Scrfi

Bst DNA polymerase (Large Fragment) was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Nt.BstNBI, ScrFI, DraI, and agarose were obtained from New England Biolabs (Ipswich, MA, USA). Syto 9 was achieved from Thermo Fisher (Waltham, MA, USA). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), hemin, dNTP mixture, and ABTS were all supplied by Sangon Biotech (Shanghai, China) Co., Ltd. LB agar and LB broth were offered by Beijing Land Bridge Technology Co., Ltd. (Beijing, China) 30% H₂O₂ was bought from Sigma-Aldrich, Inc. (Burlington, MA, USA).
The gene of invasion protein A (invA) was selected as the reference gene for amplification (GenBank accession no. NC_003197). Conventional LAMP primers were synthesized according to the previous report [32 (link)]. Inner primers incorporated with the recognition site (GAGTC) of nicking endonuclease, Nt.BstNBI, are termed as Nick-BIP and Nick-FIP. All the sequences were evaluated with IDT Oligo Analyzer 3.1 (Integrated DNA Technologies, Coralville, IA, USA). The oligonucleotides were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Sequences used in this work are listed in Table S1, and the corresponding template sequence is displayed in Figure S1.

Genomic DNA was extracted from 200 μl EDTA-anticoagulated peripheral blood sample with a DNA isolation kit (BioTeke, Peking, China) as the manufacturer's direction. DNA was stably stored at −20°C until assayed. Genotyping of the IL-31 gene polymorphism was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We designed the PCR primers with software Primer 3 (http://bioinfo.ut.ee/primer3‐0.4.0/primer3/) [27 (link)] as shown in Table 1. The 10 μl PCR reaction system was consisted of 1.0 μl DNA and 5 μl 2× Power Taq PCR Master Mix (BioTeke, Peking, China), forward and reverse primer 0.1 μl, respectively, and reserved volume was made up to 10 μl by sterilized water. The PCR condition was designed as 95°C for 4 min firstly, then 33 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and finally, 72°C for 10 min. Furthermore, the PCR products were digested in 37°C stable incubation by distinguished restriction enzyme MboII (New England Biolabs, Peking, China) for 30 minutes of rs4758680 and ScrFI (New England Biolabs, Peking, China) for 2 hours of rs7977932 as shown in Table 1, separately. Ultimately, the results were visually analyzed by 6% polyacrylamide gels in silver staining. To verify the genotyping results, DNA sequencing was performed in about 20% PCR-amplified DNA samples randomly.