGAAATAACAATACAGG-3′; Cyclo: 5′-CCTGCTTGAAACACTCTATTTTTC-3′	315	Lalonde and Gajadhar, 2016a, Lalonde and Gajadhar, 2016b
qPCR		HMPr46: 5′-TCGTGATGGGGATAGATTA-3′; HMPr43: 5′-GCTCTATTTACGCAACTTTC-3′; HMPro61: 5´-CTGGTCAGTCCAATGAGTTCACA-3´	200	Shields et al. (2013)
		Cyclo250F: 5’-TAGTAACCGAACGGATCGCATT-3’; Cyclo350RN: 5’-AATGCCACGGTAGGCCAATA-3’; ddd.IAC: 5’-CTAACCTTCGTGATGAGCAATCG-3’; ddd-IAC: 5’-GATCAGCTACGTGAGGTCCTAC-3’	101	Murphy et al., 2017, Murphy et al., 2018)
		Cyclo250F + Cyclo350RN; dd.IAC: + dd-IAC		Almeria et al. (2018)
		Cyclo250F + Cyclo350RN; dd.IAC: + dd-IAC		Assurian et al., 2020a, Assurian et al., 2020b
SNP		F1E-R2B: 5´-CAGGAGAAGCCAAGGTAGG-3´; CC719: 5´-GTAGCCTTCCGCGCTTCG-3´	298	Orlandi et al. (2003)
qPCR	hsp70	HMPr36: 5´-GGGTAAGCCACTTATTGA-3´; HMPr40: 5´-GCCTCCTTAACTTCTTTG-3´; HMPro53: 5´-CCTTCATCTTCACCAGCACCA-3´	132	Shields et al. (2013)
conventional	ITS-2	CCITS2: 5′-GCAGTCACAGGAGGCATATATCC-3′; CCITS2: 5′-ATGAGAGACCTCACAGCCAAAC-3′	116	Lalonde and Gajadhar (2008)
		CCITS2 + CCITS2		Giangaspero et al. (2015)
qPCR MCA		CCITS2 + CCITS2		Caradonna et al. (2017)
qPCR		CCITS2 + CCITS2; Probe-HEX: 5´-CGACGAACAGCCACGCACGCACTTG-3´		Sim et al. (2017)
qPCR	ITS-1	CyITS1_TT: 5 -ATGTTTTAGCATGTGGTGTGGC-3′; CyITS1_TT: 5′-GCAGCAACAACAACTCCTCATC-3′; probe CyITS1_TT-P: HEX-TACATACCCGTCCCAACCCTCGA-MGBEQ; +IAC Phocine herpesvirus-1(PhHV1)	141	Temesgen et al., 2019a, Temesgen et al., 2019b, Temesgen et al., 2020; Pineda et al. (2020)

MCA = melting curve analysis.

SNP = single nucleotide polymorphism.

^aAfter nested PCR RFPL from MnlI restriction enzyme was used to differentiate Cyclospora and Eimeria organisms.

^bAfter nested PCR RFPL from BsaBI restriction enzyme was used to differentiate Cyclospora, Eimeria, Cryptosporidium, Giardia organisms.

^cPrimers m18SeF and m18SeR not specific only to C. Cayetanensis.

^dInternal amplification control artificially designed, showing no homology to sequences in GenBank.