CMV Resistance testing (UL54 and UL97)
Fernando Lazaro
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Abstract
This protocol is a procedure for the study of antiviral resistance in Cytomegalovirus by NGS techniques.
The primers have been designed using https://primalscheme.com/ with the intention of covering the most relevant regions of the UL54 and UL97 genes.
Before start
Take into account that the quality of the results is greatly affected by the time from sample extraction to DNA amplification.
The protocol may fail if the protocol is performed from refrigerated samples or DNA.
Steps
Prepare Reagents
Q5® High-Fidelity DNA Polymerase (New England)* Agarose gel 1%
- Ethanol 70%
- Mag-Bind® TotalPure NGS (omega)
- Elution Buffer
Set up primer pools (UL97 and UL54)
UL97 Primers
| A | B | C | D |
|---|---|---|---|
| UL97_4_LEFT | TGCGCGCGGAAAGTCAG | UL97_4_RIGHT | CGGCATAACAGATCTTGTGGC |
| UL97_5_LEFT | CTCTGCGAGCTCTCTATCTCCT | UL97_5_RIGHT | AGCAGACAGCAGCCCGT |
| UL97_6_LEFT | TGGCGAGCAACAGCAGC | UL97_6_RIGHT | GCGCGCATGATCTCGCT |
| UL97_7_LEFT | TGCCACTTTGACATTACACCCA | UL97_7_RIGHT | TCCGACATGCAATAACGCCG |
| UL97_8_LEFT | TTTCCGACCCATGCCGCT | UL97_8_RIGHT | ATGCTCGCCCAGGAGACAG |
| UL97_9_LEFT | CATGGGTACGGAGGCGTTG | UL97_9_RIGHT | GGCCAACAGACGCTCCA |
UL97 Primers
UL54 Primers
| A | B | C | D |
|---|---|---|---|
| UL54_1_LEFT | TGCAAAAACTTGTCCTTGCGC | UL54_1_RIGHT | ATTCTGTAACCACCGGCGTG |
| UL54_2_LEFT | GTAGTTGCACACGGCCGAC | UL54_2_RIGHT | CGTCAATCTAACCTGCCGCA |
| UL54_3_LEFT | CGTAAAAGACCCGATCCCCG | UL54_3_RIGHT | TCTCGCTGCTCTTTGAGGATC |
| UL54_4_LEFT | CTTCATCGAGTGAGAGGCGC | UL54_4_RIGHT | AGGCTTTGGTGGCGCGT |
| UL54_5_LEFT | GCTTGACGGGCTCCACAAAA | UL54_5_RIGHT | GGCGCGGTTCATCAAAGACA |
| UL54_6_LEFT | TCCCGCGTTCCCACTACATA | UL54_6_RIGHT | CAACAAGTGGGTTTCGCAGC |
| UL54_7_LEFT | ATACGGCGCACAGGGTCTT | UL54_7_RIGHT | GTGTTTGAGCCCGAGGTGG |
| UL54_8_LEFT | AGTAGCAGAGGTTGTGAGCCA | UL54_8_RIGHT | GGTTCTGTGGCGGCTATGTT |
| UL54_9_LEFT | AAACGCCGTCCTGACTCGA | UL54_9_RIGHT (V2) | CTTGCAATCTGCGCCGTC |
| UL54_10_LEFT | GGATCTGCTGTCCGTCAAAGA | UL54_10_RIGHT | ATATTGCGGGTTCGGTGGTT |
| UL54_11_LEFT | TGTTGAGCTTATAGTTGGGCGA | UL54_11_RIGHT | CGGCCTTTGTGACCGGTTAC |
| UL54_12_LEFT | CCTTATACAGGTACTCGAGGCG | UL54_12_RIGHT | GTGCTACGAGACGGGAGGA |
| UL54_13_LEFT | AAGTGCAGCCCCGACCAT | UL54_13_RIGHT | GGATCACCACGTTCGGCTG |
| UL54_14_LEFT | CCTCGATATCACAAGTCGACGC | UL54_14_RIGHT | GGCGAACTAGTGCCCGAAC |
| UL54_15_LEFT | CCGTACCCGTAGATGGAGGT | UL54_15_RIGHT | GGGACCTATTCGTTTTCACACCTA |
| UL54_16_LEFT | ACGATAGCGCGGCGACA | UL54_16_RIGHT | CGGCGTCAGCGTTTGCA |
UL54 Primers
Pool 1 UL97 (odd primers LEFT and RIGHT)
Pool 2 UL97(even primers LEFT and RIGHT)
Use 10µL for each primer at 10micromolar (µM)
Pool 1 UL54 (odd primers LEFT and RIGHT)
Pool 2 UL54 (even primers LEFT and RIGHT)
Use 10µL for each primer 10micromolar (µM) except:
- UL54_14_LEFT/RIGHT 14: 2,5 µl
- UL54_5_LEFT/RIGHT: 5 µl
- UL54_13_LEFT/RIGHT: 5 µl
- UL54_15_LEFT/RIGHT: 20 µl
DNA extraction
Perform DNA extraction with your method of choice. Preferably from a plasma sample collected on the same day .Perform DNA extraction with your method of choice. Preferably from a plasma sample collected on the same day.
The quality of the results is greatly affected by the time from sample extraction to DNA amplification.
The protocol may fail if the protocol is performed from frozen samples or DNA.
DNA amplification
For each pool and sample, mix the following reagents (two reactions per sample):
| A | B |
|---|---|
| Reagent | Volume / sample (µl) |
| Primer Pool | 2 |
| Q5‱ Polymerase | 0,25 |
| Q5 Buffer | 5 |
| H20 | 5 |
| dNTP | 0.5 |
| Sample DNA | 12,5 |
PCR Mix for Pool 1 and 2
Set up the PCR with the following program
| A | B | C |
|---|---|---|
| Cycles | Temperature | Time |
| 1 | 98°C | 30’’ |
| 5 | 98°C | 10’’ |
| 65°C | 3’ | |
| 30 | 98°C | 10’’ |
| 65°C | 30’’ | |
| 72°C | 2’ | |
| 1 | 72°C | 2’ |
| 1 | 4°C | ∞ |
PCR program
Confirm amplification of aproximately 300bp fragments by 1% agarose gel .
Product cleaning with Mag-Bind TotalPure NGS (omega)
Add 40 uL of beads to 20 uL of amplicon. Mix.
Incubate the mixture for 5 minutes at room temperature.
Place the tube in the magnet until the solution becomes clear.
Gently remove the supernatant by pipette.
Add 180 uL of 70% ethanol. Mix without breaking the pellet.
Gently discard the ethanol by pipette.
Incubate for 2 minutes at room temperature.
Remove any remaining ethanol. Note: The pellet must not be allowed to dry excessively. If it does occur, the pellet will appear black and cracked.
Remove the tube from the magnet.
Add 25 uL of EB. Mix. Note: do not break the pellet, just peel it away from the wall of the tube.
Incubate for 2 minutes at room temperature.
Place the tube back in the magnet until the solution clears.
Transfer 20 uL of the supernatant to a new tube.
NGS sequencing
Sequence the amplicons using the sequencer of choice according to the manufacturer's instructions.
Antiviral resistance
Generate consensus sequence from the amplicons using the bioinformatics procedure of choice.
Take into account that in cases of previous exposure to antivirals or prolonged treatment, minority variants may appear.
The website http://cmv-resistance.ucl.ac.uk/herpesdrg/ is helpful for the study of resistance mutations.
