Quantifying the Replication Efficiency of Wild-Type and Mutant Viruses

摘要

Source: Manocheewa, S., et al. Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses. J. Vis. Exp. (2015)

This video demonstrates the use of reverse transcription quantitative PCR (RT-qPCR) to quantify the replication efficiency of wild-type and mutant viruses following infection of human blood immune cells.

方案

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Determine the viral variant ratio using quantitative polymerase chain reaction (qPCR)
   1. Prepare a standard serial dilution series in triplicate, diluting 10-fold in each step from 3 x 10⁶ copies/µl to 30 copies/µl of plasmid pNL4-3VifA.
   2. Set up a 96-well qPCR reaction plate. Ensure that each plate contains at least one negative control, the standard dilution series in triplicate, and duplicates of each complementary deoxyribonucleic acid (cDNA) sample.
   3. For each qPCR reaction, use 12.5 µl of qPCR master mix, 0.2 µM probe, 0.8 µM each of the forward and reverse primers and 1 µl of cDNA or the standard dilution series or water/buffer (for the negative control wells). Add water to a final volume of 25 µl. The qPCR probe is light-sensitive; keep it in a closed container.
   4. Use the VifA primer-probe to detect signals in the negative controls and the standard dilution series, and with one duplicate of the cDNA sample. Use the VifB primer-probe with the other duplicate of the cDNA sample.
   5. Set PCR cycling parameters to 50 °C for 2 min, then 95 °C for 10 min, and then 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Consult the manufacturer’s support document for the operation of the qPCR machine.
   6. Calculate the standard curve using amplification data from the standard dilution series. Compare amplification data of the cDNA samples to the standard curve to determine copy number. Consult the manufacturer’s support document for data processing.
   7. Use the growth rate calculation (GRC) web tool to calculate relative viral fitness.
      NOTE: The GRC tool accepts cDNA copy numbers or chromatogram peak heights from at least two time points as input, and outputs the net growth rate difference between the two viruses. While the tool can calculate the net growth rate from two time point data, it is strongly recommended to input data from three or more time points. Use only data obtained from time points within the exponential growth phase to obtain accurate growth rates.

材料

本文使用的材料清单

姓名	公司	目录编号	评论
pNL4-3VifA and pNL4-3VifB plasmid			Please contact Dr. James I Mullins (jmullins@uw.edu)
Deoxyribonucleoside triphosphate (dNTP)	Bioline	Bio-39026
Thermal cycler	Life Technologies		Applied Biosystems® GeneAmp® PCR system 9700
Real-time quantitative polymerase chain reaction (qPCR) machine	Life Technologies		Applied Biosystems 7300 Real-Time PCR
TaqMan® Gene expression master mix	Life Technologies	4369016
Forward, reverse primer	IDT		Custom order
Probe	Life Technologies		Custom order
Optical 96 well reaction plate	Life Technologies	I19N3Q216