Aceq qpcr probe master mix

Manufactured by Vazyme

Sourced in China

AceQ qPCR Probe Master Mix is a pre-mixed reagent for quantitative real-time PCR (qPCR) using probe-based detection. It contains all the necessary components, including DNA polymerase, dNTPs, and buffers, to perform qPCR experiments.

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Lab products found in correlation

Cfx96 real time pcr cycler, by Bio-Rad (2 mentions) Aceq qpcr probe master mix kit, by Vazyme (2 mentions) Hiscript 2 q rt supermix, by Vazyme (2 mentions) Cfx96 c1000 thermal cycler, by Bio-Rad (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions) Hiscript 3 1st strand cdna synthesis kits gdna wiper, by Vazyme (2 mentions) Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (2 mentions) Phanta high fidelity dna polymerase, by Vazyme (1 mentions) Nebuffer 2, by New England Biolabs (1 mentions) Twistamp basic kit, by Twist Bioscience (1 mentions) T7 rna polymerase, by New England Biolabs (1 mentions) Tailing reaction, by Sangon (1 mentions) Cfx96 system, by Bio-Rad (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Quantstudio 1 real time pcr system, by Thermo Fisher Scientific (1 mentions) Quantstudio real time pcr, by Thermo Fisher Scientific (1 mentions) Abi steponeplustm real time pcr system, by Thermo Fisher Scientific (1 mentions) Quantstudio 5 system, by Thermo Fisher Scientific (1 mentions) Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) Lightcycler 96 instrument, by Roche (1 mentions)

26 protocols using aceq qpcr probe master mix

Probe-based qPCR for Fusobacterium quantification

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Probe-based qPCR was performed using 2×AceQ qPCR Probe Master Mix (Vazyme, Nanjing, China) in a reaction volume of 30 μl, with 15 μl AceQ qPCR Probe Master Mix, 0.6 μl forward/reverse primer for Fn DNA (10 μm), 0.3 μl TaqMan probe for Fn DNA (10 μm), 0.6 μl forward/reverse primer for reference genes (5 μm), 0.3 μl TaqMan probe for reference genes (5 μm), and 10.5 μl DNA. The primers and TaqMan probes were designed based on the nusG gene of Fn and human reference genes (GAPDH, RPPH1, NAGK, TERT, ERV-3, and SLC O 2A1), and their sequences were shown in Table S4. PCR amplification was on a Bio-Rad CFX96 real-time PCR cycler (Bio-Rad, Hercules, CA, USA), with an initial denaturation step at 95°C for 5 min, and 40 cycles at a melting temperature of 95°C for 10 s and an annealing temperature of 60°C for 30 s. All assays were conducted in triplicate. No-template reactions were performed as negative controls. The PCR products were confirmed by sanger sequence (Figure S1).
GAPDH and TERT were selected as reference genes for normalizing qPCR as described in result part. The relative level of Fn DNA was recorded as the ratio of Q (Fn DNA) to the geometric mean of Q (GAPDH) and Q (TERT). Q was calculated according to the following formula: (Effiency+1)^-ΔCq, where △Cq = [Cq (test)-Cq (calibrator)]. The effiencies of Fn DNA, GAPDH and TERT amplification were shown in Figure 2.

Zhang X., Zhang Y., Gui X., Zhang Y., Zhang Z., Chen W., Zhang X., Wang Y., Zhang M., Shang Z., Xin Y, & Zhang Y. (2022). Salivary Fusobacterium nucleatum serves as a potential biomarker for colorectal cancer. iScience, 25(5), 104203.

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Comprehensive Molecular Diagnostic Assay

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All primers were ordered in Sangon Biotech (Shanghai, China), and the detailed sequences were listed in Supplementary Table S3. FAM-TTATT-Quencher used in fluorescent reporter assay was synthesized by Integrated DNA Technologies (IDT); FAM-TTATT-Biotin used in lateral flow strip test and probe in qPCR assay were ordered in TaKaRa Bio Inc. (Dalian, China). T7 RNA polymerase and NEBuffer 2.1 were purchased from New England Biolabs (MA, USA). The TwistAmp® Basic kit and Milenia HybriDetect 1 were purchased from TwistDx (Cambridge, UK). 2× AceQ qPCR Probe Master Mix and Phanta High-fidelity DNA polymerase were purchased from Vazyme (Nanjing, China).

Lu S., Li F., Chen Q., Wu J., Duan J., Lei X., Zhang Y., Zhao D., Bu Z, & Yin H. (2020). Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics. Cell Discovery, 6, 18.

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Optimizing Multiplex qPCR Assay Development

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The optimal reaction conditions were determined according to a previous report [33 (link)]. In brief, the 10-fold diluted plasmids constructed above from 3 × 10⁷ copies/μL to 3 × 10⁴ copies/μL were amplified as the templates. Firstly, a mixture of three diluted plasmids with a concentration of up to 1 × 10⁴ copies/μL was prepared. Secondly, four mixed pairs of customized primers and probes were added to one reaction condition and set to determine the optimal work concentrations for the development of this assay. The final volume of 20 μL included 10 μL 2 × AceQ qPCR Probe Master Mix (Vazyme, Nanjing, China), 0.1–1.8 μL of each of the primers, 0.1–0.8 μL of each of the probes, and 2 μL of templates. Thirdly, FAM, HEX, Texas Red, and Cy5 were configured as the fluorescence channels for the real-time qPCR instrument’s four fluorescence channels. Finally, a commercialized real-time PCR instrument (IDEXX, Westbrook, ME, USA) was used to collect the fluorescence signals.

Liu H., Zou J., Liu R., Chen J., Li X., Zheng H., Li L, & Zhou B. (2023). Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022. Veterinary Sciences, 10(2), 106.

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Plasma miRNA Extraction and Quantification

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miRNAs in plasma samples were extracted using a plasma miRNA isolation kit (TIANGEN Biotech, DP503) according to the manufacturer’s instructions. The isolated miRNAs were stored in nuclease-free water at − 80 ^∘C until needed. Synthetic or extracted miRNAs were first reverse transcribed into first strand cDNA using a miRNA first strand cDNA synthesis kit (Tailing Reaction) (Sangon Biotech, B432451). Reverse transcription was carried out with a total 20 µL volume containing 10 µL

2 \times

miRNA P-RT Solution mix, 2 µL miRNA P-RT Enzyme mix, 100 ng Extracted miRNAs, and add RNase-free water to 20 µL. The reverse transcription reactions were kept at 37 ^∘C for 60 min, and then at 85 ^∘C for 5 min on a Bio-rad CFX96 system. Asymmetric PCR was performed in a 20 µL system including 10 µL

2 \times

AceQ qPCR Probe Master Mix (Vazyme, Q112), 2 µL cDNA, 1 µM excess primer (specific to miRNAs), 25 nM limiting primer (universal primer), 200 nM Taqman probe (if needed), and add RNase-free water to 20 µL. The suitable cycling condition was 95 ^∘C for 5 min, 10 cycles of 95 ^∘C for 10 s and 55 ^∘C for 30 s, followed by 42 cycles of 10 s at 95 ^∘C and 30 s at 50 ^∘C.

Zhang L., Liu Q., Guo Y., Tian L., Chen K., Bai D., Yu H., Han X., Luo W., Feng T., Deng S, & Xie G. (2024). DNA-based molecular classifiers for the profiling of gene expression signatures. Journal of Nanobiotechnology, 22, 189.

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Quantifying Fusobacterium DNA in CRC

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DNA was extracted from CRC tissues using the QIAGEN DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), and amplified using 2×AceQ qPCR Probe Master Mix (Vazyme, Nanjing, China). The level of Fn DNA was calculated using the 2^−ΔCt method, in which ΔCt refers to the difference in Ct values between nusG gene of Fn and human reference gene SLCO2A1.

Zhang M., Wang Y., Yu L., Zhang Y., Wang Y., Shang Z., Xin Y., Li X., Ning N., Zhang Y, & Zhang X. (2023). Fusobacterium nucleatum promotes colorectal cancer metastasis by excretion of miR-122-5p from cells via exosomes. iScience, 26(9), 107686.

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Quantitative RT-PCR for Nipah Virus Detection

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qRT-PCR was performed using the AceQ qPCR Probe Master Mix (Vazyme, Nanjing, China) on the QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific, Shanghai, China), following manufacturer recommendations.
Briefly, the reaction system contained 10 μL of 2 × AceQ qPCR probe, 0.6 μL of 10 μM Nipah-qp forward primer, 0.6 μL of 10 μM Nipah-qp reverse primer, 0.4 μL of 10 μM Nipah-qp probe, 7.4 μL of RNase-free ddH₂O, and 1 μL of sample DNA. The amplification conditions used included an initial denaturation step of 95 °Cfor 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s.

Miao J., Zuo L., He D., Fang Z., Berthet N., Yu C, & Wong G. (2023). Rapid detection of Nipah virus using the one-pot RPA-CRISPR/Cas13a assay. Virus Research, 332, 199130.

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Multiplex qPCR for Simultaneous Detection of Avian Leukosis Viruses

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The multiplex qPCR was performed with a 10 μL AceQ^® qPCR Probe Master Mix (Vazyme Biotech) combined with all primers, probes, templates, and nuclease-free water to a final volume of 20 μL. Primer and probe concentrations for ALV-A, ALV-B, ALV-J, and ALV-K were optimized to enable the simultaneous detection of all four viruses with minimum Cq values. In addition, the optimum reaction temperature was tested from 55 °C to 65 °C, with a temperature gradient of 1 °C. The optimal conditions were defined as those that allowed for the simultaneous detection of all four viruses with minimum Cq values. All qPCRs were conducted by QuantStudio^™ Real-Time PCR (ABI, Natick, MA, USA).

Dou J., Wang Z., Li L., Lu Q., Jin X., Ling X., Cheng Z., Zhang T., Shao H., Zhai X, & Luo Q. (2023). A Multiplex Quantitative Polymerase Chain Reaction for the Rapid Differential Detection of Subgroups A, B, J, and K Avian Leukosis Viruses. Viruses, 15(9), 1789.

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Real-Time PCR Optimization and Quantification

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Real-time PCR tests were conducted using the plasmids as standards. The real-time PCR was optimized using primer and probe volumes of 1.0, 0.5, 0.3, 0.2, and 0.1 μL and annealing temperatures of 50, 55, 60, and 65°C. Each reaction had a total volume of 20 μL, including 10 μL of the AceQ ® qPCR Probe Master Mix (Vazyme, Nanjing, China), 1 μL of the standard plasmids, and optimized volumes of 10 μmol/L for each probe and two primers. The remaining 20 μL of the reaction was made up with nuclease-free water. Using the ABI StepOnePlus TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), amplification and quantification processes were carried out under the following conditions: 2 min at 37°C, 30 s at 95°C, 40 cycles of 10 s at 95°C, and 30 s at the optimal annealing temperature. The sample was deemed negative if no cycle threshold (CT) was identified after 40 amplification cycles. The initial plasmid standards comprising 10-fold serial dilutions (5.94×10 2 to 5.94×10 7 copies/μL) were used to create the standard curve. Each dilution was performed in triplicate.

Lin A., Hu X., Cui S., Yang T., Zhang Z., Li P., Guo M, & Lu Y. (2023). Development of TaqMan-based real-time PCR assay based on the E1 genefor the quantitative detection of the Getah virus. Polish journal of veterinary sciences, 26(1).

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ASFV B646L Gene Detection by qPCR

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The TaqMan real-time PCR detection of the ASFV B646L gene was carried out using a Quant Studio 5 system (Applied Biosystems, Massachusetts, USA) according to the OIE-recommended procedure described previously^{23 (link)} with the following modifications. Briefly, single-tube PCRs were prepared containing 10 μL of 2× AceQ qPCR Probe Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 0.4 μL of primer F (5′-ATAGAGATACAGCTCTTCCAG-3′, 10 μM), 0.4 μL of primer R (5′-GTATGTAAGAGCTGCAGAAC-3′, 10 μM), 0.2 μL of TaqMan probe (5′-FAM-TATCGATAAGATTGAT-MGB-3′, 10 μM), 2 μL of DNA, and 7 μL of ddH₂O. The amplification conditions used were an initial denaturation step of 95 ^oC for 3 min, followed by 45 cycles of 95 ^oC for 15 s, 52 ^oC for 10 s, and 60 ^oC for 35 s. Fluorescence information was collected at the 60 ^oC annealing extension per cycle. The cycle value (Ct) ≤ 38.0 was judged as ASFV positive.

Wang X., Ji P., Fan H., Dang L., Wan W., Liu S., Li Y., Yu W., Li X., Ma X., Ma X., Zhao Q., Huang X, & Liao M. (2020). CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus. Communications Biology, 3, 62.

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Quantifying DNA Methylation by MethyLight

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Methylation of Alu and LINE-1 was quantified by MethyLight, a methylation-specific, probe-based, real-time PCR technique [11 (link)]. PCR primers and probes are listed in Table 1. A total of 20 μL MethyLight PCR was carried out in 10 μL of AceQ™ qPCR Probe Master mix (Vazyme, Nanjing, China), 1 pmol each forward and reverse primers, 0.1 mM TaqMan probe, 0.4 μL of ROX Reference Dye 2, 50 ng of bisulfite-treated genomic DNA, and water using the following PCR program: 95 ºC for 5 minutes and then 40 cycles of 95 ºC for 10 s followed by 60 ºC for 34 s. Assays were run on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Reactions were performed in duplicate with negative controls on each plate, and the average Ct value was used in the calculations. The quantified value of DNA methylation of a target gene was normalized by β-actin. Relative copy numbers of Alu and LINE-1 were calculated according to the formula [2^-ΔCt, (ΔCt = Ct_Alu/LINE-1 - Ct_β-actin)].

Lu S., Niu Z., Chen Y., Tu Q., Zhang Y., Chen W., Tong W, & Zhang Z. (2018). Repetitive Element DNA Methylation is Associated with Menopausal Age. Aging and Disease, 9(3), 435-443.

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