M. Nosik, K. Ryzhov

I.I. Mechnikov Research Institute for Vaccines and Sera RAMS, Moscow, Russia

DESIGN OF PROBES TO DETECT POINT MUTATIONS IN HIV-1 GENOME BY MICROCHIP ASSAY

High variability is characteristic for the human immunodeficiency virus -1 (HIV-1) genome. Due to that a large number of quasispecies emerge with high rate in one organism. As a result it is quite difficult to study HIV by genotype assays such as polymerase chain reaction (PCR) and hybridization.

Currently, in HIV-1 genome there are detected regions with low variability (conservative regions) and regions with high genetic variability (high variable regions). Besides that different genetic subtypes are characteristic for HIV-1. These subtypes are unevenly distributed in different parts of the world. At present time HIV-1 subtype A prevails in the territory of Russia. However this variant of HIV-1 subtype causes less than 5% cases of HIV-1 infections worldwide. Thus there are practically no available data about subtype A genetic sequences in the world genetic database.

As the result of HIV high genetic variability new HIV-1 variants resistant to drugs emerge with high frequency in the process of antiretroviral therapy. The mutations causing the drug resistance during the certain medical treatment frequently trig the resistance to the other drugs which were not used previously. In view of this for the effective treatment timely detection of such mutations is one of the major diagnostics tasks. And in case such mutations emerge in process of antiretroviral therapy the treatment regimen could be appropriately changed.

Until very recently the cultural method was the only assay for detection of drug resistant viruses. But notwithstanding the efficiency of that method it has several disadvantages. Besides that this method is rather time-consuming it does not allow to detect which mutations in particular cause drug resistance. Lately the sequencing is widely used for the detection of drug resistant mutations. This method is more accurate and less time-consuming. However the application of this method is highly restricted by the necessity of rather expensive equipment and reagents.

Up to the date apart from sequencing there is no test kit for the detection of point mutations associated with HIV-1 resistance to antiviral drugs. The test kits based on the hybridization of nucleotide probes with HIV-1 proviral DNA could be an alternative to sequencing. This could be real time PCR or test system based on the principals of microchip technology. But by real time PCR one could detect no more than 3 mutations in one tube even when using 6-channel amplifier.

The principle of the microchip method (or biochips) consists in using the set of nucleotide probes - which differ by one nucleotide in the mutation position - for the detection of mutation associated with drug resistance. The isolated genetic material is amplified for enlarging the fragment which might have the targeted mutation. Afterwards hybridization of the enlarged fragment is performed with the set of probes. The derived duplexes are detected by fluorescence and colorimetric methods. The presence of mutation in the studied sample is confirmed by the distinct signal in the cell containing mutation associated with drug resistance. This signal indicates that an ideal duplex is formed. The signal detected in other cell indicates the absence of mutation. The absence of signal in all cells indicates that the reaction was not performed or that the set of nucleotide probes was not rightly selected. Thus in one performance one could detect the majority of all known point mutations.

The goal of that work was to calculate and design probes for performance of hybridization. For calculation of probes sequences the Stanford University HIV Drug Resistance Database was used (http://hivdb.stanford.edu/).

For excluding the false positive result it was necessary to design the set of eight probes (6 nucleotides each) for each mutation. Each probe differed by 1 nucleotide in the mutation position: 4 probes to direct sequence and 4 probes to complementary sequence. In the course of the work 96 probes to 12 mutations associated with drug resistance were designed:

*M41L AGATGG AGTTGG AGCTGG AGGTGG CCATCT CCAACT CCAGCT CCACCT

*L74V TTTGGG TGTGGG TATGGG TCTGGG CCCAAA CCCACA CCCATA CCCAGA

*Y115F CATATT CATTTT CATCTT CATGTT AATATG AAAATG AAGATG AACATG

*Q151M TCCAGA TCATGA TCCTGA TCAAGA TCTGGA TCATGA TCAGGA TCTTGA

*M184V ACATGG ACGTGG ACCTGG ACTTGG CCATGT CCACGT CCAGGT CCAAGT

*L210W TGTTGA TGTGGA TGTAGA TGTCGA TCAACA TCCACA TCTACA TCGACA

*T215Y CACCAG CTCCAG CGCCAG CCCCAG CTGGTG CTGGAG CTGGCG CTGGGG

*K219Q AAAAGC AACAGC AATAGC AAGAGC GCTTTT GCTGTT GCTATT GCTCTT

^E138K ATGAGA ATAAGA ATCAGA ATTAGA TCTCAT TCTTAT TCTGAT TCTAAT

^Y181C GTTATC GTTGTC GTTCTC GTTTTC GATAAC GACAAC GAGAAC GAAAAC

^M230L GATGGG GCAGGG GAAGGG GCTGGG CCCATC CCCTGC CCCTTC CCCAGC

**L76V CTTTGA CTGTGA CTCTGA CTATGA TCAAAG TCACAG TCAGAG TCATAG

Left column: point mutations associated with drug resistance.

Italics: probes to consensus sequence of HIV-1 subtype A.

Bold: nucleotide substitution associated with drug resistance.

*NRTI (Nucleoside RT Inhibitor Resistance Mutations):

M41L (Abacavir, Didanosine, Lamivudine, Emtricitabine)

L74V (Abacavir, Didanosine)

Y115F (Abacavir, Tenofovir)

Q151M (Abacavir, Didanosine, Tenofovir , Stavudine, Zidovudine, Lamivudine, Emtricitabine )

M184V, I (Abacavir, Didanosine, Lamivudine, Emtricitabine)

L210W (Abacavir, Didanosine, Tenofovir , Stavudine, Zidovudine)

T215Y, F (Abacavir, Didanosine, Tenofovir , Stavudine, Zidovudine)

K219Q, E (Stavudine, Zidovudine)

^NNRTI (Non-Nucleoside RT Inhibitor Resistance Mutations):

E138K (Nevirapine, Efavirenz, Etravirine, Rilpivirine)

Y181C, Y, V (Nevirapine, Efavirenz, Etravirine, Rilpivirine)

M230L (Nevirapine, Efavirenz, Etravirine, Rilpivirine)

**PI (Protease Inhibitor Resistance Mutations):

L76V (Darunavir, Fosamprenavir, Indinavir, Kaletra)

References

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