Tb green premix ex taqtm 2 tli rnaseh plus

qRT–PCR was used to explore the antibacterial adhesion and biofilm resistance of PMs@NaF-SAP at the genetic level. The assay was performed by measuring the expression of S. mutans-specific targeted genes (gtfB, gtfC, comD, comE, and luxS). The biofilms on HA discs were created as described above in the antibacterial adhesion and biofilm resistance section. Then, the bacterial cells were obtained by centrifugation at 3000g for 6 min and washed with PBS three times. RNA was extracted and purified by standard protocols optimized for biofilms (Ultrapure RNA Kit). Afterward, TB Green Premix Ex TaqTM Ⅱ (Tli RNaseH Plus, Takara Bio Inc., Japan) was used for RNA reverse transcription, and quantitative amplification was performed using HiScript III RT SuperMix for qPCR (+gDNA wiper, Nanjing Vazyme Biotech Co., Ltd.). The primers were synthesized by Wuhan Tianyi Huiyuan Bioscience & Technology, Inc., and relevant sequences are listed in Table S1. A standard curve of each primer was used to determine the relative amount of cDNA molecules, and the relative expression was calculated with 16 S rRNA as a reference. PCR results were analyzed with Bio–Rad CFX Manager.

Total RNA of each sample was extracted using an RNA Isolation Kit (TianGen, Beijing, China). cDNA was synthesized with approximately 1 µg of total RNA in a final volume of 20 μL using PrimeScript^TM RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The qRT-PCR reactions were performed using TB Green Premix Ex TaqTM Ⅱ (TliRNaseH Plus) (TaKaRa, Dalian, China) on a Step One Plus RT-PCR system (Applied Biosystems, Waltham, MA, USA). The reaction program was described by Zhan et al. [37 (link)]. The relative expression levels of randomly selected QmbHLH genes were calculated using the 2^−∆∆Ct method with the QmUBQ gene as a reference gene [38 (link)]. The specific primers of selected genes are shown in Table S2. Three independent replications were performed for each sample.

K. pneumoniae was incubated with FLD (32 µg/mL) for 24 h at 37 °C, and the control group was cultured in an FLD-free medium. Total bacterial RNA was extracted using the RNAiso Plus kit (Takara Bio Inc.) according to the manufacturer’s instructions. RNA quantity was measured by a NanoDrop One^C spectrophotometer (Thermo Scientific, USA). Further, single-stranded cDNA was synthesized using the PeimeScript^TM reagent kit with gDNA Eraser (Takara Bio Inc.) according to the manufacturer’s instructions. qRT-PCR was performed in a QuantStudio (TM) 6 Flex System using TB Green^® Premix Ex Taq^TM Ⅱ (Tli RNaseH Plus) (Takara Bio Inc.) according to the manufacturer’s instructions. The expression of genes was assessed by qRT-PCR with the 23S rRNA gene as the reference gene. The primer sequences used in this experiment were synthesized by Tsingke Biotech and are shown in Table 1. Cycling parameters were as follows: pre-denaturation stage at 95 °C for 30 s; polymerase chain reaction stage at 95 °C for 5 s, 40 cycles at 60 °C for 34 s, and one melting curve stage at 95 °C for 15 s, followed by 65 °C for 1 min and 95 °C for 15 s. The 2^−ΔΔCt method was used to assess relative changes in gene transcription levels.