Schematic representation of the RLB hybridization process. (a) Oligoprobes are covalently coupled to the membrane in lines using a line-blotter apparatus; (b) the membrane is then rotated 90°; (c) the PCR amplicons are added perpendicularly to the probes; (d) amplicons come into contact with all the probes in a macroarray format; (e) schematic representation of the hybridization and detection reactions; (f) example of the RLB hybridization signal on an autoradiography film
RLB hybridization is a highly specific and sensitive diagnostic tool that can be used to identify piroplasms in different sample types like blood and tissues from different animal hosts and ticks, both as single and mixed infections . The use of generic 18S rRNA gene primers and the inclusion of a catchall Theileria and Babesia control probe allow for the discovery of novel species or genotypes; a generic hybridization signal with the catchall probe in the absence of species-specific hybridization signals would indicate that a new species or genotype is present. Sequencing analysis of the amplicon would then be needed to characterize it, and, if required, a new specific probe could be designed and added to the panel of probes used in the array. In fact, this strategy has helped in the description of novel species/genotypes [4–6].
RLB hybridization methods were first used for bovine piroplasm detection in 1999  and soon extended to other piroplasm species [4, 5, 7, 8]. Nowadays, RLB hybridization has become the molecular technique of choice for the simultaneous detection and identification of several Babesia and Theileria species in hosts and tick vectors [9–15]. More recently, the piroplasm RLB membrane-based assay for bovine piroplasm detection has been transferred to a DNA bead-based suspension array test using the Luminex® xMAP technology that provides higher throughput screening and more flexibility in array preparation .
Miniblotter ® 45 line-blotter apparatus and foam cushions.
Hybridization oven, glass cylinders, and hybridization mesh sheets.
Shaking water bath.
Plastic container (e.g., Tupperware).
Negatively charged nylon membrane (e.g., Biodyne® C, Pall Corporation, NY, USA).
16 % EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride): Weight 3.2 g of EDAC and add 20 ml of ultrapure water.
20× SSPE: Weight and mix 175.3 g of NaCl, 27.6 g of NaH2PO4, and 7.4 g of EDTA. Add 800 ml of water, adjust the pH to 7.4 with 10 M NaOH, and bring volume to 1 L with water. Sterilize in the autoclave for 15 min at 121 °C.
2× SSPE: Dilute 100 ml of 20× SSPE into 900 ml water.
100 mM NaOH: Weight 4.2 g of NaOH and bring volume to 1 L with water.
10 % SDS: Weight 100 g of SDS, add 800 ml of water, mix well, and bring volume to 1 L with water.
1 % SDS: Dilute 100 ml of 10 % SDS into 900 ml of water.
2× SSPE/0.1 % SDS: Mix 495 ml of 2× SSPE and 5 ml of 10 % SDS.
2× SSPE/0.5 % SDS: Mix 950 ml of 2× SSPE and 50 ml of 10 % SDS.
0.5 M EDTA: Weight 46.5 g of EDTA-Na2 and bring volume to 250 ml with water.
20 mM EDTA: Mix 960 ml of water and 40 ml of 0.5 M EDTA, and adjust pH to 8.0 with NaOH.
Indian ink: Make up a 1/100 dilution in 2× SSPE.
500 mM NaHCO3: Weight 4.2 g of NaHCO3, add 90 ml of water, adjust pH to 8.4 with NaOH, and bring volume to 100 ml with water.
Streptavidin-horseradish peroxidase (HRP) conjugate.
Chemiluminescent substrate (e.g., ECL or SuperSignal West Dura): The substrate consists of two solutions to be mixed prior to incubation.
Film developer and fixer: Dilute with distilled water following manufacturer’s instructions.
Autoradiography films 18 × 24 cm.
Oligonucleotide probes (see Table 1), containing a N-(trifluoroacetamidohexyl-cyanoethyl-N,N-diisopropyl-phosphoramidite [TFA])-C6 amino linker.
Sequence and concentration of oligonucleotide probes used for piroplasm species identification by RLB
Catchall probe (Theileria spp. + Babesia spp.)
TAA TGG TTA ATA GGA (A/G)C(A/G) GTT G
CGT TTT TTC CCT TTT GTT GG
CAG GTT TCG CCT GTA TAA TTG AG
B. caballi (genotype B)
GTT GCG TTG TTC TTG CTT TTT GCT T
B. caballi (genotype A)
CGG GTT ATT GAC TTC GCT TTT TCT T
B. canis subsp. canis
CGT TGA CGG TTT GAC CAT TTG GT
B. canis subsp. vogeli
GTG TTC GAG TTT GCC ATT CGT T
TTA TGG CCC GTT GGC TTA T
GTT AAT ATT GAC TAA TGT CGA G
TTG CCC GAC TCG GCT ACT TG
TCC GAC TTT GGT TGG TGT
ATC TCG CTT CCG AGC GTT TTT T
ATT GGA GTA TTG CGC TTG CTT TTT
GTG TGC CTC TTT TGG CCC ATC
GCG CGC GGC CTT TGC GTT ACT
CCG AAC GTA ATT TTA TTG ATT TGG C
CCT CTG GGG TCT GTG CA
GGC TTA TTT CGG (A/T)TT GAT TTT
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