Linegene9660 system

TRIzol reagent (Invitrogen) was used to purify total RNA from peripheral blood lymphocytes in accordance with the manufacturer's protocol. Peripheral blood was collected on the second day after admission, and peripheral blood lymphocytes were extracted according to the manufacturer's instructions (Solarbio). A QuickDrop spectrophotometer was used to assess the concentration and quality of the RNA. Each sample was reverse‐transcribed into cDNA and analyzed using the SYBR‑Green Real‑Time PCR kit (Vazyme). The 2‐∆∆Cq method was used for the relative quantification of IRAK4 mRNA. A series of dilutions (1 × 10⁷, 1 × 10⁶, 1 × 10⁵, and 1 × 10⁴ copies/μl of a DNA fragment) derived from EV71 were used to create a standard curve for calculating the copy numbers of viral RNA in various samples. Quantitative RT‐PCR was performed using a Linegene9660 system (Thermo Fisher Scientific). The specific primers and cycling programs are listed in Table 1.

Peripheral blood lymphocytes were extracted according to the manufacturer's instructions (Solarbio) on the second day after admission. Total RNA was purified by TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocol, and the concentration and quality of the RNA were assessed by A QuickDrop spectrophotometer. The SYBR‑Green Real‑Time PCR kit (Vazyme) was used to reverse‐transcribe the mRNA into complementary DNA (cDNA). A standard curve for calculating the copy numbers of viral RNA was built by a series of dilutions (1 × 107, 1 × 106, 1 × 105, and 1 × 104 copies/μl of a DNA fragment) in various samples. The ligation cycling step consisted of 95°C for 15 min and 40 cycles of thermal cycling at 95°C for 10 s and 60°C for 60 s. Quantitative RT‐PCR was performed using a Linegene9660 system (Thermo Fisher Scientific). The specific primers used were as follows: EV71‐S: GTTCTTAACTCACATAGCA, EV71‐A: TTGCAAAAACTGAGGGTT (Table 1).