Individual tablets of TRUVADA (tenofovir/emtricitabine; Gilead Sciences) or raltegravir (Merck) were crushed into fine powder and manufactured as 5BXL by TestDiet based on previously published 26 (link); 27 (link). HIV-1 RNA was extracted from plasma (Viral RNA Mini Kit, Qiagen). RNA was reverse transcribed and quantitatively detected by real-time PCR using TaqMan® Fast Virus 1-Step PCR kit (ThermoFisher Scientific) and QuantStudio 6 Flex PCR system (Applied Biosystems) (detection limit ≈400 copies/ml)23 (link); 28 (link).
Taqman fast virus 1 step pcr kit
Manufactured by Thermo Fisher Scientific
The TaqMan® Fast Virus 1-Step PCR kit is a real-time PCR assay designed for the detection of viral nucleic acids. It enables fast, sensitive, and reliable detection of viral targets in a one-step RT-PCR format.
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4 protocols using taqman fast virus 1 step pcr kit
1
Quantifying HIV-1 RNA from Crushed ARV Tablets
2
Quantifying HIV-1 RNA from Plasma
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Individual tablets of TRUVADA (tenofovir/emtricitabine; Gilead Sciences) or raltegravir (Merck) were crushed into fine powder and manufactured as 5BXL by TestDiet based as previously published (Halper-Stromberg et al. 2014 ; Cheng et al. 2018 ). HIV-1 RNA was extracted from plasma (Viral RNA Mini Kit, Qiagen). RNA was reverse transcribed and quantitatively detected by real-time PCR using TaqMan® Fast Virus 1-Step PCR kit (ThermoFisher Scientific) and QuantStudio 6 Flex PCR system (Applied Biosystems) (detection limit ≈400 copies/ml) (Li et al. 2014 , 2017 ).
3
Quantitative Detection of HIV-1 RNA
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HIV-1 RNA was purified from the plasma with the QIAamp® Viral RNA Mini Kit. The RNA was then reverse transcribed and quantitatively detected by real-time PCR using the TaqMan® Fast Virus 1-Step PCR kit (ThermoFisher Scientific). The primers used for detecting the HIV Gag gene were (5′-GGTGCGAGAGCGTCAGTATTAAG-3′ and 5′-AGCTCCCTGCTTGCCCATA-3′). The probe (FAM-AAAATTCGGTTAAGGCCAGGGGGAAAGAA-QSY7) used for detection was ordered from Applied Biosystems, and the reactions were set up following the manufacturer’s guidelines and were run on the QuantStudio 6 Flex PCR system (Applied Biosystems). The detection limit of the real-time PCR reaction is four copies per reaction. Accordingly, the limit of detection of the assay with 50 µl of blood is 400 copies/ml in humanized mice.
4
Quantitative HIV-1 Detection in Plasma
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HIV-1 genomic RNA was purified from plasma by using the QIAamp Viral RNA Mini Kit. The RNA was reverse-transcribed and quantitatively detected by real-time PCR with the TaqMan Fast Virus 1-Step PCR kit (Thermo Fisher Scientific). We used the forward primer 5’-GGTGCGAGAGCGTCAGTATTAAG-3’ and the reverse primer 5’-AGCTCCCTGCTTGCCCATA-3’ to detect the HIV gag gene. The probe (FAM-AAAATTCGGTTAAGGCCAGGGGGAAAGAA-QSY7) used for detection was ordered from Applied Biosystems, and the reactions were set up following the manufacturer’s guidelines and were run on the QuantStudio 6 Flex PCR system (Applied Biosystems). The limit of detection of each real-time PCR was 4 copies per reaction. Accordingly, owing to the relatively small volume of plasma in mice (about 50–100 μL total blood), the limit of detection was 400 copies/mL plasma. The copy numbers below the detectable limit were set as 1. Triplicate reactions were performed for each sample.
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