DNA in E. coli was isolated with reference to the Molecular Cloning (Green and Sambrook 2012 ). Bacterial RNA was extracted by RNAprep Pure Cell/Bacteria Kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Then, reverse transcription of RNA was conducted to generate complementary DNA (cDNA) using PrimeScript RT reagent Kit with genomic DNA (gDNA) Eraser (Takara Bio Inc., Dalian, China). The abundances of ampR and efflux pump gene (acrA) in DNA and cDNA were quantified by quantitative real-time polymerase chain reaction (qPCR) using SuperReal PreMix (Probe) Kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) on LightCycler 96 platform (Roche, Basel, Switzerland). Here, multidrug efflux pump AcrAB-TolC encoded gene acrA was chosen as it can pump out a broad range of antibiotics from cell compartment and plays a vital role in the development of antibiotic resistance in bacteria (Nolivos et al. 2019 (link); Poole 2005 (link); Xuan et al. 2017 (link)). The abundance of ARGs was calculated as the ARGs copies divided by the total concentration of DNA for ampR (copies/ng DNA) or RNA for acrA (copies/ng RNA) (Hu et al. 2021 (link); Ruiying et al. 2021 (link)). Detailed descriptions of DNA extraction, RNA reverse transcription, and qPCR test are presented in the Supporting Information (SI) (S1-S4) .
Superreal premix probe kit
Manufactured by Tiangen Biotech
Sourced in China
The SuperReal PreMix (Probe) Kit is a reagent kit designed for real-time PCR (polymerase chain reaction) analysis. The kit contains a pre-mixed solution that includes all the necessary components for the amplification and detection of target DNA sequences using fluorescent probe-based detection methods.
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Lab products found in correlation
2 protocols using superreal premix probe kit
1
Quantifying Antibiotic Resistance Genes in E. coli
2
Quantitative DNA Analysis of Gut Microbiome
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Total DNA was extracted from cecum digesta with the Stool DNA Kits (Omega Bio-Tek, Doraville, CA, USA), executed by real-time quantitative PCR using the CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA). The total bacteria application program entailed 95 °C for 25 s, followed by 40 cycles of 95 °C for 5 s and 64.5 °C for 25 s. Lactobacillus, E. coli, Bacillus and Bifidobacterium were tested using the SuperReal PreMix (Probe) kit (Tiangen Biotech Co., Ltd., Beijing, China). All results are included for 95 °C for 15 min, followed by 50 cycles of 95 °C for 3 s, and 53 °C for 25 s. All primers used are showed in Table S3 .
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