Molecular detection and genotypic characterization of Toxoplasma gondii infection in bats in four provinces of China

Qin, Si-Yuan; Cong, Wei; Liu, Ye; Li, Nan; Wang, Ze-Dong; Zhang, Fu-Kai; Huang, Si-Yang; Zhu, Xing-Quan; Liu, Quan

Multilocus PCR-RFLP was conducted to genetically characterize T. gondii in bats, using 11 genetic markers (i.e., SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) as described previously [12, 18] . Briefly, the target sequences were first amplified by multiplex PCR using external primers for all 11 markers. Nine reference strains, namely GT1, PTG, CTG, MAS, TgCgCa1, TgCatBr5, TgCatBr64, TgRsCr1, and TgWtdSc40, were used as the positive controls ( Table 1 ). The PCR amplification was performed using a thermal cycler (PTC 200, Bio-RAD). The T. gondii B1-positive DNA sample was incubated at 95°C for 5 min to activate the DNA polymerase, then 30 cycles of PCR at 95°C for 30 s, 55°C for 60 s and 72°C for 90 s, and then at 72°C for 7 min. Then 1 μl of the products were used as template DNA for nested PCR with internal primers for each marker, respectively. The nested PCR products were digested with restriction enzymes for 2 h, and the restriction fragments were resolved in 2.5% agarose gel to distinguish single nucleotide polymorphisms (SNPs) using a gel document system (UVP GelDoc-It™ Imaging System, Cambridge, UK).