Human T-Cell Lymphotropic Virus type I Pro Viral Load Assay

Transcription

Human T-Cell Lymphotropic
Virus type I Pro Viral Load
Assay
Stirling Dick
NRL, Melbourne, Australia
NRL and HTLV
NRL developed an HTLV I/II Proviral DNA
Real Time PCR
Multiplex PCR targeting HTLV I and II and
Albumin as the HK gene.
Ability to detect all subtypes, queried
Need for HTLV VL assay
HTLV-I Epidemiology
15 to 25 million people world wide infected
with HTLV-1
Life long infection
95% carriers remain asymptomatic
5% associated with severe diseases
Inflammatory syndromes (HTLV-1 associated
myelopathy/tropical spastic paraparesis (HAM/TSP)etc)
Neoplastic diseases (lymphoma, Adult T-cell leukaemia)
Opportunistic infections (infective dermatits, arthritis or
Strongyloides stercoralis hyperinfection etc.)
HTLV
Positive sense, single-stranded RNA virus
Retroviridae family and Deltaretrovirus
genus
HTLV-I subtypes A, B, C, D, E, F and G
HTLV-II subtypes A, B and D
HTLV-III
HTLV-IV
HTLV-I
HTLV-I subtypes
Subtype A (Caribbean, North America,
Japan)
Subtype B,D,E,F,G (Central Africa)
Subtype C (Australo-Melanesian found
in PNG, Solomon Islands and Australia)
HTLV-I Genome
HTLV-I Replication
HTLV-I infects CD4+ T cells
(CD8+ T) cells acting as a
possible reservoir
Entry is thought to involve
the glucose transporter as a
receptor.
The single stranded RNA is
converted to double
stranded DNA by reverse
transcriptase
Inserted into the host cell
genome by integrase
Phylogenetic trees for HTLV-I
Low degree of genetic variation (0.5 to 3%) in
the cosmopolitan strains of the virus
Subtype C shows a divergence of 8.5% at the
nucleotide level
Amino acid sequence varying between 3 to 11% for
the structural genes
Amino acid sequence varying between 8.5 to 25%
for the regulatory and pX regions
More closely related to HTLV-II than HTLV-I
Diagnostic assays are based on the prototype
virus HTLV-IAKT (cosmopolitan strain)
Phylogenetic trees for HTLV-I
HTLV-I transmission
HTLV remains cell associated and is
transmitted by cell to cell contact during
early infection
Modes of transmission include
Breast feeding from an infected mother
Exposure to contaminated blood products
Sexual contact
In the later stages it is replicated by
clonal expansion of the host cells by
mitosis
Diagnosis and monitoring infection
Anti-HTLV antibody assays
Proviral DNA
Aim of NRL’s HTLV work
Improve on assay sensitivity
Examine extraction efficiency
Examine HTLV-I sequences
Determine conserved primer and probe
targets
Development of a Viral Load Assay
Requirement for a VL assay
Examination of Nucleic Acid
Extraction Methods
Effect of NA extraction efficiency on an
assay
Examination of several methods
Roche MagNA Pure LC Roche MagNA Pure LC Qiagen EZ1 Qiagen QIACube –
DNA Isolation Kit
TNA Isolation Kit
DNA Blood Kit
DNA Blood Mini Kit
Automated Platforms
Increase standardisation
Ease of use
Extraction Methods; Extraction
Technologies
Magnetic glass Particle
Silica spin column
Extraction Methods; Samples
Cultured 8E5 cells containing 1 copy of HIV-1
DNA per cell and 2 copies of the albumin gene.
A series of 10 dilutions was made from 1x101 to
1x107 8E5 cells/mL
Reproducibility; 10 Replicates of 5x102, 1x105,
1x107 8E5 cells/mL
Quality;3 Replicates of 5x102, 1x105, 1x107 8E5
cells/mL in 5x109 erythrocytes / mL
Post extraction qPCR on Stratagene Mx 3000p
Extraction; Results
Dilution series on each platform
Average Albumin Ct of platform vs Cell count
45
35
40
Ct Value
40
30
25
Qiacube. QIAmp DNA Blood Mini
Mag NA Pure DNA Isolation
EZ1 DNA Blood
Mag NA Pure TNA Isolation
20
35
30
Qiacube. QIAmp DNA Blood Mini
Mag NA Pure DNA Isolation
EZ1 DNA Blood
Mag NA Pure TNA Isolation
25
15
Cells/mL
7
10
6
1
X
10
5
X
1
1
X
10
4
10
3
X
1
1
X
10
X
5
10
2
2
10
2
X
5
2.
1
X
10
1
10
1
5
X
10
0
X
1
7
10
6
X
1
X
1
1
10
5
10
4
Cells/mL
X
10
3
X
1
1
X
10
X
5
10
2
2
10
2
X
5
2.
X
10
1
10
1
X
5
X
10
1
0
20
1
Average Ct
Average HIV-1 Ct of platform vs Cell count
Extraction; Results
Replicates
HIV-1 Ct using different extraction platforms
Albumin Ct using different extraction platforms
50
40
5 x 10^2
1 x 10^5
1 x 10^7
5 x 10^2
1 x 10^5
1 x 10^7
40
Albumin Ct
HIV-1 Ct
30
30
20
20
10
10
0
QiaCube
MagNA Pure DNA
EZ1
MagNA Pure TNA
Extraction Platform
0
QiaCube
MagNA Pure DNA
EZ1
MagNA Pure TNA
Extraction Platform
Extraction; Results
Replicates
DNA recovered using different extraction platforms
1000
5 x 10^2
1 x 10^5
1 x 10^7
DNA ug/ml
100
10
1
0.1
0.01
QiaCube
MagNA Pure DNA
EZ1
MagNA Pure TNA
Extraction Platform
Extraction; Results
Blood
HIV-1 Ct using different extraction platforms
Albumin Ct using different extraction platforms
50
30
5 x 10^2
1 x 10^5
1 x 10^7
25
Albumin Ct
HIV Ct
40
30
20
10
5 x 10^2
1 x 10^5
1 x 10^7
20
15
10
5
0
0
QiaCube
MagNA Pure DNA
EZ1
MagNA Pure TNA
Extraction Platform
QiaCube
MagNA Pure DNA
EZ1
MagNA Pure TNA
Extraction Platform
Extraction; Conclusions
The Qiagen QIA Cube results suggest
slightly better efficiency and a greater
dynamic range
Good reproducibility amongst each
platform
Good quality extract production amongst
each platform
Examination of HTLV-I Sequences
NCBI BLAST search conducted comparing
as many HTLV-I subtypes as possible
Data base contains approximately 20 full
length HTLV-I genomes & several partial
sequences
Compare HTLV-II sequence for future
multiplexing assay
Sequencing HTLV-I Gag region
Gag region selected for sequencing
Design primers to amplify region of
interest
Determine if amplification has produces a
amplicon of the correct size 760bp
Agarose Gel Electrophoresis
Results 760 bp amplicon visible
1
2
3
4
5
6
7
Lane 1 100bp ladder
Lane 2 MT2 Extract +
1500bp
1000bp
Lane 3 PBMC Extract Lane 4 Z370093
500bp
Lane 5 Z365967
Lane 6 Z379025
Lane 7 Neg Extract
Sequencing HTLV-I Gag region
Design sequencing primers
Sequence region of interest using BDT
Produce electropherograms
Align sequenced region to determine
highly conserved areas
HTLV-I Gag region
5’GCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCTCTGC
CGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTA
AGTTCGGAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTA
GAGCCCACTTAGATTCGGTCGGCTCTCCACGCTTTGCCTGACCCTGCT
TGCTCAACTCCACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGCTACCAA
TCGAAAGTTCCGCCCCCTTCCCTTTCATTCACGTCTGATTGCCGGCTT
GGCCCACGGCCAAGCACCGGCACCTCTACTGGCCCGGAGCCAGTGA
CAGCCCATTCTATACCTCTCTCCAGGAGAGAAACATAGAACACAGTTG
GGGGCTCGTCCGGGATTCTAACGAGCGCCCCTTTATTCCCTAGGCAAT
GGGCCAAATCTTCCCTCGTAGCGCTAACCCTATCCCGCGGCCGCCCC
GGGGGCTGGCCACTCATCACTGGCTTAATTTCCTCCAGGCGGCATAC
CGCCTGGAGCCCGGTCCTTCCAGTTACGATTTTCATCAGTTAAAAACA
GTTCTTAAAATGGCCCTAGAAACGCCAGTCTGGATGTGTCCCATTAACT
ACTCCCTCCTAGCTAGTCTACTCCCAAAAGGATACCCTGGCCAGGTAA
ATGAAATTTTACAGGTACTCATCCAAACCCAAACCCAGATCCCGTCCCA
CCCCGCGCCGCCGCCGCCGTCATCCCCCACCCACGACCCCCCAGATT
CTGATCCACAGATCCCCCCTCCCTATGTTGAGCCTACAGCCCCCCAAG
TCCTTCCAGTCATGCACCCACACGGTGTCCCTCCCACGCACCGCCCG
TGGCAAATGAAAGACCTACAGGCCTATTAAGCAGGAAGTCTCC 3’
Forward and reverse Gag region primers RED GRN
Forward sequence primers 1 & 2 PNK
Sequence Alignment
Z365967
Z370093
gi|50404473|gb|AY563954.1|
gi|254273153|dbj|AB513134.1|
gi|1488238|gb|L02534.1|HTVMEL5
Z379025
CACGCTTTGCCTGACCCTGCTTGCTCAACTCTGCGTCTTTGTTTCGTTTT
CACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTT
CACGCTTTGCCTGACCCTGCTTGCTCAACTCCACGTCTTTGTTTCGTTTT
CACGCTTTGCCTGACCCTGCTTGCTCAACTCCACGTCTTTGTTTCGTTTT
57
66
481
599
597
69
Z365967
Z370093
gi|50404473|gb|AY563954.1|
gi|254273153|dbj|AB513134.1|
gi|1488238|gb|L02534.1|HTVMEL5
Z379025
CTGTTCTGCGCCGCTACAGATCGAAAGTTCCACCCCTTTCCCTTTCATTC
CTGTTCTGCGCCGTTACAGATCGAAAGTTCCACCCCTTTCCCTTTCATTC
CTGTTCTGCGCCGCTACCAATCGAAAGTTCCGCCCCCTTCCCTTTCATTC
CTGTTCTGCGCCGCTACCGATCGAAAGTTCCGCCCCTTTCCCTTTCATTC
107
116
531
649
647
119
Z365967
Z370093
gi|50404473|gb|AY563954.1|
gi|254273153|dbj|AB513134.1|
gi|1488238|gb|L02534.1|HTVMEL5
Z379025
ACGACTGACTGCCGGCTTGGCCCACGGCCAAGTACCGGCGACTCCGTTGG
ACGACTAACTGCCGGCTTGGCCCACGGCCAAGTGCCGGCGACTCCGTTGG
ACGTCTGATTGCCGGCTTGGCCCACGGCCAAGCACCGGCACCTCTACTGG
ACGTCTGACTGCCGGCTTGGCCCACGGCCAAGCACCGGCACCCTTACTGG
157
166
581
699
697
169
Z365967
Z370093
gi|50404473|gb|AY563954.1|
gi|254273153|dbj|AB513134.1|
gi|1488238|gb|L02534.1|HTVMEL5
Z379025
CTCGGAGCCCGGCGACAGCCCATTCTATAGCA-CTCTCCAGG-AGAGAAA
CTCGGAGCC-AGCGACAGCCCATTCTATAGCAACTCTCCAGG-AGAGAAA
CTCGGAGCC-AGCGACAGCCCATTCTATAGCA-CTCTCCAGG-AGAGAAA
CTCGGAGCC-AGCGACAGCCCATCCTATAGCA-CTCTCCAGG-AGAGAAA
CCCGGAGCC-AGTGACAGCCCATTCTATACCT-CTCTCCAGG-AGAGAAA
CTCGGAGCC-AGCGACAGCCCATTCTATACCT-CTCTCCAGGGAGAGAGA
205
214
628
746
744
217
Designing the Assay Target Region;
Primers and probe
Determine suitability of Gag region as
assay target
Based on alignment of the sequenced Gag
region and data base sequences
Design assay 15-30 bp primer and probe
set over a highly conserved region of 50–
150bp
Design considerations, Tm, primer dimers,
GC clamps, GC content, unintentional PCR
amplicons etc.
Assay Primers and Probe
5’GCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCTCTGC
CGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTA
AGTTCGGAGCTCAGXXXXXXXXXXXXCCTTTGTCXXXXXXXXXXTTAGA
GCCCACTTAGATTCGGTCGGCTCTCCACGCTTXXXXXXXXXXXXXXXG
CTCAACTCCACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGCTACCAATC
GAAAGTTCCGCCCCCTTCCCTTTCATTCACGTCTGATTGCCGGCTTGG
CCCACGGCCAAGCACCGGCACCTCTACTGGCCCGGAGCCAGTGACAG
CCCATTCTATACCTCTCTCCAGGAGAGAAACATAGAACACAGTTGGGG
GCTCGTCCGGGATTCTAACGAGCGCCCCTTTATTCCCTAGGCAATGGG
CCAAATCTTCCCTCGTAGCGCTAACCCTATCCCGCGGCCGCCCCGGG
GGCTGGCCACTCATCACTGGCTTAATTTCCTCCAGGCGGCATACCGCC
TGGAGCCCGGTCCTTCCAGTTACGATTTTCATCAGTTAAAAACAGTTCT
TAAAATGGCCCTAGAAACGCCAGTCTGGATGTGTCCCATTAACTACTC
CCTCCTAGCTAGTCTACTCCCAAAAGGATACCCTGGCCAGGTAAATGA
AATTTTACAGGTACTCATCCAAACCCAAACCCAGATCCCGTCCCACCC
CGCGCCGCCGCCGCCGTCATCCCCCACCCACGACCCCCCAGATTCTG
ATCCACAGATCCCCCCTCCCTATGTTGAGCCTACAGCCCCCCAAGTCC
TTCCAGTCATGCACCCACACGGTGTCCCTCCCACGCACCGCCCGTGG
CAAATGAAAGACCTACAGGCCTATTAAGCAGGAAGTCTCC 3’
HTLV-I FWD Primer CAGXXXXXXXXXXXXCCT
HTLV-I REV Primer CTTXXXXXXXXXXXXXXXGCT
HTLV-I Probe
[6FAM] GTCXXXXXXXXXXXXXXXGCC[BHQ1]
Testing Primer and Probe Sets
Optimise q PCR reaction.
Run HTLV-I Primers and Probes as a
uniplex
Run HTLV-I Primers and Probes as a
multiplex
HTLV-I Quantification; Dilution
Series
Preparation of a dilution series 1x106 –
1x101 SP cells/mL
SP Cell contain 1 copy of HTLV-I
provirus/cell & 2 copies of Alb gene/cell
Assume a similar extraction efficiency for
samples and dilution series
HTLV-I Quantification; Proviral Load
Samples will generate a Ct value for
HTLV-I and Alb; generate standard curve
HTLV-I Ct will indicate the number of
proviral copies in the sample
Alb Ct will indicate the number of
leukocytes in the sample
Normalise proviral load by expressing as
copies / number of leukocytes
Assay Validation
Sensitivity
Dynamic range
Subtype detection
Specificity
False reactivity
Reproducibility
Inter Assay
Intra Assay
Controls
Positive – Standards ( cell culture )
Negative
Acknowledgments
NHMRC Project Grant
Dr. Lloyd Einsiedel, provision of samples
Dr. Kim Wilson and staff at NRL, provision
of technical support

Human T-Cell Lymphotropic Virus type I Pro Viral Load Assay

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