WO2017099801A1 - Compositions, kits et méthodes pour détecter le virus vih - Google Patents

Compositions, kits et méthodes pour détecter le virus vih Download PDF

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WO2017099801A1
WO2017099801A1 PCT/US2015/065312 US2015065312W WO2017099801A1 WO 2017099801 A1 WO2017099801 A1 WO 2017099801A1 US 2015065312 W US2015065312 W US 2015065312W WO 2017099801 A1 WO2017099801 A1 WO 2017099801A1
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aceln
primer
acid sequence
nucleic acid
acein
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PCT/US2015/065312
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Changchun Liu
Scott SHERRIL-MIX
Haim H. Bau
Frederic D. Bushman
Karen E. OCWIEJA
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The Trustees Of The University Of Pennsylvania
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Priority to US15/534,835 priority Critical patent/US10731227B2/en
Priority to PCT/US2015/065312 priority patent/WO2017099801A1/fr
Publication of WO2017099801A1 publication Critical patent/WO2017099801A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present inventions relate generally to compositions, kits, and methods for detecting HIV virus in a sample.
  • compositions comprising: a primer having the nucleic acid sequence of ACeIN-F3_c, a primer having the nucleic acid sequence of ACeIN-B3_a, a primer having the nucleic acid sequence of ACeIN-B3_b, a primer having the nucleic acid sequence of ACeIN-FIP_e, a primer having the nucleic acid sequence of ACelN- FIP_f, a primer having the nucleic acid sequence of ACelN-BIP, a primer having the nucleic acid sequence of ACelN-LF; and a primer having the nucleic acid sequence of ACelN-LB.
  • compositions comprising: a primer having the nucleic acid sequence of ACeIN-F3_c, a primer having the nucleic acid sequence of ACeIN-B3a, a primer having the nucleic acid sequence of ACeIN-B3b, a primer having the nucleic acid sequence of ACelN-FIPe, a primer having the nucleic acid sequence of ACelN-FIPf, a primer having the nucleic acid sequence of ACelN-BIP-song, a primer having the nucleic acid sequence of ACelN-LF; and a primer having the nucleic acid sequence of ACelN-LB.
  • HIV human immunodeficiency virus
  • RT- LAMP reverse transcription-based loop mediated isothermal amplification
  • the present disclosure also provides methods of detecting human immunodeficiency virus (HIV) nucleic acids in a sample comprising contacting a reaction mixture comprising a reverse transcription-based loop mediated isothermal amplification assay of the previously disclosed composition, magnesium, dNTPs, a reaction buffer, a DNA polymerase and a sample to be tested for presence of HIV nucleic acids and incubating the reaction mixture under DNA polymerase reactions conditions so as to produce a reaction product comprising amplified HIV nucleic acids and detecting a reaction product.
  • HIV human immunodeficiency virus
  • immunodeficiency virus in a patient comprising obtaining a sample from said patient and performing reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on the sample using the previously disclosed compositions.
  • RT-LAMP reverse transcription-based loop mediated isothermal amplification
  • the present disclosure provides methods of detecting human immunodeficiency virus (HIV) in a patient comprising obtaining a sample from said patient and contacting a reaction mixture comprising a reverse transcription-based loop mediated isothermal amplification assay composition of claim 1 or claim 2, magnesium, dNTPs, a reaction buffer, a DNA polymerase and the sample to be tested for presence of HIV nucleic acids, incubating the reaction mixture under DNA polymerase reactions conditions to produce a reaction product comprising amplified HIV nucleic acids, and detecting a reaction product.
  • HSV human immunodeficiency virus
  • a further aspect of the present disclosure includes methods of monitoring a response to a medication in a subject in need thereof, comprising obtaining a first sample from the subject at a first time point, obtaining a second sample from the subject a second time point following administration of a medication to the subject, determining the amount of human
  • HIV immunodeficiency virus
  • the determining comprising, performing reverse transcription-based loop mediated isothermal amplification (RT-LAMP) on a sample containing HIV using the primers of claim 1 or claim 2, and comparing the amount of HIV in the first and second samples, wherein a decrease in the amount of HIV from the first sample relative to the second sample indicates treatment of HIV infection.
  • R-LAMP reverse transcription-based loop mediated isothermal amplification
  • kits comprising a primer having the nucleic acid sequence of ACeIN-F3_c, a primer having the nucleic acid sequence of ACelN- B3_a, a primer having the nucleic acid sequence of ACeIN-B3_b, a primer having the nucleic acid sequence of ACelN-FIP e, a primer having the nucleic acid sequence of ACelN-FIP f, a primer having the nucleic acid sequence of ACelN-BIP, a primer having the nucleic acid sequence of ACelN-LF, a primer having the nucleic acid sequence of ACelN-LB, and packaging for said primers.
  • kits comprising a primer having the nucleic acid sequence of ACeIN-F3_c, a primer having the nucleic acid sequence of ACeIN-B3a, a primer having the nucleic acid sequence of ACeIN-B3b, a primer having the nucleic acid sequence of ACelN-FIPe, a primer having the nucleic acid sequence of ACelN- FlPf, a primer having the nucleic acid sequence of ACelN-BIP-song, a primer having the nucleic acid sequence of ACelN-LF, a primer having the nucleic acid sequence of ACelN-LB, and packaging for said primers.
  • Figure 1 shows a summary of amplification results for all the RT-LAMP primer sets tested in this study. The data is shown as a heat map, with more intense coloring indicating shorter amplification times (key at bottom). Primer sets tested are named along the left of the figure. Primer sequences, and their organization into LAMP primer sets, are cataloged in tables S I and S2. The raw data and averaged data are collected in tables S3 and S4. ACeIN-26 primer set (highlighted) had one of the best performances across the subtypes and a relatively simple primer design.
  • Figure 2 shows bioinformatic analysis to design subtype-agnostic RT-LAMP primers.
  • the x-axis shows the coordinate on the HIV genome
  • the y-axis shows the proportion matching the consensus in each 21 base segment of the genome (points).
  • the black line shows a 101 base moving average over these proportions.
  • the vertical shading shows the region targeted for LAMP primer design that was used as input into the EIKEN primer design tool. Numbering is relative to the HIVg 9 .6 sequence.
  • Part (B) shows aligned genomes, showing the locations of the ACeIN26 primers. Sequences in the shaded region in A are shown, with DNA bases color-coded as shown at the lower right.
  • Each row indicates an HIV sequence and each column a base in that sequence. Horizontal lines separate the HIV subtypes (labeled at right). Arrows indicate the strand targeted by each primer. Primers targeting the negative strand of the virus are shown as reverse compliments for ease of viewing.
  • Figure 3 Performance of the AceIN26 primer set with different starting RNA concentrations. Tests of each subtype are shown as rows. In each lettered panel, the left shows the raw accumulation of fluorescence signal (y-axis) as a function of time (x-axis); the right panel shows the threshold time (y-axis) as a function of log RNA copy number (x-axis) added to the reaction.
  • Figure 4 Examples of time course assays, displaying replicate tests of RT- LAMP primer set ACeIN26 tested over six HIV subtypes. A total of 5000 RNA copies were tested in each reaction. Time is shown on the x-axis, fluorescence intensity on they-axis. Replicates are distinguished using an arbitrary code. Z-factor values and standard deviations are shown on each panel.
  • Figure 6 shows real-time monitoring of RT-LAMP amplification of HIV subtype C sample (sample ID: 4160) nominally containing 1 ,000, 500, and 0 (negative control) copies per sample on the microfluidic chip.
  • the actual number of RNA copies may be lower due to the age of the sample and possible RNA degradation.
  • Figure 7 shows real-time monitoring of RT-LAMP amplification of HIV subtype C sample nominally containing 500 copies per sample on benchtop thermal cycler. 16 replicate tests were run in parallel. The actual number of RNA in the aliquots may have been lower due to the age of the sample and possible RNA degradation.
  • Figure 8 shows real-time monitoring of RT-LAMP amplification of five HIV subtype C samples (sample ID 6053, 6057, 1108, 1113 and 1115) on the microfluidic chips.
  • R-LAMP reverse transcription-based loop mediated isothermal amplification
  • Primer binding sites are chosen so that a series of strand displacement steps allow continuous synthesis of DNA without requiring thermocy cling. Reaction products can be detected by adding a dye to reaction mixtures that fluoresces only when bound to DNA, allowing quantification of product formation by measurement of fluorescence intensity.
  • RT-LAMP assays for HIV-1 have been reported previously to show high sensitivity and specificity for subtype B, the most common HIV strain in the developed world. Assays have also been developed for HIV -2. However, a complication arises in using available RT-LAMP assays due to the variation of HIV genomic sequences among the HIV subtypes, so that an RT- LAMP assay optimized on one viral subtype may not detect viral RNA of another subtype. Tests presented below show that available RT-LAMP assays are efficient for detecting subtype B, for which they were designed, but often performed poorly on other subtypes, some of which are abundant world-wide.
  • the present disclosure provides development of an RT-LAMP assay capable of detecting HIV-1 subtypes A, B, C, D, and G.
  • bioinformatic analysis was carried out to identify regions conserved in all the HIV subtypes. 44 different combinations of RT- LAMP primers targeting this region were tested in over 700 individual assays, allowing identification of primer sets (ACeIN-26 and ACeIN-35) that were optimal for detecting the subtypes tested.
  • Optimized RT- LAMP assay may be useful for quantifying HIV RNA copy numbers in point-of- care applications in the developing world, where multiple different subtypes may be encountered.
  • Example 1 Testing published RT-LAMP primer sets against multiple HIV subtypes.
  • RNA samples from multiple HIV subtypes were assessed. Viral stocks from HIV subtypes A, B, C, D, F, and G, were obtained, and the numbers of virions per ml were quantified and RNA was extracted. RNAs were mixed with RT-LAMP reagents which included the six required RT-LAMP primers, designated F3, B3, FIP, BIP, LF and LB. Reactions also contained the intercalating fluorescent EvaGreen Tm dye, which yields a fluorescent signal upon DNA binding. DNA synthesis was quantified as the increase in fluorescence over time, which yielded a typical curve describing exponential growth with saturation (examples are presented elsewhere herein). Results are expressed as threshold times (Tt) for achieving 10% amplification with 5000 HIV RNA template copies.
  • Tt threshold times
  • Table 1 shows the primer sequences used.
  • B-PR primers were mixed, which detected clade F (albeit with limited efficiency) with the B-CA and B-RT primers ( Figure 1 and Table 3 and 4). In neither case did this provide coverage of all four clades tested. Primer sets targeting additional regions of the HIV genome were thus sought.
  • Table 2 shows the HIV RT-LAMP primer sets studied.
  • ACeIN-3 work ACelN-BIP, ACelN-LF, , ACelN-LB
  • ACeIN-4 work ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACeIN-7 work ACelN-BIP, ACelN-LF, ACelN-LB
  • ACeIN-8 work ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe T, ACelN- 10 work FlPf T, ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPg, ACelN-FIPh, 11 work ACelN-BIP, ACelN-LF b, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPg, ACelN-FIPh, 12 work ACelN-BIP T, ACelN-LF b, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPg T, ACelN- 13 work FlPh T, ACelN-BIP, ACelN-LF b, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPg T, ACelN- 14 work FlPh T, ACelN-BIP T, ACelN-LF b, ACelN-LB
  • ACelN- This ACeIN-F3 b, ACeIN-B3a, ACeIN-B3b, ACelN-FIPi, ACelN-BIP, 15 work ACelN-LF c, ACelN-LB
  • ACelN- This ACeIN-F3 b, ACeIN-B3a, ACeIN-B3b, ACelN-FIPi, ACelN- 16 work BIP T, ACelN-LF c, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPj, ACelN-FIPk, 17 work ACelN-BIP, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPj, ACelN-FIPk, 18 work ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 b, ACeIN-B3a, ACeIN-B3b, ACelN-FIPi T, ACelN- 19 work BIP, ACelN-LF c, ACelN-LB
  • ACelN- This ACeIN-F3 b, ACeIN-B3a, ACeIN-B3b, ACelN-FIPi T, ACelN- 20 work BIP T, ACelN-LF c, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPj T, ACelN- 21 work FlPk T, ACelN-BIP, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3, ACeIN-B3a, ACeIN-B3b, ACelN-FIPj T, ACelN- 22 work FlPk T, ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACelN- This ACelN-FIPb, ACelN-FIPe, ACelN-FIPf, ACelN-FIPj, ACelN- 23 work FlPk, ACelN-BIP, ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACeIN-F3, ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPa, 23+B-PR work ACelN-FIPb, ACelN-FIPe, ACelN-FIPf, ACelN-FIPj, ACelN- FlPk, ACelN-BIP, ACelN-BIP T, ACelN-LF, ACelN-LB, B-PR- F3, B-PR-B3, B-PR-FIP, B-PR-BIP, B-PR-LF, B-PR-LB
  • ACelN- This ACeIN-F3 cL, ACeIN-B3a L, ACeIN-B3b L, ACelN-FIPe, 24 work ACelN-FIPf, ACelN-BIP LT, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPa, ACelN- 25 work FlPb, ACelN-BIP, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf,
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf, 27 work ACelN-BIP T, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPj, ACelN-FIPk, 28 work ACelN-BIP, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPj, ACelN-FIPk,
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe L, ACelN-
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe L, ACelN- 31 work FlPf L, ACelN-BIP LT, ACelN-LF, ACelN-LB
  • ACeIN-F3 c ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf,
  • ACelN- This ACelN-BIP, ACelN-LF, ACelN-LB, B-PR-F3, B-PR-B3, B-PR- 26+B-PR work FIP, B-PR-BIP, B-PR-LF, B-PR-LB
  • ACeIN-F3 c ACeIN-B3a, ACeIN-B3b, ACelN-FIPe L, ACelN-
  • ACeIN-F3 c ACeIN-B3a, ACeIN-B3b, ACelN-FIPj, ACelN-FIPk,
  • ACelN- This ACelN-BIP, ACelN-LF, ACelN-LB, B-PR-F3, B-PR-B3, B-PR- 28+B-PR work FIP, B-PR-BIP, B-PR-LF, B-PR-LB
  • ACelN- This ACeIN-F3 cL, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN- 32 work FlPf ACelN-BIP, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 c, ACeIN-B3a L, ACeIN-B3b L, ACelN-FIPe, 33 work ACelN-FIPf, ACelN-BIP, ACelN-LF, ACelN-LB
  • ACelN- This ACeIN-F3 cL, ACeIN-B3a L, ACeIN-B3b L, ACelN-FIPe, 34 work ACelN-FIPf, ACelN-BIP, ACelN-LF, ACelN-LB
  • ACeIN-F3 c ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf,
  • ACelN- ACelN-BIP ACelN-LF, ACelN-LB, A-PR-F3, B-PR-F3, C-PR-
  • ACeIN-F3 c ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf,
  • ACelN- This ACelN-BIP, ACelN-LF, ACelN-LB, F-IN-F3, F-IN-B3a, F-IN- 26+F-IN work B3b, F-IN-FIP, F-IN-BIP, F-IN-LF, F-IN-LB
  • ACeIN-F3 c ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf,
  • ACelN- This ACeIN-F3 c, ACeIN-B3a, ACeIN-B3b, ACelN-FIPe, ACelN-FIPf, 35 work ACelN-BIP-song, ACelN-LF, ACelN-LB
  • Table 3 shows average threshold times. Reactions contained 5000 copies of HIV-1
  • the threshold time (T t ) is defined as the reaction time that elapses until the threshold signal increases 10% of maximum fluorescence intensity (Imax) above the baseline level.
  • Table 4 shows all tkeshold times generated in this study.
  • ACeIN-1 (“Ace” for "all clade”, and “IN” for “integrase”), targeted the HIV M coding region and contained multiple bases at selected sites to broaden detection (Figure 1).
  • ACeIN ⁇ 2 and -3 have primers with slightly different landing sites. Tests showed that the mixture of primers allowed amplification with a shorter threshold time than did either alone ( Figure 1).
  • a new primer set was designed to target the CA coding region (Figure 1, ACeCA) but found that the set only amplified clade B, and not efficiently.
  • ACeIN3-6 were altered by inserting a polyT sequence between the two different sections of FIP and BIP in various combinations, a modification introduced with the goal of improving primer folding, but these designs performed quite poorly (Figure 1).
  • ACeIN-26, 28 and 30 primers were tested combined with the B-PR primer (a slightly modified version of the row 3 primer) but no improvement was seen and efficiency may even have fallen for some subtypes.
  • a primer set was also designed that matched exactly to the problematic subtype F, and this set was mixed with the ACeIN-26 primers.
  • Subtypes A, B, C, D, and G were detected efficiently and showed z-factors above 0.5, but subtype F was detected only with higher template amounts. Subtype F is estimated to comprise only 0.59% of all infections globally, so perhaps inefficient detection is still acceptable.
  • RT-LAMP assay Today, rapid assays are available that can report infection by detecting anti-HIV antibodies in oral samples, allowing simplified assays, but the nucleic- acid based method presented here has additional possible uses. Combining the RT-LAMP assay with simple point of care devices for purifying blood plasma and quantitative analysis of accumulation of fluorescent signals is envisioned. In one implementation of the technology, cell phones could be used to capture and analyze results. Together, these methods will allow assessment of parameters beyond just the presence/absence of infection. Quantitative RT-LAMP assays should allow tracking of responses to medication, detection in neonates (where immunological tests are confounded by presence of maternal antibody), and early detection before seroconversion.
  • the A-UG strain contains subtype A sequences over the target region of ACeIN26, and so was used here to represent subtype A.
  • Viral stocks were prepared by transfection and infection. Culture supernatants were cleared of cellular debris by centrifugation at 1500g for 10 min. The supernatant containing virus was then treated with 100 U DNase (Roche) per 450 ul virus for 15 min at 30°C. RNA was isolated using QiaAmp Viral RNA mini kit (Qiagen GmbH, Hilden, Germany). RNA was eluted in 80 ⁇ of the provided elution buffer and stored at -80°C.
  • RNA copies Concentration of viral RNA copies was calculated from p24 capsid antigen capture assay results provided by the University of Pennsylvania CFAR or the NIH AIDS-reagent program. In calculating viral RNA copy numbers, it was assumed that all p24 was incorporated in virions, all RNA was recovered completely from stocks, 2 genomes were present per virion, 2000 molecules p24 were present per viral particle, and the molecular weight of HIV-1 p24 was 25.6 kDa.
  • RT-LAMP reaction mixtures (15 ⁇ ) contained 0.2 ⁇ of primers F3_c, B3_a, and B3_b; 0.8 ⁇ FIP e, FIP_f, LoopF and LoopB; and 1.6 ⁇ BIP; 7.5 ⁇ , OptiGene
  • Amplification was measured using the 7500-Fast Real Time PCR system from Applied Biosystems with the following settings: 1 minute at 62°C; 60 cycles of 30 seconds at 62°C and 30 seconds at 63°C. Data was collected every minute. Product structure was assessed using dissociation curves which showed denaturation at 83°C. Products from selected amplification reactions were analyzed by agarose gel electrophoresis and showed a ladder of low molecular weight products.
  • AcelN-BIP primer in ACeIN-26 primer set was modified to better match the HIV subtype C sequence.
  • AcelN-BIP primer in ACeIN-26 primer set i) GGAYTATGGAAAACAGATGGCAGCCATGTTCTAATCYTCATCCTG (SEQ ID NO: 63)
  • Example 9-HIV Viral Load Test of HIV Subtype C Clinical Sample [0059]
  • the Penn-designed assay for HIV clade C is compatible with clinical samples of HIV patients from Botswana. Since the samples were over three years old, some of the RNA in the samples may have degraded and it was not possible to accurately verify the quantitative aspects of the assay.
  • Table 5 shows information of six plasma samples that were tested.
  • the viral loads were determined by quantitative PCR.
  • HIV RT-LAMP The sequences of the HIV RT-LAMP are the same as previously reported (RT-LAMP) primers, ACeIN-26 primer set) with a slight modification in the BIP primer.
  • Viral RNA was extracted from plasma with a benchtop centrifuge using the QIAamp viral RNA mini kit (QIAGEN, Inc.). Briefly, 140 ⁇ , of plasma were mixed with 560 ⁇ , AVL buffer containing carrier RNA in a 1.5 mL micro-centrifuge tube by pulse-vortexing for 15 seconds followed by incubation at room temperature for 10 min. 560 of absolute ethanol were added and mixed by pulse-vortexing for 15 seconds. The lysate were loaded in the QIAamp spin- column mounted on 2 mL collection tubes and centrifuged at 8000 rpm for 1 min. The column was then washed by 500 ⁇ . of wash buffers WB1 and WB2.
  • viral RNA was eluted using 60 ⁇ . of AVE buffer.
  • sample IDs 6053, 6057, 1108, 1113 and 1115, 420 of plasma was lysed and eluted with 60 of AVE buffer to obtain a relatively high target concentration.
  • the viral RNA was tested on a microfluidic chip.
  • the extracted plasma containing the HIV virus was amplified in a microfluidic chip.
  • the chip contains three independent multifunctional, 5.0 mm long, 1.0 mm wide, 3.0 mm deep, and -15.0 in volume amplification reactors. Each of these reactors is equipped with a flowthrough Qiagen silica membrane (QIAamp Viral RNA Mini Kit) at its entry port.
  • QIAamp Viral RNA Mini Kit Qiagen silica membrane
  • the 140 ⁇ . of plasma collected with our plasma separator was mixed with 560 ⁇ . of lysis buffer (QIAamp Viral RNA Mini Kit, Qiagen, Valencia, CA) and inserted into one of the amplification reactors.
  • high chaotrophic salts such as guanidinium chloride
  • the silica membrane was washed with 500 ⁇ . of wash buffer 2 (AW2) containing 70% ethanol, followed by air drying for 30s.
  • RT-LAMP master mixture which contains all the reagents necessary for the RT-LAMP, 0.5 ⁇ EvaGreen@ fluorescence dye (Biotium, Hay ward, CA), and 8 units of RNase inhibitor (Life Technologies), was injected into each reaction chamber through the inlet port. Subsequently, the inlet and outlet ports were sealed using transparent tape (Scotch brand cellophane tape, 3M, St. Paul, MN) to minimize evaporation during the amplification process.
  • the nucleic acid chip was placed on a portable heater and heated to 63 °C for approximately 60 min. The fluorescence excitation and detection were carried out with a handheld, USB-based, fluorescence microscope (AM4113T- GFBW Dino-Lite Premier, AnMo Electronics, Taipei, Taiwan).
  • Figure 6 shows examples of real time RT-LAMP curves of HIV subtype C sample containing 1,000, 500, and 0 (negative control) copies per sample obtained with our microfluidic chip. These samples were prepared by diluting the clinical sample (Sample ID: 4160) with HIV negative plasma. The HIV RNA was extracted by the isolation silica membrane, embedded in our microfluidic chip from sample lysate, and the RNA extracted by the membrane served as a template for RT-LAMP.
  • Figure 7 depicts real time RT-LAMP curves of 16 replicate purified HIV RNA samples that are extracted from sample 4160. Each sample contains 500 copies that were carried out on the benchtop.
  • Table 2 summarizes the results of the HIV subtype C RT-LAMP assay carried out in our microfluidic chip and in a benchtop thermal cycler. The nominal sensitivity of our chip is 500 copies per reaction.
  • Table 6 shows HIV subtype C RT-LAMP assay in our microfluidic chip and in a benchtop thermal cycler.
  • the table documents the number of positive results normalized with the number of tests.
  • Figure 8 shows real time RT-LAMP curves of five HIV subtype C samples (samples ID: 6053, 6057, 1108, 1113, and 1115) on our microfluidic chips.

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Abstract

L'invention concerne des compositions comprenant une amorce possédant la séquence d'acides nucléiques ACeIN-F3_c, une amorce possédant la séquence d'acides nucléiques ACeIN-B3_a, une amorce possédant la séquence d'acides nucléiques ACeIN-B3_b, une amorce possédant la séquence d'acides nucléiques ACelN-FIP e, une amorce possédant la séquence d'acides nucléiques ACelN-FIP f, une amorce possédant la séquence d'acides nucléiques ACelN-BIP (ou ACelN-BIP-song), une amorce possédant la séquence d'acides nucléiques ACelN-LF ; et une amorce possédant la séquence d'acides nucléiques ACelN-LB. L'invention concerne également des méthodes de détection des acides nucléiques du virus de l'immunodéficience humaine (VIH) dans un échantillon, comprenant la réalisation sur un échantillon d'une amplification isotherme facilitée par boucle à base de transcription inverse (RT-LAMP) à l'aide des compositions décrites précédemment.
PCT/US2015/065312 2014-12-12 2015-12-11 Compositions, kits et méthodes pour détecter le virus vih WO2017099801A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6503705B2 (en) * 1992-05-14 2003-01-07 Leland Stanford Junior University Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome
US20040034888A1 (en) * 1999-05-06 2004-02-19 Jingdong Liu Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
WO2008121992A2 (fr) * 2007-03-30 2008-10-09 Research Foundation Of State University Of New York Virus atténués utiles pour des vaccins
WO2014003583A2 (fr) * 2012-06-26 2014-01-03 University Of The Philippines Diliman Détection de pathogènes
WO2015118491A1 (fr) * 2014-02-06 2015-08-13 University Of The Witwatersrand, Johannesburg Procédé de détection du vih

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6503705B2 (en) * 1992-05-14 2003-01-07 Leland Stanford Junior University Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome
US20040034888A1 (en) * 1999-05-06 2004-02-19 Jingdong Liu Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
WO2008121992A2 (fr) * 2007-03-30 2008-10-09 Research Foundation Of State University Of New York Virus atténués utiles pour des vaccins
WO2014003583A2 (fr) * 2012-06-26 2014-01-03 University Of The Philippines Diliman Détection de pathogènes
WO2015118491A1 (fr) * 2014-02-06 2015-08-13 University Of The Witwatersrand, Johannesburg Procédé de détection du vih

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Title
DATABASE GenBank [o] 15 February 2015 (2015-02-15), Database accession no. KR 017777.1 *
ISOTHERMAL MASTER MIX, 2012, pages 1 - 3, Retrieved from the Internet <URL:http://www.optigene.co.uk/support> *
OCWIEJA, KE ET AL.: "A Reverse Transcription Loop-Mediated Isothermal Amplification Assay Optimized to Detect Multiple HIV Subtypes", PLOS ONE, vol. 10, no. 2, 12 February 2015 (2015-02-12), pages 1 - 11, XP055390141 *
TONGO, M ET AL., HIV1 ISOLATE BS55 FROM CAMEROON GAG PROTEIN (GAG) GENE , COMPLETE CDS; POL PROTEIN (POL) GENE , PARTIAL CDS; AND VIF PROTEIN (VIF). VPR PROTEIN (VPR), TAT PROTEIN (TAT), REV PROTEIN (REV), VPU PROTEIN (VPU), ENVELOPE GLYCOPROTEIN (ENV), AND NEF PROTE *

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