Genered

The total RNA was extracted from the metacestode vesicles and mouse liver tissue using an RNAsimple Total RNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Subsequently, the RNA was reverse-transcribed into cDNA utilizing a FastKing gDNA Dispelling RT SuperMix Kit (Tiangen Biotech).
The presence or absence of host cell contamination in metacestode vesicles and germinal cells was determined using E. multiloculus GAPDH and mouse GAPDH as indicators. Specific primers were synthesized by Shanghai Sangon Biotech with the following sequences: EmGAPDH, forward: 5′-TTTCGCAGCCGATGCCCGAT-3′, reverse: 5′-CACCGCTGTGAGAGCAGCCA-3; mouse GAPDH, forward: 5′-CCACCATGGAGAAGGCCGGG-3′, reverse: 5′-ATGTTCTGGGCAGCCCCACG-3. The RT-PCR reaction was performed using a Mastercycler Nexus PCR apparatus to a final volume of 50 μL: 10 μL 2 × Taq PCR MasterMix, 0.6 μL (10 μM) for each primer, 2 μL cDNA, 6.8 μL ddH₂O. Amplification was carried out under the following conditions: initial denaturation at 94 °C for 3 min; followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s; with a final extension at 72 °C for 3 min. PCR products were separated via 2% agarose gel electrophoresis and visualized under UV light after staining with GeneRed (Tiangen Biotech).

Metacestode vesicles (without host cell contamination) were detected by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from metacestode vesicles and host tissue (normal mouse liver) using the RNAsimple Total RNA Kit (TianGen Biotech, Beijing, China) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using the FastKing gDNA Dispelling RT SuperMix Kit (TianGen Biotech). PCR was performed using primers for the specific amplification of E. multilocularis GAPDH [27 (link)] and mouse GADPH (forward: 5′-CGTGGGGCAGCCCAGAACAT-3′; reverse: 5′-GAGCAATGCCAGCCCCAGCA-3′).
The RT-PCR reaction was performed using the Mastercycler Nexus PCR apparatus in a final volume of 50 μl: 25 μl 2× Taq PCR Mastermix (TianGen Biotech), 1.2 μl of each primer (10 μM), 2 μl cDNA product, 20.6 μl ddH₂O. Amplification was performed using the following conditions: 3 min at 94 °C followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 20 s, and a final extension step at 72 °C for 3 min. PCR products were separated by 2% agarose gel electrophoresis and stained with GeneRed (TianGen Biotech) for visualization under UV light. Metacestode germinal cells were subsequently identified in the same manner.

Metacestode vesicles (without host cell contamination) were detected by real-time polymerase chain reaction (RT-PCR). Total RNA was extracted from metacestode vesicles and host tissue (normal mouse livers) using an RNAsimple Kit (TianGen Biotech, Beijing, China) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized using a FastKing gDNA Dispelling RT SuperMix Kit (TianGen Biotech). PCR was performed using specific primers for amplification of E. multilocularis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [19 (link)] and mouse GAPDH (forward, 5′-CGTGGGGCAGCCCAGAACAT-3′; reverse, 5′-GAGCAATGCCAGCCCCAGCA-3′).
RT-PCR was performed using a MasterCycler Pro S (Applied Biosystems, Foster City, CA) in a final volume of 20 μL, including 10 μL of 2 × Taq PCR MasterMix (TianGen Biotech), 0.6 μL of each primer (10 μM), 2 μL of cDNA product, and 6.8 μL of double-distilled water. Amplification was performed using the following conditions: 3 min at 94 °C followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and a final extension step at 72 °C for 3 min. The PCR products were separated by 2% agarose gel electrophoresis and stained with GeneRed (TianGen Biotech) for visualization under ultraviolet light. Metacestode germinal cells were subsequently identified in the same manner.