Preparation of the plasmid standard and positive sample
Each plasmid bearing 18S rRNA sequences of B. microti, B. divergens, B. duncani, B. motasi Hebei, and B. crassa (accession number HQ285838 for B. duncani, FJ944826 for B. divergens, AY260176 for B. crassa, KF410825 for B. microti, and DQ159074.1 for B. motasi Hebei) were used as positive controls to assess the sensitivity and specificity of the qPCR-HRM detection method. Briefly, PCR-amplified fragments of each Babesia strain were purified using the Zymolean™ Gel DNA Recovery Kit (ZYMO, Los Angeles, USA), cloned into pGEM-T Easy vector (Promega, USA), and transformed into Escherichia coli DH5α competent cells (Takara Biotech Co., Ltd., Dalian). Three clones from each sample were selected and cultured in 3 ml LB medium containing 100 µg/ml ampicillin (Merck, Darmstadt, Germany) for 12 h, and plasmid extraction was performed using a Plasmid Miniprep Kit (Axygen, USA). Sequencing was performed using Big Dye Terminator Mix (Genscript, Nanjing, China). The prepared plasmid was diluted to 107~10− 1 copies/µl using an eluent (Axygen, USA).
The strains of B. motasi Hebei and B. duncani, as well as B. crassa genomes were obtained from the Vectors and Vector-Borne Diseases (VVBD) Laboratory, Lanzhou Veterinary Research Institute (LVRI). Six-month-old sheep that were negative for piroplasm infection as determined using microscopy and PCR were purchased from Jingtai County, Gansu Province, China [44]. Splenectomy was performed on sheep one month before the experiment. The suspension of B. motasi Hebei, preserved in liquid nitrogen, was rapidly thawed in 37°C water, and then 50 ml (~ 1 × 109 infected erythrocytes) was injected into the splenectomy sheep through the jugular vein. Three-month-old LVG golden Syrian hamsters were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. Similarly, after rapid resuscitation of frozen B. duncani in a 37°C water bath, five hamsters were inoculated intraperitoneally with 200 µL of the B. duncani suspension (~ 2.0×106 infected red blood cells). Blood smears were made by collecting blood four days after infection in hamsters and seen days after infection in sheep. After Giemsa staining of blood smears, parasitemia was calculated by ME. Approximately 3000 red blood cells (RBCs) were counted per blood smear to calculate the percentage of parasitized RBCs. When parasitemia was 8–10%, whole blood with different parasitemia levels was collected into EDTA anticoagulant tubes. Total DNA was extracted from 200 µL of blood using a commercial DNA extraction kit (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Negative control DNA was isolated from the whole blood of healthy humans and sheep and piroplasm-free tick tissue, which was examined by blood smear ME and nested PCR [44]. The DNA concentration was assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The extracted DNA was stored at − 20°C until use. The study was approved by the LVRI Animal Ethics Committee of the Chinese Academy of Agricultural Sciences. All procedures were performed in accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China.
Target gene sequence selection and primer design
Sequences of the 18S rRNA gene of the zoonotic Babesia spp. (accession numbers HQ285838 for B. duncani, FJ944826 for B. divergens, AY260176 for B. crassa, KF410825 for B. microti, and DQ159074 for B. motasi Hebei) were retrieved from GenBank. BLAST analysis of the 18S rRNA gene sequences of B. microti, B. divergens, B. duncani, B. motasi Hebei, and B. crassa on the NCBI website was performed using the BLASTn program. This analysis demonstrated that the sequences of the strains Babesia sp. KO1(DQ346955) and Babesia sp. KCDC-1(MK930513), isolated from two human cases in Korea, were highly similar to the sequences of B. motasi Hebei from China [19]. The conserved and variable regions of each Babesia species were identified after sequence alignment using DNAMAN v.2.0. Primers were designed based on a conserved region of the Babesia 18S rRNA gene to obtain a consistent reference sequence of Babesia, however there was at least one base difference between the distinct species of Babesia. Several pairs of universal primers were designed for qPCR-HRM analysis using Primer v.5.0, in order to distinguish the five Babesia spp. (Table 1).
Table 1
Primers targeting Babesia 18S rRNA
Primer name
|
Primer sequences
(5’ to 3’)
|
Babesia-1F
|
gtt ggg ggc att cgt att t
|
Babesia-1R
|
tag ttc gtc ttt aac aaa tct
|
Babesia-2F
|
gac tcc ttc agc acc ttg a
|
Babesia-2R
|
cca gaa ccc aaa gac ttt g
|
SP-F
|
acg aga cct taa cct gct aa
|
SP-R
|
cac aga cct gtt att gcc tta
|
SP-2F
|
gaa tac ttc agc atg gaa taa t
|
SP-2R
|
gtt aaa tac gaa tgc ccc caa c
|
SP-3F
|
ttg ctt ctt aga ggg act
|
SP-3R
|
aga cct gtt att gcc tta
|
HRM-F1
|
ttc cgt taa cga acg aga cc
|
HRM-R1
|
Agt ccc tct aag aag caa
|
HRM-F2
|
Tag gga ttg gag gtc gtc att t
|
HRM-R2
|
Acc caa aga ctt tga ttt ctc
|
HRM-F3
|
gac aag aaa taa caa tac agg gc
|
HRM-R3
|
Gct ggc acc aga ctt gcc ctc ca
|
Several pairs of primers were designed to target Babesia 18S rRNA in order to screen a pair of primers capable of simultaneously amplifying five Babesia species
Evaluation of primer efficiency at various annealing temperatures
To ensure that the primers could amplify the target regions of all five Babesia species simultaneously without producing nonspecific bands or primer dimers, which would interfere with the judgment of the results in subsequent qPCR-HRM analysis, we performed a series of gradient PCRs to determine the optimal annealing temperature for the primers. For this, 20 µL PCR mix was prepared containing 10 µL Premix Taq DNA polymerase (Takara, Dalian, China), 1 µL DNA template, 1 µL each primer (10 µM), and 7 µL RNase-free water. Gradient PCR was performed using a T100™ Thermal Cycler (Bio-Rad, USA). The mixture was heated at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at a gradient temperature of 54°C to 61°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. After gradient PCR amplification, the PCR products were resolved by electrophoreses using 1.5% (w/v) agarose gels to evaluate primer specificity and amplification efficiency at different annealing temperatures.
Performing qPCR-HRM analysis
The qPCR amplification was performed in 20 µl reaction containing 10 nM forward and reverse primers (SPF, SPR), 10 µL Forget-me-Not™ qPCR Master Mix (Biotium, CA, USA), 0.5 µL 40 ×Template Buffer, 6.5 µL of RNase-free water, and 1µl DNA sample. The qPCR-HRM assays were performed using a Rotor-Gene Q6000 Real-Time PCR system (Qiagen, Sydney, Australia) with an initial denaturation step at 95°C for 2 min, followed by 45 cycles of 95°C for 5s, and 55°C for 30 s. High Resolution Melting (HRM) was performed from 75°C to 85°C using a 0.2°C step with a 2 s hold time at each acquisition step. Finally, melting curves were normalized using High-Resolution Melt software v.2.3.1 (Qiagen).
Evaluation of analytical specificity and sensitivity of the qPCR‑HRM
The specificity of the assay was assessed using 20 ng/µL plasmids of zoonotic Babesia which were diluted by healthy human blood genomes, including B. divergens, B. motasi Hebei, B. crassa, B. duncani, and B. microti, to ensure that a pair of primers could clearly distinguish the five Babesia species in a single reaction. Five replicates were made for each plasmid, and the standard Tm value and GCP of each Babesia strain were obtained from five independent qPCR-HRM experiments. Genomic DNA extracted from the whole blood of healthy humans was used as a negative control.
The analytical sensitivity was assessed using10 fold serial dilutions of the five plasmid DNA standards, ranging from 107 to 1 copy number(s)/µL. This process was repeated three times to ensure the accuracy of the results. Standard curves were plotted using the software (v.2.3.1; Qiagen) to evaluate the efficiency of the assay.
Evaluation of the qPCR-HRM assay using experimental and clinical samples
The performance of the qPCR-HRM assay was evaluated using standard positive samples (experimentally infected animal specimens and plasmids), as well as clinical samples. We determined the positive and negative standard samples through microscopic examination, nested PCR, and sequencing. A total of 200 laboratory-positive specimens were prepared. Briefly, plasmids of B. divergens and B. microti were added to the DNA samples of healthy people or ticks at different concentrations to prepare artificial positive samples. Forty samples were prepared for each Babesia species. Blood samples were collected from laboratory animals infected with B. duncani and B. motasi Hebei, and 40 DNA samples were prepared for each. And 40 B. crassa genomes preserved by the team. Blood samples were collected from 492 patients with a history of tick bite who had visited the Second Hospital of Lanzhou University (May 2017 to July 2019) residing in the Gannan Dibetan Autonomous Prefecture (Gansu Province). All samples were collected using approved protocols and after obtaining written informed consent. The collected blood samples were analyzed by microscopy. The aforementioned 692 clinical specimens were randomized, relabeled, and blindly detected by qPCR-HRM. Simultaneously, the qPCR products of all samples were purified with a gel recovery kit, and 18S rRNA sequencing was performed to verify the qPCR-HRM results.