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The reverse transcription PCR (RT-PCR) was conducted using the Quant Studio 7 Flex PCR System (Thermo Fisher Scientific, Cat# 4485700).The reaction conditions were as follows: initial denaturation at 95 C for 20 s, followed by 40 cycles at 95 C for 15 s denaturation, 60 C for 30 s annealing, and 72 C for 30 s extension

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The samples were subjected to heat treatment at temperatures ranging from 40 C, 50 C, 60 C, 70 C, 80 C and 90 C for 3 min, followed by a 3-minute cooling period at 4 C in a thermocycler