Molecular diagnosis of alveolar echinococcosis in patients based on frozen and formalin-fixed paraffin-embedded tissue samples

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Table 3

Molecular targets tested, PCR conditions, and references.

PCR name (target gene)	Primer name	Primer sequence (5′ – 3′)	Size (bp)	Originally designed on	Species targeted and specific detection	Ref.	Cycling conditions
A-16S*	Em rrn-F	CTGTGATCTTGGTGTAGTAGTTGAGATTT	84	Echinococcus multilocularis	Echinococcus multilocularis	[19]	10 min/95 °C, 40 cycles: 30 s/94 °C, 30 s/60 °C, 10 s/72 °C
(16S-rrnL)	Em rrn-R	GGCTTACGCCGGTCTTAACTC					10 min/95 °C, 40 cycles: 30 s/94 °C, 30 s/60 °C, 10 s/72 °C
	Hydrolysis probe	FAM-TGGTCTGTTCGACCTTTTTAGCCTCCAT-TAMRA					(RT-cycles) 2 min/50 °C, 10 min/95 °C, 40 cycles: 75 s/95 °C, 1 min/60 °C
B1-2-Multiplex TRACHSEL	Cest1	TGCTGATTTGTTAAAGTTAGTGATC	395	Echinococcus multilocularis	Echinococcus multilocularis	[35]	(Multiplex PCR) 40 cycles: 30 s/94 °C, 90 s/58 °C, 10 s/72 °C
	Cest2	CATAAATCAATGGAAACAACAACAAG	395	Echinococcus multilocularis	Echinococcus multilocularis
(nad1 and	Cest4	GTTTTTGTGTGTTACATTAATAAGGGTG	117	Echinococcus granulosus	Echinococcus granulosus
12S-rrnS)	Cest5	GCGGTGTGTACMTGAGCTAAAC	117	Echinococcus granulosus	Sensu stricto
	Cest3	YGAYTCTTTTTAGGGGAAGGTGTG	267	Taenia spp.	Taenia spp.
	Cest5	GCGGTGTGTACMTGAGCTAAAC	267	Taenia spp.	Taenia spp.
C-JB11.5	ND1 JB11.5	TTATGGTAGATATTATAG	183	Echinococcus granulosus	Echinococcus spp., 1	[5]	40 cycles: 30 s/94 °C, 30 s/50 °C, 30 s/72 °C
(nad1)	ND1 JB12.5	CACACACATAAAACAAGC	183	Echinococcus granulosus	mutation between G6/G7	[5]	40 cycles: 30 s/94 °C, 30 s/50 °C, 30 s/72 °C
D-EMH15 (12S-rrnS)	EM-H15	CCATATTACAACAATATTCCTATC	200	Echinococcus multilocularis	Echinococcus multilocularis	[12]	40 cycles: 30 s/95 °C, 30 s/55 °C, 30 s/72 °C
	EM-H17	GTGAGTGATTCTTGTTAGGGGAAG	200	Echinococcus multilocularis	Echinococcus multilocularis	[12]	40 cycles: 30 s/95 °C, 30 s/55 °C, 30 s/72 °C
E-EM29	EM29	GATTTGCTGATTTGTTAAAGTTAGTGATC	252	Echinococcus multilocularis	Echinococcus multilocularis	[30]	45 cycles: 30 s/95 °C, 45 s/56 °C, 60 s/72 °C
(nad1)	EM281	AGAACTTAAAAACGAATATTTATTGTAACT	252	Echinococcus multilocularis	Echinococcus multilocularis	[30]	45 cycles: 30 s/95 °C, 45 s/56 °C, 60 s/72 °C
F-EG1	Eg1f	CATTAATGTATTTTGTAAAGTTG	255	Echinococcus granulosus	Echinococcus granulosus	[31]	40 cycles 30 s/94 °C, 30 s/53 °C, 45 s/72 °C
(12S-rrnS)	Eg1r	CACATCATCTTACAATAACACC	255	Echinococcus granulosus	Sensu stricto	[31]	40 cycles 30 s/94 °C, 30 s/53 °C, 45 s/72 °C
G-12S	12S-Echino-F	AAAKGGTTTGGCAGTGAGYGA	268	Echinococcus spp.	Echinococcus spp.	[28]	40 cycles: 30 s/94 °C, 30 s/55 °C, 1 min/72 °C
(12S-rrnS)	12S-Echino-R (Cest5)	GCGGTGTGTACCTGAGCTAAAC	268	Echinococcus spp.	Echinococcus spp.	[28]	40 cycles: 30 s/94 °C, 30 s/55 °C, 1 min/72 °C
H-COX1	CO1-F	TTTTTTGGGCATCCTGAGGTTTAT	446	Fasciola hepatica	Echinococcus spp., 1	[4]	30 cycles: 30 s/94 °C, 40 s/52 °C, 45 s/72 °C
(cox1)	CO1-R	TAAAGAAAGAACATAATGAAAATG	446	Fasciola hepatica	mutation between G6/G7	[4]	30 cycles: 30 s/94 °C, 40 s/52 °C, 45 s/72 °C
I-JB11	ND1 JB11	AGATTCGTAAGGGGCCTAATA	529	Fasciola hepatica	Echinococcus spp., 4	[5]	40 cycles: 30 s/94 °C, 30 s/50 °C, 60 s/72 °C
(nad1)	ND1 JB12	ACCACTAACTAATTCACTTTC	529	Fasciola hepatica	mutations between G6/G7	[5]	40 cycles: 30 s/94 °C, 30 s/50 °C, 60 s/72 °C
J-Alea	Alea-F	CCTAAAAATGTCTATGATTGGTCCACTA	167	Random nucleic sequence	Alea plasmid	[20]	(qPCR) 2 min/50 °C, 10 min/95 °C, 40 cycles: 75 s/95 °C, 1 min/60 °C
	Alea-R	GGGAGTACCTTGCCATACAAAATT
	Alea-probe	VIC-TTAAATCAACTCCTAAATCCGCGCGATAGG-TAMRA

Target used for real-time quantitative PCR.

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