Table 2 Summary of the gene targets, primers, PCR conditions employed for the amplification and genetic analysis of Spirocerca spp.; and the outgroups used for the phylogenetic analyses

18S

~750

Nem18S-F (5'-CGC GAA TRG CTC ATT ACA ACA GC-3') and Nem18S-R (5'-GGG CGG TAT CTG ATC GCC-3') (400 nM) [23]

95 °C for 5 min; 35 cycles of 95 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min; 72 °C for 5 min

Spirocerca lupi (AY751497.1 and Q674750.1), Spirocerca sp. (AY751498.1), Cylicospirura petrowi (KM434335.1), Gongylonema pulchrum (AB495401.2), Gongylonema nepalensis (AB646109.1), Oxyspirura petrowi (LC316613.1), Dirofilaria immitis (AF182647.1), Dirofilaria repens (AB973229.1), Loa loa (XR002251421.1), Wuchereria bancrofti (AY843436.1), Litosomoides sigmodontis (AF227233.1), Thelazia lacrymalis (DQ503458.1), Thelazia callipaeda (AB538282.1)

~870

Nem18S-F2 (5'-CGA AAG TCA GAG GTT CGA AGG-3') and Nem18S-R2 (5'-AAC CTT GTT ACG ACT TTT GCC C-3') (400 nM) [5]

95 °C for 5 min; 35 cycles of 95 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min; 72°C for 5 min

ITS1

610

rDNA2 (5'-TTG ATT ACG TCC CTG CCC TTT-3') [24] and rDNA1.58S (5'-GCC ACC TAG TGA GCC GAG CA-3') (375 nM) [25]

94 °C for 5 min; 35 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min; 72 °C for 5 min

C. petrowi (KM434335.1)

~100

ITS inner-F (5'-GCT ATC TTG TAA AAA CGG TG-3')^a and M13-R (5'-CAG GAA ACA GCT ATG AC-3') (200 nM)

94 °C for 5 min; 35 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min; 72 °C for 5 min

450

C18 (5'-GTT TCC GTA GGT GAA CCT GC-3') and 5818 (5'-ACG ARC CGA GTG ATC CAC-3') (400 nM) [26]

94 °C for 4 min; 40 cycles of 94 °C for 45 s, 59 °C for 30 s and 72 °C for 2 min; 72 °C for 5 min

cox1

650 (Fragment A)

NTF (5'-TGA TTG GTG GTT TTG GTA A-3') and NTR (5'-ATA AGT ACG AGT ATC AAT ATC-3') (200 nM) [28]

95 °C for 2 min; 49 cycles of 95 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min; 72 °C for 7 min

S. lupi from China (KC305876.1) [27], Spirocerca sp. from Denmark (KJ605484) [13], C. petrowi (KF719952.1), Cylicospirura felineus (GQ342967.1) [61], Cylicospirura subaequalis (GQ342968.1) [60]

~300

NTInt (5'-GGC TAG ACA ACT CTA AAC G-3')^a and NTF (5'-TGA TTG GTG GTT TTG GTA A-3') (200 nM) [28]

95 °C for 2 min; 49 cycles of 95 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min; 72 °C for 7 min

394 (Fragment B)

JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') (400 nM) [30]

95 °C for 5 min; 30 cycles of 95 °C for 1 min, 54 °C for 1 min and 72 °C for 1 min; 72 °C for 5 min

S. lupi from China (KC305876.1) [27], S. lupi obtained from domestic dogs and black-backed jackals from South Africa (KY495493.1-KY495505.1) [2] and from the Andean fox from Peru (KY634868.1-KY634870.1) [11]