Table 2.
PCR oligonucleotide sequences used for detection of various Babesia species in the tick-vector reported in different studies. The identity of Babesia parasites amplified with non-specific primers was confirmed through sequence analysis.
| Babesia Species Detected | PCR Format | Target Gene or Region | Primers Name | Product Size | Primer Sequence | References |
|---|---|---|---|---|---|---|
|
B. bigemina and B. bovis |
nPCR |
SpeI-AvaI rap-1 |
BiIA/BiIB BiIAN/BiIBN BoF/BoR BoFN/BoRN |
278 bp 170 bp 356 bp 291 bp |
5′-CATCTAATTTCTCTCCATACCCCTCC-3′ 5′-CCTCGGCTTCAACTCTGATGCCAAAG-3′ 5′-CGCAAGCCCAGCACGCCCCGGTGC-3′ 5′-CCGACCTGGATAGGCTGTGTGATG-3′ 5′-CACGAGGAAGGAACTACCGATGTTGA-3′ 5′ CCAAGGAGCTTCAACGTACGAGGTCA 3′ 5′-TCAACAAGGTACTCTATATGGCTACC-3′ 5′-CTACCGAGCAGAACCTTCTTCACCAT-3′ |
[38,40,41,42,43] |
| B. bovis | nPCR | msa-1 | external forward external reverse internal forward internal reverse |
ND 212 bp |
5′-TTCGACCAGACCAAATTGT-3′ 5′-CGCATCAAAAGA CTCAACA-3′ 5′-GCCCTGATCTATTTAATGCA-3′ 5′-CCCCGTATAAACATGCTTC-3′ |
[35,36] |
| B. bovis | qRT-PCR | msa-1 | Forward/reverse Probe |
150 bp | 5′-GATGCGTTTGCACATGCTAAG-3′ 5′-TGAGAGCACCGAAGTACCCG-3′ 5′-CACGCTCAAGTAGGAAATTTTGTTAAACCTGGA-3′ |
[35,36] |
| B. bovis | nPCR | rap-1 | BoF/BoR BoFN/BoRN |
354 bp 291 bp |
5′-CACGAGGAAGGAACTACCGATGTTGA-3′ 5′ CCAAGGAGCTTCAACGTACGAGGTCA 3′ 5′-TCAACAAGGTACTCTATATGGCTACC-3′ 5′-CTACCGAGCAGAACCTTCTTCACCAT-3′ |
[53] |
|
B. bigemina and B. bovis |
nPCR | 18S rRNA | KB-16/KB-17 KB-18/KB-19 KB-24/KB-25 |
ND 262 bp 217 bp |
5′-CATCAGCTTGACGGTAGGG-3′ 5′-GTCCTTGGCAAATGCTTTC-3′ 5′-GATGTACAACCTCACCAGAGTACC-3′ 5′-CAACAAAATAGAACCAAGGTCCTAC-3′ 5′-GGGGGCGACCTTCAC-3′ 5′-CTCAATTATACAGGCGAAAC-3′ |
[44] |
|
B. bigemina and B. bovis |
PCR | ssrDNA | A/B C/B |
118 bp 225 bp |
5′-TGTCCTCGTTTGCTTCTTAGAGGGACTCCT-3′ 5′-CCGACACGATGCACACTAAACATTACCCAA-3′ 5′-TTGGCATGGGGGCGACCTTCACCCTCGCCC-3′ 5′-CCAAAGTCAACCAACGGTACGACAGGGTCA-3′ |
[45] |
| B. bigemina | qPCR | 18S rDNA | RTBbF/RTBbR | ND | 5′- AGCTTGCTTTCACAACTCGCC -3′ 5′- TTGGTGCTTTGACCGACGACAT -3′ |
[51] |
| B. bigemina | qPCR | 18S rDNA | Forward/reverse | ND | 5′- AATAACAATACAGGGCTTTCGTCT -3′ 5′- AACGCGAGGCTGAAATACAACT -3′ |
[52] |
|
B. bovis, and B. bigemina |
PCR-RFLP | 18S rDNA | PiroA/Piro B RE: MspI BoxI |
400 bp 250, 150 bp 290, 110 bp |
5′-AATACCCAATCCTGACACAGGG-3′ 5′-TTAAATACGAATGCCCCCAAC-3′ |
[47] |
|
B. bigemina and B. bovis |
qPCR | mitochondrialcytochrome b | Cbisg 1 and 2 Cbosg 1 and 2 |
88 bp 88 bp |
5′-TGTTCCAGGAGATGTTGATTC-3′ 5′-AGCATGGAAATAACGAAGTGC-3 5′-TGTTCCTGGAAGCGTTGATTC-3′ 5′-AGCGTGAAAATAACGCATTGC-3′ |
[49,50] |
| B. divergens | PCR | 18S rRNA | BAB GF2/GR2 | 559 bp | 5′-GYYTTGTAATTGGAATGATGG-3′ 5′-CCAAAGACTTTGATTTCTCTC-3′ |
[55] |
| Bovine Babesia spp. | PCR | 18S rRNA | ND | 422–440 bp | 5′-GTTTCTGMCCCATCAGCTTGAC-3′ 5′-CAAGACAAAAGTCTGCTTGAAAC-3′ |
[56] |
| B. divergens | PCR | 18S rRNA | ND | 353 bp | 5′-GTTTCTGMCCCATCAGCTTGAC-3′ 5′-CAATATTAACACCACGCAAAAATTC-3′ |
[56] |
| Babesia sp. genotype EU1 | PCR | 18S rRNA | ND | 362 bp | 5′-GTTTCTGMCCCATCAGCTTGAC-3′ 5′-AGACAAGAGTCAATAACTCGATAAC-3′ |
[56] |
| B. orientalis | Semi-nested PCR | 18S rRNA | P1/B-R2 B-P2/B-R2 |
ND 257 bp |
5′-AACCTGGTTGATCCTGCCAGTAGT-3′ 5′-CACACGCACAACGCTGAA-3′ 5′-TGAGAAACGGCTACCACA-3′ 5′-CACACGCACAACGCTGAA-3′ |
[57] |
| B. occultans | PCR/RLB hybridization | 18S rRNA (V4 variable region) |
RLB-F2/RLB-R2 Probe |
460 bp | 5′-GACACAGGGAGGTAGTGACAAG-3′ 5′-biotin-CTAAGAATTTCACCTCTGACAGT-3′ 5′-GTGTGCCTCTTTTGGCCCATC-3′ Species-specific containing a C12 amino linker in 5′ |
[60] |
| B. ovata | PCR | β-tubulin | ND | ND | 5′-ACACTGTGCATCCTCACCGTCATAT-3′ 5′-CTCGCGGATCTTGCTGATCAGCAGA-3′ |
[61] |
| B. vogeli | PCR-RFLP | 18S rRNA | PiroA/PiroB RE: TaqI |
400 bp 203, 171, 26 bp |
5′-AATACCCAATCCTGACACAGGG-3′ 5′-TTAAATACGAATGCCCCCACC-3′ |
[46,70,83] |
| B. vogeli | Semi-nested PCR | 18S rRNA | 455-459/793-772 BCV/793-772 |
339 bp 192 bp |
5′-GTCTTGTAATTGGAATGATGGTGAC-3′ 5′-ATGCCCCCAACCGTTCCTATTA-3 5′-GTTCGAGTTTGCCATTCGTT-3′ 5′- ATGCCCCCAACCGTTCCTATTA-3′ |
[68,70] |
| B. vogeli | PCR | 18S rRNA | BCV-F/Ba721R | 422 bp | 5′- GTGTTCGAGTTTGCCATTCG-3′ 5′-CCCAGAACCCAAAGACTTTGATTTCTCTCAAG-3′ |
[79] |
| B. vogeli | PCR-RFLP | 18S rRNA | BJ1/BN2 RE: ApoI |
489 bp 367, 122 bp |
5′-GTCTTGTAATTGGAATGATGG-3′ 5′-TAGTTTATGGTTAGGACTACG-3′ |
[71] |
| B. vogeli and B. canis | PCR | cox1 | BFor/BvRev/BcRev | 450 bp 750 bp |
5′-GCATCTGGAATAGCTAGTGC-3′ 5′-CTGCTTCTAAACCAGAAGTG-3′ 5′-TGGAAATGACCTACAACATAC-3′ |
[75] |
| B. gibsoni | PCR | 18S rRNA | PiroA/PiroB | 408 bp | 5′-AATACCCAATCCTGACACAGGG-3′ 5′-TTAAATACGAATGCCCCCACC-3′ |
[77] |
|
B. gibsoni and B. vogeli |
nPCR (non-specific) |
18S ssrRNA | 5-22F/1661R 455-479F/793-722R (generic primers) |
293–338 bp | 5′-GTTGATCCTGCCAGTAGT-3′ 5′-AACCTTGTTACGACTTCTC-3′ 5′-GTCTTGTAATTGGAATGATGGTGAC-3′ 5′-ATGCCCCCAACCGTTCCTATTA-3′ |
[78] |
|
B. gibsoni, B. vogeli, B. canis |
PCR/RLB hybridization | 18S rRNA (V4 variable region) |
RLB-F2/RLB-R2 Probes |
460 bp | 5′-GACACAGGGAGGTAGTGACAAG-3′ 5′-biotin-CTAAGAATTTCACCTCTGACAGT-3′ Oligonucleotides probes (species-specific) linked to N-terminal N-(trifluoracetamidohexyl-cyanoethyl, N, N-diisopropyl phosphoramidite [TFA])-C6 amino |
[81,82,84,86] |
|
B. gibsoni and B. vogeli |
Multiplex PCR | 18S rRNA | BAGI F/BAGI R BAB1 F/BAB4 R |
590 bp 488 bp |
5′- TTGGCGGCGTTTATTAGTTC-3′ 5′- AAAGGGGAAAACCCCAAAAG-3′ 5′- GTGAACCTTATCACTTAAAGG-3′ 5′- CAACTCCTCCAC GCAATCG-3′ |
[73,74] |
| B. canis | PCR | 18S rRNA | BcW-A/BcW-B | 500 bp | 5′-CATCTAAGGAAGGCAGCAGG-3′ 5′- TTAATGGAAACGTCCTTGGC-3′ |
[87] |
| B. canis | PCR | 18S rRNA | PiroA/PiroB | 408 bp | 5′-AATACCCAATCCTGACACAGGG-3′ 5′-TTAAATACGAATGCCCCCACC-3′ |
[69] |
| B. canis | PCR (non-specific) |
18S rRNA | BJ1/BN2 (generic primers) |
ND | 5′-GTCTTGTAATTGGAATGATGG-3′ 5′-TAGTTTATGGTTAGGACTACG-3′ |
[72,88,89,90] |
| B. canis | PCR nPCR |
18S rRNA | BS1/PiroC PiroA/PiroC |
ND | 5′-GACGGTAGGGTATTGGCCT-3′ 5′-CCAACAAAATAGAACCAAAGTCCTAC-3′ 5′-ATTACCCAATCCTGACACAGGG- 3′ 5′-CCAACAAAATAGAACCAAAGTCCTAC-3′ |
[92,93] |
| B. venatorum | PCR (non-specific) |
18S rRNA | BJ1/BN2 (generic primers) |
411–452 bp | 5′-GTCTTGTAATTGGAATGATGG-3′ 5′-TAGTTTATGGTTAGGACTACG-3′ |
[72,98,99] |
| B. odocoilei | Semi-nested PCR | 18S rDNA | Piro_18S_300F/Piro_18S_1688R Cocci_18S_595F/Piro_18S_1688R |
1393 bp 1147 bp |
5′-GACGGTAGGGTATTGGCCTA-3′ 5′-CGACTTCTCCTTCCTTTAAGTGATAAG-3′ 5′-CCGCGGTAATTCCAGCTCCAAT-3′ 5′-CGACTTCTCCTTCCTTTAAGTGATAAG-3′ |
[100] |
| B. caballi | nPCR | BC48 | BC48F1/BC48R3 BC48F11/BC48R31 |
530 bp 430 bp |
5′-ACGAATTCCCACAACAGCCGTGTT-3′ 5′-ACGATTTCGTAAAGCGTGGCCATG-3′ 5′-GGGCGACGTGACTAAGACATG-3′ 5′-GTTCTCAATGTCAGTGACATCCGC-3′ |
[105,106] |
| B. caballi | PCR/RLB hybridization | 18S rRNA (hypervariable V4 region) | RLB-F2/RLB-R2 Genotypes A Genotypes B |
ND | 5′-GACACAGGGAGGTAGTGACAAG-3′ biotin-5′-CTAAGAATTTCACCTCTGACAGT-3′ 5′-GTTGCGTTGTTCTTGCTTTTTGCTT-3′ 5′-CGGGTTATTGACTTCGCTTTTTCTT-3′ |
[109] |
| B. divergens/B. venatorum/B. microti | PCR/RLB hybridization |
18S rRNA | Bath-F/Bath-R (generic primers) |
ND | 5′-TAAGAATTTCACCTCTGACAGTTA-3′ 5′-ACACAGGGAGGTAGTGACAAG-3′ |
[113] |
| B. motasi | PCR (non-specific) |
18S rRNA | BTH 1F/BTH 1R GF2F/GR2R (generic primers) |
561 bp | 5′-CCTGAGAAACGGCTACCACATCT-3′ 5′-TTGCGACCATACTCCCCCCA-3′ 5′-GTCTTGTAATTGGAATGATG-3′ 5′-CCAAAGACTTTGATTTCTCTC-3′ |
[114] |
| B. motasi | PCR (non-specific) |
Cytochrome b (COB) | COB F/COB R (generic primers) | 550 bp | 5′-CCATAGCAATTAATCCAGCTA-3′ 5′-ACCTTGGTCATGGTATTCTGG-3′ |
[114] |
| B. motasi | PCR (non-specific) |
Cytochrome c (COX-3) | COX3 F/COX3 R (generic primers) | 552 bp | 5′-TCAACAAAATGCCAATATGT-3′ 5′-AAGTGCATCTTTGGGAGAAG-3′ |
[114] |
| B. microti | nPCR | β-tubulin | Tubu93 F/Tubu897 R Tubu192 F/Tubu782 R |
551 bp | 5′-GAYAGYCCCTTRCAACTAGAAAGAGC-3′ 5′-CGRTCGAACGAACATTTGTTGHGTCARTTC-3′ 5′-ACHATGGATTCTGTTAGATCYGGC-3′ 5′-GGGAADGGDATRAGATTCACAGC-3′ |
[114] |
| B. microti | RT-PCR | 18S rRNA | ND Probe |
ND | 5′-AACAGGCATTCGCCTTGAAT-3′ 5′-CCAACTGCTCCTATTAACCATTACTCT-3′ 6FAM-CTACAGCATGGAATAATGA-MGBNFQ |
[115] |
| B. duncani | nPCR | β-tubulin | F34/R323 BtubFn/BtubRn |
ND 175–181 bp |
5′-TGTGGTAACCAGATYGGWGCCAA-3′ 5′-TCNGTRTARTGNCCYTTRGCCCA-3′ 5′-TCWGACGAGCACGGCATYGA-3′ 5′-CCAGGCTCCAARTCCATYAA-3′ |
[116] |
| B. ovis | PCR | ssrRNA | Bbo-F/Bbo-R | 549 bp | 5′-TGGGCAGGACCTTGGTTGTTCT-3′ 5′-CCGCGTAGCGCCGGCTAAATA-3′ |
[124,130] |
| B. ovis | qPCR (non-specific) | 18S | Bab_18s_F/Bab_18s_R Bab_18s_P |
ND | 5′-TTGGGGGCATTCGTANTNRAC-3′ 5′-TTCTTGATTAATGAAAACGTCTTG-3′ 6FAM-AAGACGAACTACTGCGAAAGCATTTGC-TAMRA |
[131] |
| B. ovis | PCR (non-specific) | 18S rRNA | CRYPTOF/CRYPTOR | ND | 5′-AACCTGGTTGATCCTGCCAGT-3′ 5′-GCTTGATCCTTCTGCAGGTTCACCTAC-3′ |
[131,132] |
| B. ovis | PCR qPCR |
BoSPD BoSPD |
SDP forward/SDP reverse SDP forward/SDP reverse |
486 bp 141 bp |
5′-ATGTTGGCCAAGTATCTTGCC-3′ 5′-CTACGTCAATTTGGCCTTGAACTC-3′ 5′-TAATGACGCAGACCTGATGG-3′ 5′-GTTTGATCACCCTCGGAAAC-3′ |
[125,133,134] |
ND: data not defined; RE: Restriction enzyme.