WO2015118491A1 - Procédé de détection du vih - Google Patents

Procédé de détection du vih Download PDF

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WO2015118491A1
WO2015118491A1 PCT/IB2015/050906 IB2015050906W WO2015118491A1 WO 2015118491 A1 WO2015118491 A1 WO 2015118491A1 IB 2015050906 W IB2015050906 W IB 2015050906W WO 2015118491 A1 WO2015118491 A1 WO 2015118491A1
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seq
nos
hiv
viral load
primers
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PCT/IB2015/050906
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Clement Brian PENNY
Andrea Olga PAPADOPOULOS
Denise Hilary EVANS
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University Of The Witwatersrand, Johannesburg
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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

Definitions

  • This invention relates to a method for testing detectable and undetectable HIV-1 viral load of HIV-1 positive patients.
  • HIV viral load testing is used as a means of monitoring disease progression in HIV positive patients.
  • the viral load result provides an indication of response to or failure of antiretroviral treatment.
  • Successful treatment suppresses viral replication to the extent that an undetectable (less than 400 copies HIV RNA/ml) viral load is maintained, while treatment failure or lack of response to treatment is indicated by a detectable (equal or above 400 copies/ml) viral load which can also be suggestive of progression to AIDS.
  • HIV-1 patient viral load is determined by Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a nucleic acid amplification reaction which requires large and expensive thermocycling devices because the test requires three different temperatures for multiple cycles to amplify the sample nucleic acid.
  • RT-PCR Real Time Reverse Transcriptase Polymerase Chain Reaction
  • the result comes about by detection of amplification, which is done by means of a fluorescent reagent attached to an oligonucleotide probe having a complimentary sequence to the target nucleic acid. As each strand of nucleic acid is synthesised, the fluorescent signal is detected.
  • the reagents and equipment required for the test make it expensive to perform and complex to maintain. It takes five to six hours to obtain a test result. This creates setbacks for disease monitoring of HIV- positive patients on antiretroviral therapy in resource-limited settings.
  • a method of detecting HIV nucleic acids in a sample comprising: amplifying by isothermal amplification at least a portion of HIV-1 using a reverse transcription loop-mediated isothermal amplification (RT-LAMP) primer set selected from the group consisting of SEQ ID NOS: 1 -6, SEQ ID NOS: 7-12, SEQ ID NOS:13-18, SEQ ID NOS: 19-23 and 18, SEQ ID NOS: 24-27 and SEQ ID NOS: 28-33, or primer sets which have nucleic acid sequences which are at least 90% identical to any one of SEQ ID NOS: 1 -6, 7-12, 13-18, SEQ ID NOS: 19-23 and 18, SEQ ID NOS: 24-27 and SEQ ID NOS: 28-33; then visually detecting the amplified product by observing a colour change of hydroxyl-napthol blue colour indicator:
  • R-LAMP reverse transcription loop-mediated isothermal amplification
  • BIP acatacaaactaaagaactatttttttccaaatagggtctctgc (SEQ ID NO: 2);
  • F3 tttcaccagtgctgcagttaaggca (SEQ ID NO: 3);
  • Loop F tactactccctgactttggg (SEQ ID NO: 5);
  • Loop B aattcaaaattttcgggtttatt (SEQ ID NO: 6).
  • FIP tctttattcatggattctaattacatacagacaatggcagt (SEQ ID NO: 7);
  • F3 actaaaattagcaggaagatggc (SEQ ID NO: 9);
  • Loop F tggggattgtagggaat (SEQ ID NO: 1 1 );
  • Loop B caggggggaaagaataatagata (SEQ ID NO: 12);
  • FIP gataccagcccaacaggttttagacaatggcagtaatttcac (SEQ ID NO: 13);
  • F3 actaaaattagcaggaagatggc (SEQ ID NO: 15);
  • Loop F gccttaactgcagcactag (SEQ ID NO: 17);
  • Loop B ggagtagtagaatccatgaataaagaatt (SEQ ID NO: 18); or
  • FIP gatacctgcccaccaacaggttttagacaatggtagtaatttcac (SEQ ID NO: 19);
  • F3 actaaaattagcaggaagatggc (SEQ ID NO: 21 );
  • Loop F gccttaactctagcactgg (SEQ ID NO: 23);
  • Loop B ggagtagtagaatccatgaataaagaatt (SEQ ID NO: 18).
  • cccgtgagaaacatccgg SEQ ID NO: 24;
  • tccccagatagccagagag (SEQ ID NO: 25); cagaccacacctcctggaacgaagactgctgacacagaagg (SEQ ID NO: 26); and agtggtcaaccctcagatgctgggctcagatctggtctacct (SEQ ID NO: 27).
  • FIP tctgcagcctcctcattgattgtatcatcatgttaaatacagtggggggac (SEQ ID NO: 28);
  • BIP agtgagaatgtatagccctgtcagccagcagaaccggtctacatagtctct (SEQ ID NO: 29);
  • F3 gcactatcagagggagcca (SEQ ID NO: 30);
  • Loop F ttaacatttgcatggc (SEQ ID NO: 32);
  • Loop B aagggccaaaggaatctttt (SEQ ID NO: 33).
  • the colour change of the hydroxy-napthol blue may be observable with the unaided eye.
  • the method may detect an HIV viral load of greater than 40 copies/ml in the sample, greater than 400 copies/ml in the sample, or greater than 1 ,000 copies/ml in the sample.
  • the primer set may amplify a target in the integrase region of the po/ gene of HIV-1 , a target in the p24 region of the gag gene of HIV-1 or a target in the Long Terminal Repeat (LTR) region of HIV-1 .
  • the method may comprise the following steps:
  • RNA or plasma from an HIV positive patient a test mixture including:
  • nuclease-free molecular grade water viii. nuclease-free molecular grade water
  • test mixture b) heating the test mixture to from about 55 °C to about 65°C for no longer than about two hours;
  • the HIV-RNA of the positive control in step (d) may have 1000 copies/ ml or greater.
  • the HIV-RNA of the positive control in step (d) may have 400 copies/ ml or greater.
  • the test mixture may be heated to a second temperature after step (b) to stop the reaction, wherein the second temperature is higher than the first temperature.
  • the RT-LAMP mixture may comprise 2 units of reverse transcriptase and 6 to 8 mM MgS0 4 .
  • the reverse transcriptase may be any reverse transcriptase that functions above 60 °C, such as AMV Reverse Transcriptase.
  • the reverse transcriptase may be replaced with water
  • the HIV-1 specific LAMP primers may be replaced with water
  • the BSt polymerase enzyme may be replaced with water.
  • kit for performing the method described above comprising:
  • an HIV-1 specific LAMP primer set selected from the group consisting of SEQ ID NOS: 1 -6, SEQ ID NOS: 7-12, SEQ ID NOS:13-18, SEQ ID NOS: 19-23, SEQ ID NOS: 24-27 and SEQ ID NOS: 28-33, or primer sets which have nucleic acid sequences which are at least 90% identical to any one of SEQ ID NOS: 1 -6, 7-12, 13-18, SEQ ID NOS: 19-23, SEQ ID NOS: 24-27 and SEQ ID NOS: 28-33; and c) HNB dye.
  • the kit may further include one or more of the following reagents: reverse transcriptase, BSt polymerase, deoxy-nucleotide tri-phosphates; magnesium sulphate, molecular biology-grade betaine, a suitable BSt polymerase reaction buffer and/ or nuclease-free molecular grade water.
  • the kit may further include a positive control sample (reference sample) of plasma or RNA from a patient with an HIV-1 viral load which is equal to or greater than the detectable viral load.
  • the kit may further include a positive control sample (reference sample) of RNA obtained from HIV-1 positive plasma, RNA or tissue culture which is equal to or greater than the detectable viral load and a positive control which is less than the detectable viral load.
  • the kit may further include a negative control sample of plasma or RNA from an HIV-negative subject or other tissue culture.
  • the kit may further include instructions for performing the method of the invention and analysing the results.
  • a primer set comprising primers which have nucleotide sequences which are at least 90% identical to the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 -6, 7-12, 13-18, 19-23 and 18, 24-27 and 28-33.
  • FIGURES Figure 1 shows a schematic representation of initiation of Loop-mediated isothermal amplification showing loop-formation and strand-displacement (Notomi et al., 2000).
  • Figure 2 shows the relation between turbidity of the LAMP reaction mixture and its DNA yield during continuous amplification reaction process (Mori et al., 2001 ).
  • Figure 3 shows a schematic diagram of the method of the present invention.
  • Figure 4 shows hydroxy-naphthol blue interaction with free Mg 2 + allowing for colour change from violet to sky-blue in an RT-LAMP reaction.
  • C and D The effect of Reverse transcriptase on available Mg 2+ and reaction colour in negative reactions: C) 1 -7: 10mM MgS0 4 ; 1 ,3,5 and 7: 10U AMV RT; 2,4, and 6: 0U AMV RT. D) 1 -6: 7mM MgS0 4 and 2U AMV RT.
  • Figure 5 shows the effect of different inner primer template on HNB induced colour progression using 6mM MgS0 4 . 1 -2: no primer-template control; 3-4:1 .6 ⁇ ; 5-6: 3.6 ⁇ ; 7-8: 16 ⁇ .
  • Figure 6 shows the effect of different template concentration on HNB induced colour progression for 5 minute and 60 minute LAMP reactions respectively.
  • Figure 7 shows the use of sub-type C integrase-targeted primers in the RT-LAMP HIV-1 viral load assay.
  • A The amplification of HIV-1 RNA produced a colour change from violet to blue whereas all negative controls remained violet; NTC: No template control.
  • B Analysis of RT-LAMP amplification products by 2% TBE agarose gel electrophoresis; NTC: No template control.
  • Figure 8 shows the specifity of integrase-targeted RT-LAMP primers for HIV-1 amplification by BSe XI restriction digestion analysis on a 2% TBE agarose gel.
  • DIG Digested RT-LAMP product.
  • UNDIG Undigested RT-LAMP product.
  • the present invention provides a qualitative method for testing detectable and undetectable HIV-1 viral load of HIV-1 positive patients.
  • the method makes use of the RT-LAMP assay (Reverse Transcriptase Loop Mediated Isothermal Amplification) and a colour indicator to visually indicate whether the HIV viral load of the patient being tested is above or below a certain threshold (hereinafter referred to as a "detectable viral load").
  • the colour indicator which is used in the method is preferably hydroxy-naphthol blue (HNB), a metal-chelating colorimetric dye.
  • HNB hydroxy-naphthol blue
  • the assay can thus provide a semi-quantitative, colorimetric result read-out.
  • a detectable HIV viral load is currently regarded in South Africa as being 400 copies/ml or above, but it will be appreciated by a person skilled in the art that what is regarded as a detectable viral load may change with time or from country to country. Therefore, the method of the present invention may be set to test for detectable viral loads of anywhere from 40 to 1000 copies/ml, such as 50 copies/ml or above, 100 copies/ml or above, 200 copies/ml or above, 300 copies/ml or above, 400 copies/ml or above, 500 copies/ml or above, 600 copies/ml, 800 copies/ml or above, or even 1 000 copies/ml or above.
  • the LAMP reaction takes place by oligonucleotide-primer directed addition of deoxyribo-nucleotides to a growing chain of DNA, catalysed using a high-strand displacement activity polymerase (Notomi et al., 2000, Japanese patent 3313358, which is incorporated herein by reference).
  • heat denaturation brings about template and daughter strand displacement. This serves to initiate each cycle of PCR, after which oligonucleotide primers can bind to the now single-stranded DNA.
  • This step is not required in the LAMP reaction as the BSt Polymerase catalysing the reaction displays characteristically high strand displacement activity, which is further mediated by the interaction between 2 distinct pairs of primers, namely outer primers (F3 and B3) and loop-generating inner primers (FIP and BIP).
  • the inner primers consist of 2 sequences, 1 complimentary to the target and 1 which is identical to a sequence further downstream, forming a 5' overhang when the first piece binds. The overhang will subsequently loop back and bind to its complement on the daughter strand).
  • outer primers which are much like the forward and reverse primers used in PCR, will bind upstream to the inner primers, displacing the stem-loop structured, daughter strand) ( Figure 1 ). Therefore, with no need for heat denaturation at the start of each reaction, LAMP is run at a temperature equivalent to the annealing step of PCR and terminated at 85 °C within an average incubation period of 1 to 2 hours.
  • LAMP has been adapted for RNA amplification with the addition of a robust reverse transcriptase (RT) with the LAMP reaction mixture, allowing for simultaneous cDNA synthesis and amplification in a single step (Notomi et al., 2000; Buates et al., 2010; Cardoso et al., 2010).
  • RT reverse transcriptase
  • the activity of AMV RT is optimal at temperatures between 50 ⁇ and 65 °C, which fits in with the LAMP incubation temperature range required by BSt Polymerase.
  • the LAMP reaction produces magnesium pyrophosphate, an insoluble by-product, as a result of the interaction between magnesium ions and phosphates of the deoxy-ribonucleotides (dNTPs), both of which are present in the reaction mixture.
  • dNTPs deoxy-ribonucleotides
  • amplification can be detected by observing the precipitate of the by-product within the reaction tube, measuring the turbidity of the reaction ( Figure 2), or using fluorescent probes and intercalating dyes to detect the nucleic acid product itself.
  • the HNB dye is used to detect the magnesium pyrophosphate, bringing about a colour-change reaction from violet to blue that can be seen with the unaided eye. This allows for a simple result read-out of the test, which is important in resource-limited settings that are characterised by a shortage of skilled laboratory staff.
  • RT-LAMP assays have previously been developed for detecting the absence or presence in a sample of a number of pathogens, including HIV-1 (Curtis et al. (2008)) and combined with HNB for the detection of H1 N1 (Ma et al., 2010).
  • pathogens including HIV-1 (Curtis et al. (2008)) and combined with HNB for the detection of H1 N1 (Ma et al., 2010).
  • HNB H1 N1
  • the RT-LAMP reaction is applied in combination with hydroxy-naphthol blue (HNB) to distinguish between undetectable (e.g. less than 1 ,000 or even less than 400 copies RNA per ml plasma) and detectable (400 copies per ml and above, e.g. 1 ,000 copies per ml and above) HIV-1 viral load.
  • HNB hydroxy-naphthol blue
  • the reaction is stopped after a determined time(generally 1 - 2 hours), at which time a colour change will indicate detectable viral load (Figure 3).
  • the reaction can also be used to diagnose HIV-1 infection (e.g. using the primer set with primers of SEQ ID NOS: 28-33).
  • Primers targeting three different HIV-1 conserved regions were designed and/or identified.
  • the primers targeting gag have been used for RT-LAMP HIV-1 detection by Curtis et al (2008), while the primers targeting the pol and LTR regions have been specifically designed for the present invention using online tools (Table 1 ).
  • Table 1 Online tools used to aid design of RT-LAMP primers against the HIV-1 Pol gene
  • kits for performing the method of the invention will be provided.
  • the kit could include the LAMP reagents and HNB, and instructions for performing the method of the invention.
  • the kit could further include a positive control or reference sample with a detectable HIV-1 viral load (e.g. greater than or equal to 400 copies/ ml, such as greater than 1 ,000 copies/ ml).
  • the kit could also include a negative control sample.
  • the present invention is particularly suited for use in resource-poor settings, such as rural areas in South Africa, where access to expensive and high technology equipment such as thermocyclers and spectrophotometers is limited and skilled laboratory staff are in short supply.
  • the reaction of the present invention takes place at a constant temperature and makes use of relatively inexpensive reagents (such as HNB), it is significantly cheaper than RT-PCR, requiring only pipettes, a bench-top centrifuge, a heating block and possibly a fridge or freezer (thermocyclers and expensive reagents are not required).
  • LAMP reagents and HNB can be stored together as a readily available test mixture, the test is also simpler to perform than conventional RT-PCR tests.
  • Individual or combined reagents, buffers, primers and controls may be stable at room temperature or may require refrigeration. Lyophilized reagents, buffers, primers or controls which require no refrigeration may be provided.
  • the present invention can also provide a result within 2 hours, which means that patients, who may not be telephonically contactable or who may live far from a clinic and may not have access to transport, do not have to wait at the clinic for a long period or return to the clinic on another day to receive their results and possibly to have their treatment adjusted.
  • Oligonucleotide primers including published primers and self-designed primers were designed for LAMP according to the recommendations of Notomi et al. (2000) for the outer and inner primer sets and Nagamine et al. (2002) for the Loop primer pair.
  • primer sets were assessed, including integrase-1 , -2, -3, integrase-DU179, Long Transcript Repeat (LTR)-1 , and published primers targeting p24 (Curtis et al., 2009). Once primer sequences were obtained and synthesised, each set was applied to the RT-LAMP reaction and tested for specificity to HIV-1 using restriction digestion analysis.
  • Integrase-1 The integrase-1 primer set (SEQ ID NOS: 1 -6) was designed against the integrase gene region of the Pol gene of South African HIV-1 isolate 05ZAJF04 (GenBank accession no. GQ872538.1 ). Primers iF3 and iB3 were obtained using NCBI primer blast (Ye et al., 2012), while the inner (iFIP and iBIP) and loop primers (iLoopF and iLoopB) were self-selected according to the guidelines of Notomi et al. (2000) and Nagamine et al. (2002).
  • lntegrase-2 The second set of primers (SEQ ID NOS: 7-12) targeting the integrase region was designed against a sub-type C reference sequence (GenBank accession no. AF286227) listed in the Los Alamos reference sequence database.
  • lntegrase-DU179 The third and fourth sets of primers (SEQ ID NOS: 13-18 and SEQ ID NOS: 19-23 and 18, respectively) were designed against the sequence of isolate DU179 (GenBank accession no. AY043174.1 .), for specific RT-LAMP testing on the DU179 culture supernatant sample.
  • LAMP primer design software Primer Explorer 3 (Fujitsu Ltd.) was used.
  • Primer Explorer 3 generated a list of possible primer sets for the outer (DF3 and DB3) and inner primer pairs (DFIP and DBIP).
  • a single set of primers was selected by a process of elimination by assessing each set for self- and cross-hybridisation using the jPCR software (Kalender, Lee and Schulman, 201 1 ).
  • Loop primers, DIoopF and DIoopB were self-selected for the chosen set of primers according to the guidelines of Nagamine et al. (2002) and the final set of 6 primers was assessed using jPCR (Kalender et al., 201 1 ) and for specificity to HIV-1 across the nucleotide database of the nucleotide BLAST alignment tool (Altschul et al., 1990).
  • FIP cagcttcctcattgatggtttctttttaacaccatgctaaacacagt (SEQ ID NO: 34)
  • Loop F tttaacatttgcatggctgcttgat (SEQ ID NO: 38)
  • Loop B gagatccaaggggaagtca (SEQ ID NO: 39)
  • LTR-1 LTR-1 DNA (and not RNA), to ascertain if they could be used in the assay to diagnose or detect HIV-1 , rather than in a semi-quantitative assay to determine viral load.
  • FIP cagaccacacctcctggaacgaagactgctgacacagaagg
  • BIP agtggtcaaccctcagatgctgggctcagatctggtctacct (SEQ ID NO: 41 )
  • the assay was first optimized so as to ensure that it is capable of distinguishing between detectable and undetectable viral load consistently.
  • This initial optimisation phase consisted of designing and testing LAMP oligonucleotide primers; determining concentrations of various reaction components for optimal RT-LAMP amplification; extrapolating the minimum duration of the reaction upon which a colour change from violet to blue indicates a detectable viral load (at least 1000 copies HIV RNA/ml); and applying the optimised reaction conditions to semi-quantify viral load results of 27 South African HIV positive samples.
  • the RT-LAMP viral load assay was developed using 3 sources of live HIV virus - whole blood of infected individuals, WHO 2nd international HIV-1 standard plasma (NIBSC) and HIV-1 culture supernatant isolate DU179, a gift from the Lynn Morris virology lab (The Centre for HIV & Sexually Transmitted Infections, NICD, Africa). Whole blood was also collected from 5 HIV-negative, healthy volunteers.
  • LAMP reaction mixtures containing BSt polymerase; reaction buffer (Tris-HCI, (NH 4 ) 2 S0 4 , KCI, MgS0 4 , Triton X-100); dNTPs; MgS0 4 ; betaine; hydroxy-naphthol blue dye and LAMP inner primer were made up to volumes of 25 ⁇ or 50 ⁇ with nuclease- free water and incubated across a range of incubation temperatures from 58°C to 64°C (more specifically, from 59 °C to 62 °C, and even more specifically at 60 °C) for up to 2 hours followed by inactivation at 85 °C for five minutes.
  • RT-LAMP reactions were performed as above with the addition of AMV RT, LAMP outer primers and loop-targeting primers.
  • Negative control reactions were performed by excluding AMV RT, or primers or by inactivation following five-minute incubation. Mg 2 P 2 0 7 by-product was precipitated by pulse centrifugation
  • RT-LAMP is performed optimally at the annealing temperature of the F3 and B3 primer sequences (Notomi et al., 2000). For this reason, some primer sets were subjected to RT LAMP incubation at a temperature as low as 55 °C, though generally RT-LAMP is performed between 60 °C and 65 °C. The RT-LAMP reaction was found to be quite robust with regards to temperature, performing consistently at different incubation temperatures.
  • the DU179 primers successfully amplified HIV at 59°C, 60 °C and 62 ⁇ ⁇ .
  • RNA was best extracted from plasma in serum separator tubes (SST), followed by concentration of viral particles in 1 ml of plasma by high speed centrifugation. The viral pellet is then resuspended in 200 ⁇ PBS as starting material for the Qiagen Viral RNA mini procedure and processed according to the manufacturer's protocol, but with exclusion of the optional step of drying out the column prior to elution. All plasma and RNA should be stored at - 70 °C.
  • HNB detection is more suitable than DNA intercalating dyes such as Sybr Green or Pico Green, which will detect any double-stranded DNA produced in a reaction. From the results shown in Figures 4 to 6, it can be seen that HNB can distinguish between samples of low and high amounts of template, and therefore can be implemented semi- quantitatively or qualitatively in a LAMP-based viral load assay.
  • RNA obtained from culture supernatant isolate DU179 was added to an RT-LAMP reaction mixture containing BSt polymerase; reaction buffer (Tris-HCI, (NH 4 ) 2 S0 4 , KCI,
  • MgS0 4 Triton X-100
  • dNTPs dNTPs
  • MgS0 4 betaine
  • hydroxy-naphthol blue dye Thermoscript enhanced AMV RT, nuclease-free water and six primers (FIP, BIP, F3, B3, Loop F, Loop B) targeting the integrase-region of the pol gene of an HIV-1 sub-type C reference sequence.
  • the 25 ⁇ volume reaction was incubated at 59 °C for 60 minutes followed by inactivation at 85 °C for five minutes.
  • RT-LAMP was performed with the same primer set as above but adjusted at single nucleotides to match the integrase sequence of culture supernatant isolate DU179.
  • RNA obtained from culture supernatant isolate DU179 was added to an RT-LAMP reaction mixture containing BSt polymerase; reaction buffer (Tris-HCI, (NH4)2S04, KCI, MgS04, Triton X-100); dNTPs; MgS04; betaine; hydroxy-naphthol blue dye, Thermoscript enhanced AMV RT, nuclease-free water and six primers (FIP, BIP, F3, B3, Loop F, Loop B) targeting the integrase-region of the pol gene of an HIV-1 sub-type C reference sequence.
  • the 25 ⁇ volume reaction was incubated at 59 °C for 60 minutes followed by inactivation at 85 °C for five minutes.
  • the amplification product was purified using a commercial silica-column affinity technique and stored in Tris-HCI prior to digestion with a commercial restriction endonuclease, Sse XI.
  • the qualitative detection of the HIV-1 DU179 was demonstrated, evident as a HNB-induced colour-change from violet to blue while the employed negative controls remained violet, following application of the optimisations that were carried out.
  • the result of agarose gel analysis in figure 8 of the RT-LAMP product produced by the integrase-targeted primers, specific to HIV isolate DU179 displays a change from the ladder banding pattern of the undigested product to two concentrated bands of the digested product, showing the RT-LAMP assay was able to amplify HIV RNA specifically.

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Abstract

La présente invention concerne une méthode de test semi-qualitative d'une charge virale de VIH-1 chez des patients VIH-1 positifs. Le procédé fait appel à l'analyse RT-LAMP (Transcriptase inverse et amplification isotherme induite par boucle) et au bleu d'hydroxy-naphtol (HNB), un indicateur coloré, afin d'indiquer visuellement si la charge virale du VIH du patient testé est inférieure ou égale à un certain seuil. L'invention porte sur la conception d'ensembles d'amorces destinés à être utilisés dans cette méthode qui amplifient une cible dans la région de l'intégrase du gène pol du VIH-1, du gène gag du VIH-1 ou dans la région de la longue répétition terminale (LTR) du VIH-1.
PCT/IB2015/050906 2014-02-06 2015-02-06 Procédé de détection du vih WO2015118491A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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CN106119413A (zh) * 2016-07-01 2016-11-16 浙江省疾病预防控制中心 一种艾滋病病毒多重荧光pcr检测试剂盒及检测方法
WO2017099801A1 (fr) * 2015-12-11 2017-06-15 The Trustees Of The University Of Pennsylvania Compositions, kits et méthodes pour détecter le virus vih
CN107326102A (zh) * 2017-08-01 2017-11-07 广西中医药大学附属瑞康医院（广西中西医结合医院） 一种艾滋病试剂盒
US10731227B2 (en) 2014-12-12 2020-08-04 The Trustees Of The University Of Pennsylvania Compositions, kits, and methods to detect HIV virus
CN113249516A (zh) * 2020-02-10 2021-08-13 华南理工大学 一种2019-nCoV的引物组和2019-nCoV核酸检测试剂盒

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