WO2013116039A1

WO2013116039A1 - Amplification primers and probes for detection of hiv-1

Info

Publication number: WO2013116039A1
Application number: PCT/US2013/022503
Authority: WO
Inventors: Thomas Leitner
Original assignee: Advanced Liquid Logic Inc
Priority date: 2012-01-31
Filing date: 2013-01-22
Publication date: 2013-08-08
Also published as: EP2809784A4; EP2809784A1

Abstract

The present invention relates to amplification oligonucleotides (primers) and detection probes for the detection of human immunodeficiency virus type-1 (HIV-1) in a test sample. The primers and probes may be used in an assay for the detection and/or quantitation of HIV-1 nucleic acid. In various embodiments, the primers and detection probes may be used for real-time reverse transcription polymerase chain reaction (RT-PCR) assays for detection of HIV-1 nucleic acid. The detection probes may, for example, be fluorescently-labeled TaqMan® probes. In one embodiment, amplification primers and probes may be specific for the pol region of the HIV-1 genome. In another embodiment, the amplification primers and probes may be specific for the env region of the HIV-1 genome. "Cocktails" of multiple amplification primers and/or probes may be used to achieve maximal coverage of different HIV-1 subtypes.

Description

Amplification Primers and Probes for Detection of HIV-1

Government Interest

This invention was made with government support under Contract No. HHSN272200900030C awarded by National Institutes of Health. The United States Government has certain rights in the invention.

Background

Human immunodeficiency virus type 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV- 1 shows considerable genetic variability among different isolates. A combination of a rapid replication cycle, replication errors, high viral loads and in vivo selective pressure all contribute to the genetic diversity of HIV- 1. Four groups (M, O, N and P) of HIV-1 have been described based on their genetic relatedness. Group M is further divided into 9 subtypes (A, B, C, D, F, G, H, J and K) and circulating recombinant forms (CRFs).

Detection of HIV- 1 using a variety of assays and reagents has been described. In one example, nucleic acid-based assays such as reverse transcriptase polymerase chain reaction (RT-PCR) assays have been used to amplify HIV- 1 nucleic acid for early detection of HIV- 1 infection. However, the genetic variability of HIV- 1 presents particular challenges for diagnostic RT-PCR assays which may be limited by their inability to detect all genotypes with equal efficiency. The selection of oligonucleotides to be used as primers and probes in the amplification and detection of nucleic acid sequences is critical for the sensitivity and specificity of the assay. Consequently, there is a need for oligonucleotide primer sets and detection probes for specifically and sensitively amplifying and detecting all HIV-1 groups as wells the various subtypes within or derived from these groups.

Brief Description of the Drawings

Figure 1 shows an example of a plot of genetic variation across the HIV genome;

Figures 2A and 2B show examples of a plot of entropy in region 3000-4000 and a schematic diagram of the landmarks of the HIV- 1 genome, respectively; Figures 3A and 3B show examples of a plot of entropy in region 5000-6000 and a schematic diagram of the landmarks of the HIV- 1 genome, respectively;

Figures 4A and 4B show examples of a plot of entropy in region 9000-10000 and a schematic diagram of the landmarks of the HIV- 1 genome, respectively;

Figure 5 illustrates a table of PCR forward and reverse primer sequences for sub-regions poll . l, pol2.1, env 1.1, and env 1.3; and

Figure 6 illustrates a table of probe constructs for sub-regions poll . l, poll .2, pol2.1, and envl . l .

Description

The present invention provides amplification oligonucleotides (primers) and detection probes for the detection of human immunodeficiency virus type-1 (HIV- 1) in a test sample. The primers and probes may be used in an assay for the detection and/or quantitation of HIV- 1 nucleic acid. In various embodiments, the primers and detection probes may be used for real-time reverse transcription polymerase chain reaction (RT-PCR) assays for detection of HIV- 1 nucleic acid. The detection probes may, for example, be fluorescently-labeled TaqMan® probes. In one embodiment, amplification primers and probes may be specific for the pol region of the HIV- 1 genome. In another embodiment, the amplification primers and probes may be specific for the env region of the HIV- 1 genome. Amplification primers and probes of the invention may be used to detect all subtypes of HIV-1, including groups M, N, and O, as well as major circulating recombinant forms. "Cocktails" of multiple amplification primers and/or probes may be used to achieve maximal coverage of different HIV- 1 subtypes.

4.1 Genetic Variation of HIV-1

HIV- 1 is one of the most variable organisms known to man. HIV- 1 evolves at a rate a million times faster than humans and other higher organisms. This high rate of evolution is determined by a combination of replication errors, large population size within a human host, a rapid replication cycle, and strong selection pressures. As the selection pressure varies in mode and strength across the genome, the resulting genetic variation varies significantly across HIV- l 's genome. Figure 1 shows an example of a plot 100 of genetic variation across the HIV genome. The Shannon entropy for all HIV-1/CPZ full genome sequences in the 2009 'web alignment' (www.hiv.lanl.gov) was calculated by a bias-corrected maximum likelihood method¹. Each class (subtype, group, or recombinant form) with >10 sequence representatives has its own line, and the remaining sequences are combined into "others". The entropy was calculated as the mean in a window of 25 nt (i.e., the approximate size of a primer) spaced 100 nt apart. The following HIV-1 classes (groups, subtypes, and recombinants) were considered in this analysis:

[1] "A" "Α "A2" "B" "C" "D" "Fl"

[8] "F2" "G" "H" "J" "K" "U" "01_AE"

[15 02 AG" "03 AB" "04_cpx" "05 DF" "06_cpx" "07 BC" "08 BC

[22 09_cpx" "10 CD" "ll cpx" "12 BF" "13_cpx" "14 BG" "15 01B

[29 A2D" "17 BF" "18_cpx" "19_cpx" "20 BG" "21 A2D" "22 01A1

[36 '23 BG" "24 BG" "25_cpx" "26 AU" "27_cpx" "28 BF" "29 BF

[43 31 BC" "32 06A1" "33 01B" "34 01B" "35 AD" "36_cpx" "37_cpx'

[50 "38 BF1" "39 BF" "40 BF" "42 BF" "43 02G" "44 BF" "45_cpx"

[57 '46 BF" "0102A" "01A1" "01ADF2" "01AF2U" "01B" "01BC

[64 "01C" "01DU" "0206" "0209" "0225" "02A" "02A1"

[71 "02A1U" "02B" "02C" "02D" "02G" "02GK" "02O"

[78 "02U" "06A1" "0708" "1819" "26C" "26CU" "A1A2D"

[85 A1B" "AIC" "AICD" "AICDGKU" "AICG" "AID" "A1DHK'

[92 "A1DK" "A1F2" "A1G" "A1GHU" "A1GJ" "A1GU" "A1U" [99] "A2C" "A2CD" "A2G" "AC" "ACD" "AD" "AF2"

[106] "AF2G" "AG" "AGKU" "AGU" "AHJU" "AKU" "BC" [1 13] "BCF1 " "BCU" "BF" "BF1 " "BFG" "BG" "CD" [120] "CU" "DG" "DO" "O" "N" "P" "CPZ"

All epidemiologically relevant classes worldwide that have been described by full-genome sequences were included in the analysis. The 2009 HIV database 'web alignment' formed the basis for the alignment used in the analysis. Entropy per class was investigated to ensure that all classes would be detected in a design that averaged across all sequences. The entropy plot includes SIV CPZ, the ancestor of HIV- 1 , for reference purpose only. SIV CPZ is included to deliminate the most extreme variation conceivable.

Based on the overall genetic variation across the entire genome, three large genomic regions of interest for primer/probe design were identified (1) region 3000-4000, start of the pol gene; (2) region 5000-6000, end of the pol gene; and (3) region 9000-10000, at the junction of gpl20 and gp41 in the env gene. To further analyze these regions of interest, SIV CPZ was removed from each regional alignment, and sequences with big indels (i.e., insertions or deletions), uncalled nucleotide positions (N's), and otherwise suspected sequencing artifacts were removed. Subsequently, each regional alignment was manually checked and unnecessary gaps were removed before further analysis. Within each of these 3 regions (3000-4000 = poll, 5000-6000 = pol2 and 9000-10000 = envl), a full resolution entropy analysis was performed (i.e., moving the 25 nt primer window 1 nt across each regional alignment). The full resolution entropy analysis was used to identify smaller sub-regions (designated poll .1, poll .2, pol2.1, pol2.2, envl . l, env 1.2, and env 1.3) for primer and probe design.

Figures 2A and 2B show examples of a plot 200 of entropy in region 3000-4000 and a schematic diagram 250 of the landmarks of the HIV-1 genome, respectively. Referring to Figure 2B, schematic diagram 250 shows the gene locations and coordinates for the HIV-1 reference strain HXB2. The HXB2 genome is used as a universal HIV-1 coordinate system (the standard in the HIV database www.hiv.lanl.gov). Open reading frames (e.g., pol, env) are shown as rectangles. A target region 252 for primer/probe design corresponds to HXB2 coordinates 2238-3138. Figures 3A and 3B show an example of a plot 300 of entropy in region 5000-6000 and another view of schematic diagram 250 of the landmarks of the HIV- 1 genome, respectively. Referring to schematic diagram 250 of Figure 3B, target region 252 for primer/probe design corresponds to HXB2 coordinates 4127-5060.

5 Figures 4A and 4B show an example of a plot 400 of entropy in region 9000-10000 and yet another view of schematic diagram 250 of the landmarks of the HIV-1 genome, respectively. Referring to schematic diagram 250 of Figure 4B, target region 252 for primer/probe design corresponds to HXB2 coordinates 7308-81 15.

4.2 Primer and Probe Designs

[0 All primers and probes were designed to detect all known HIV-1 groups, subtypes and recombinants. The alignment underlying the design of primers and probes is described in reference to Figures 1 through 4B. Primers and probes were identified at a 5% and 10% detection level. This level indicates at which percentage nucleotide variation was included per alignment position in the design. This level was used to generate degenerated primers to capture HIV-1

[ 5 genetic variation at the specified level.

Figure 5 illustrates a table 500 of PCR forward and reverse primer sequences for sub-regions poll . l, pol2.1, envl . l, and envl .3. Individual primer constructs were selected for genetic variation, complexity, and melting temperature (Tm).

Figure 6 illustrates a table 600 of probe constructs for sub-regions poll . l, poll .2, pol2.1, and ^0 envl .1. Individual probe constructs were selected for complexity, melting temperature (Tm), and the distance from the forward primer 3'-end to the start (5'-end) of the probe (F dist).

Referring to Figures 5 and 6, primer and probe sequences are written 5' to 3'. If the primers and probes include genetic variation, this was indicated using the standard IUPAC codes for multi- state characters, e.g. R = A and G, M = A and C, Y = C and T, K = G and T, V= A and C and G, ^5 W = A and T. Primer and probe constructs were labeled as follows: F = forward primer, R = reverse primer, P = probe; detection level 5% or 10%; The numbers following the F or R designation correspond to the HXB2 genome, which is used as a universal HIV-1 coordinate system (i.e., the standard in the HIV database ww.hiv.lanl.gov). For example, F 10.2329.2355 is a forward primer designed to detect sequences with nucleotide variation per site that has been empirically observed at or above 10% in the underlying alignment. This primer spans positions 2329 through 2355 in the HXB2 genome. The fact that the first coordinate is smaller than the second also indicates that this is a forward primer. Conversely, reverse primers will have the larger coordinate first, e.g., R5.2424.2397. Probes follow the same labeling system. All amplicon lengths are <150 bp. Because HIV-1 is genetically very diverse, HIV-1 targets amplified using the same primer pairs may not yield amplicons of the same length.

The genetic variation per primer/probe is indicated by the estimated Shannon entropy. Lower entropy values indicate less genetic variation. The complexity is the number of individual sequences that a primer/probe may be deconvoluted into. Primers/probes were selected for a complexity equal to or less than 64. Too great a complexity means many different primers, possible primer dilution and increased risk of non-specific amplification. Complexity and entropy are closely linked; in general, higher entropy means higher complexity. Thus, at a lower detection level (5% vs 10%), complexity usually increases, but not always (e.g., pol2.1 primers).

The melting temperature (Tm) was calculated as a mean of all deconvoluted individual sequences as they would appear on the plus-strand. The Tm was calculated according to the thermodynamic nearest neighbor model². The Tm indicates a target annealing temperature and may be used as an initial start-point for determining reaction annealing temperatures. Note that for the reverse primers this is a further approximation. In a preferred embodiment, primer pair Tm's were designed to be within 4° C of each other in a 60-64° C annealing temperature range. Because all Tms are approximations, small deviations from this range may occur. Probes were designed to have a Tm >5° C higher than matching primers.

Referring to Figure 5, up to 5 sets of primer combinations were designed for each sub-genomic region (i.e., poll . l, pol2.1, envl . l, envl .3). Some primers may be included in several sets. Bolded sequences overlap with other sequences in table 500. In a preferred embodiment, pol primers were constructed with G/C at the 3 '-end to improve extension. For env primers, this constraint was relaxed due to more genetic variation in this part of the genome. Risk of primer dimerization was assessed through a sliding alignment procedure. Primers and probes within sets selected to have <10 consecutive matches. Only primers in sets 3-5 of envl. l are shown. Primers in sets 1 and 2 (not shown) involved a rare length variation at the forward primer 3 '-end. Tm's for envl . l primer sets 3-5 may be further adjusted (i.e., lowered) by removing the 5' nucleotides of the primers. Referring to Figure 6, column "F dist" lists the distance from the forward primer 3'-end to the start (5'-end) of the probe. In a preferred embodiment, detection probes are TaqMan® probes. To avoid quenching of the fluorophore, the first 5'-base of the probe is not a "G". Probes were designed to be located 2-10 bp from one of the primers, on the same strand. Probes were designed to have a Tm >5° C higher than matching primers.

Primers and probes with different detection levels may be used in combination. For example, primers with a 5% detection level may be combined with a probe with a 10% detection level. Alternatively, primers and probes with the same detection level (e.g., 5%) may be used. Primers at lower detection level may be used to detect more HIV-1 classes and at lower template concentrations, and once detected the probes will find the amplicons with less degeneracy.

The annealing temperature may be selected such that all forms of HIV- 1 , including those that have not yet been sequenced, may be detected. In one example, primers and probes may be selected at 10% detection level and the Tm adjusted to detect all HIV-1 forms. Primers and probes at 10% detection level may have less misannealing and less effects of primer dilution.

All primer and probe designs were evaluated against the human genome using BLAST (megablast) against the NCBI Genome-reference only and default small fragment parameter adjustments. E-value, chromosome, strand orientation, and number of consecutive matches of the primer/probe lengths were considered. The number of consecutive matches indicates how stable the binding is at increased annealing temperatures. Matches with <15 consecutive matches are in general not a problem at temperatures >62 °C; worst case was around 20 matches. However, all probe Tm's were about >70 °C where problems are even less likely. Also, matches to the human genome occurred on different chromosomes, and/or on the same strands, which make amplicons nearly impossible. At low temperatures it is possible that some primers/probes could anneal and be consumed if extension is allowed even if they would not form specific amplicons. Nonspecific amplification may be avoided using a hot start PCR protocol. None of the designed primers/probes shown in Figures 5and 6 had significant matches to the human genome.

References

1 Miller, G. 1955. Note on the bias of information estimates. Info. Theory Psychol. Prob.

Methods II-B:95-100. 2 Watkins & SantaLucia, NAR 2005, Vol 33, pp. 6258-6267

Concluding Remarks

The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the invention. Other embodiments having different structures and operations do not depart from the scope of the present invention. The term "the invention" or the like is used with reference to certain specific examples of the many alternative aspects or embodiments of the applicants' invention set forth in this specification, and neither its use nor its absence is intended to limit the scope of the applicants' invention or the scope of the claims. This specification is divided into sections for the convenience of the reader only. Headings should not be construed as limiting of the scope of the invention. The definitions are intended as a part of the description of the invention. It will be understood that various details of the present invention may be changed without departing from the scope of the present invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

The Claims We claim

1. An oligonucleotide primer selected from the group consisting of SEQ ID NOS: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, and 19.

2. The oligonucleotide primer of claim 1, wherein the oligonucleotide primer is specific for the pol region of the HIV- 1 genome and is selected from the group consisting of SEQ ID NOS: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1 1.

3. The oligonucleotide primer of claim 2, wherein the oligonucleotide primer is SEQ ID NO: l l .

The oligonucleotide primer of claim 1 , wherein the oligonucleotide primer is specific for the env region of the HIV- 1 genome and is selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18, and 19.

The oligonucleotide primer of claim 4, wherein the oligonucleotide primer is selected from the group consisting of SEQ ID NOS: 12 and 16.

An oligonucleotide probe selected from the group consisting of SEQ ID NOS:20, 21, 22, and 23.

The oligonucleotide probe of claim 6, wherein the oligonucleotide probe is specific for the pol region of the HIV- 1 genome and is selected from the group consisting of SEQ ID NOS: 20, 21, and 22.

The oligonucleotide probe of claim 7, wherein the oligonucleotide probe is SEQ ID NO: 22.

9. The oligonucleotide probe of claim 6, wherein the oligonucleotide probe is specific for the env region of the HIV- 1 genome and is SEQ ID NO:23.

10. A method for detecting human immunodeficiency virus type-1 (HIV-1) in a test sample comprising the steps of:

(a) providing the test sample;

(b) amplifying an HIV- 1 nucleic acid within the test sample using an oligonucleotide primer selected from the group consisting of SEQ ID NOS: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, and 19; and

(c) detecting the presence of the HIV- 1 nucleic acid using an oligonucleotide probe; wherein detecting the presence of the HIV- 1 nucleic acid is an indication of the presence of HIV- 1 in the test sample.

1 1. The method of claim 10, wherein the oligonucleotide primer is specific for the pol region of the HIV-1 genome and is selected from the group consisting of SEQ ID NOS: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1 1.

12. The method of claim 1 1, wherein the oligonucleotide primer is SEQ ID NO: l 1.

13. The method of any one of claims 1 1 or 12, wherein the oligonucleotide probe is specific for the pol region of the HIV- 1 genome and is selected from the group consisting of SEQ ID NOS:20, 21, and 22.

14. The method of claim 13, wherein the oligonucleotide probe is SEQ ID NO:22.

15. The method of claim 10, wherein the oligonucleotide primer is specific for the env region of the HIV-1 genome and is selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18, and 19.

16. The method of claim 15, wherein the oligonucleotide primer is selected from the group consisting of SEQ ID NOS: 12 and 16.

17. The method of any one of claims 15 to 16, wherein the oligonucleotide probe is specific for the env region of the HIV-1 genome and is SEQ ID NO:23.

18. The method of any one of claims 10 to 17, wherein amplifying the HIV-1 nucleic acid sequence is performed using real-time reverse transcription polymerase chain reaction (RT-PCR).

19. The method of any one of claims 10 to 18, wherein the oligonucleotide probe is fluorescently- labeled.

20. A method for detecting human immunodeficiency virus type-1 (HIV-1) in a test sample comprising the steps of:

(a) providing the test sample;

(b) amplifying at least one HIV-1 nucleic acid within the test sample using at least one or a combination of oligonucleotide primers selected from the group consisting of SEQ ID NOS: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, and 19; and

(c) detecting the presence of the HIV- 1 nucleic acid using at least one or a combination of oligonucleotide probes; wherein detecting the presence of the HIV- 1 nucleic acid is an indication of the presence of HIV- 1 in the test sample.

21. The method of claim 20, wherein at least one or a combination of the oligonucleotide primers are specific for the pol region of the HIV-1 genome and are selected from the group consisting of SEQ ID NOS: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1 1.

22. The method of claim 21 , wherein the oligonucleotide primer is SEQ ID NO: 1 1.

23. The method of any one of claims 21 or 22, wherein at least one or a combination of the oligonucleotide probes are specific for the pol region of the HIV-1 genome and are selected from the group consisting of SEQ ID NOS:20, 21, and 22.

24. The method of claim 23, wherein the oligonucleotide probe is SEQ ID NO:22.

25. The method of claim 20, wherein at least one or a combination of the oligonucleotide primers are specific for the env region of the HIV- 1 genome and are selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18, and 19.

26. The method of claim 25, wherein the oligonucleotide primers are selected from the group consisting of SEQ ID NOS: 12 and 16.

27. The method of any one of claims 25 to 26, wherein at least one of the oligonucleotide probes is specific for the env region of the HIV-1 genome and is SEQ ID NO:23.

28. The method of any one of claims 20 to 27, wherein amplifying the HIV-1 nucleic acid sequence is performed using real-time reverse transcription polymerase chain reaction (RT-PCR).

29. The method of any one of claims 20 to 28, wherein the oligonucleotide probes are fluorescently-labeled probes.