KR101542507B1 - Hiv-1 - -1 - Google Patents

Abstract

The use of the primer set and the premix composition for amplification of HIV-1 infection or antiviral-derived antiretroviral-related resistance gene of ADIS according to the present invention can provide a wide range of HIV -1-related resistance gene sequences can be analyzed.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for amplification of an antiretroviral-related resistance gene from a nucleic acid extracted from a plasma, which is an HIV-1 infection, and a premix composition containing the primer set and amplified antimicrobial composition.

The present invention relates to a primer set for amplification of an antiretroviral-related resistance gene from a nucleic acid extracted from plasma, which is an HIV-1 infection, and a premix composition containing the primer set.

In HIV-1-infected and AIDS patients, antiretroviral resistance test results are used as useful guidelines in HIV-infected hospital for monitoring progress of antiretroviral therapy. The National Institutes of Health is conducting an antiretroviral resistance test using the in house method established by Stanford University in the past. In addition, the ViroSeq kit is used in some university hospitals or clinical examination centers have.

However, in the conventional in house method, there is a disadvantage that reproducibility may occur depending on the experimenter, and when the experiment is immature, the PCR is not smoothly performed and the frequency of detection is different according to the sample .

Furthermore, in the case of commercialized kits, the problem of too high a unit price has not been solved because the number of medical insurance for current inspection items can not be solved. Therefore, the present inventors have found that a pre-mix kit for HIV-1 resistant gene amplification, which has a higher detection rate than that of the conventional in-house method and solves the reproducibility problem due to the change of the experimenter, Thereby completing the present invention.

An object of the present invention is to provide a method for amplifying an HIV-1 drug resistance gene superior to the conventional method by performing RT-PCR and 2nd PCR using a primer specific to a target gene of HIV-1.

It is another object of the present invention to provide a premix composition for amplifying the HIV-1 viral antiretroviral resistance gene and a kit for amplifying a target gene.

In one embodiment of the present invention, there is provided a primer set for RT-PCR for the amplification of antiretroviral-related resistance gene derived from HIV-1 infection comprising SEQ ID NO: 1 and SEQ ID NO:

In one embodiment of the present invention, there is provided a 2 ^nd PCR primer set for cDNA amplification for amplification of antiretroviral-related resistance gene derived from HIV-1 infection comprising SEQ ID NO: 3 and SEQ ID NO: 4.

In one embodiment of the present invention, there is provided a primer set for SEQ ID NO. 1 and SEQ ID NO. 2, or a primer set for SEQ ID NO. 3 and SEQ ID NO. 4 for a PCR for HIV-1 drug resistance gene amplification.

In one embodiment of the present invention, there is provided a method of amplifying cDNA comprising the steps of: synthesizing cDNA with a primer set for RT-PCR of SEQ ID NO: 1 and SEQ ID NO: 2; And a step of amplifying the target gene with the PCR primer set of SEQ ID NO: 3 and SEQ ID NO: 4. In this embodiment, there is provided a method for amplifying a gene of HIV-1 virus, wherein the target gene is a gag-pol gene. In this embodiment, the cDNA synthesis step is carried out at 50 ° C for 30 minutes, the pre-modification is carried out at 94 ° C for 10 minutes, and the annealing step is carried out at 55 ° C for 20 seconds. Amplification method. In this embodiment, the amplification step of the target gene is carried out by using 2ul of the first RT-PCR product, and the annealing step is carried out at 60 ° C or 65 ° C for 30 seconds. do.

In one embodiment of the present invention, cDNA is synthesized with a primer set for RT-PCR including the primers of SEQ ID NO: 1 and SEQ ID NO: 2; ^And an amplification step of HIV-1 gene of interest with a 2 ^nd PCR primer set comprising the primers of SEQ ID NO: 3 and SEQ ID NO: 4, wherein the amplified HIV-1 gene is subjected to base sequence analysis and then analyzed for resistance to antiretroviral agents The method comprising the steps of: In the above embodiments, the antiretroviral agent is used as a combination therapy and is any one or more selected from the group consisting of a protease inhibitor, a nucleic acid-based reverse transcriptase inhibitor and a non-nucleic acid-based reverse transcriptase inhibitor. The method comprising the steps of:

A "biomarker" is a molecular indicator of a specific biological characteristic, biochemical characteristic or aspect that can be used to measure the progress of a disease or the effect of treatment. Proteins and nucleic acids are exemplary biomarkers. In particular, nucleic acids have been identified in most body fluids, including blood, urine and cerebrospinal fluid, and have been successfully applied for use as diagnostic biomarkers for disease.

To date, various methods have been developed for measuring retroviruses, which can be classified into three broad categories. The first method is to measure the amount of genome RNA of virus using reverse transcriptase activity or dot blot hybridization reaction or semi-quantitative RT-PCR method, And measuring the amount of virus in the supernatant. The second method is indirectly measuring the concentration of the virus by measuring the expression of the gene introduced by the retrovirus into the target cell. In this method, target cells are selectively cultured in a culture medium containing a specific drug using various marker genes capable of distinguishing between a cell into which a gene is introduced and a cell not containing the gene, thereby calculating the number of colonies, And the concentration of the virus is determined by determining the number of infected cells by staining. The third method is to measure the retrovirus concentration by measuring the amount of retroviral genomic DNA inserted into the genome of the target cell. Southern blot analysis is generally used for this purpose. This method has the advantage of being able to measure the infectivity of retroviruses and selecting the probe used for the hybridization reaction to be able to measure the concentration of general retrovirus regardless of the inserted gene, It is difficult to obtain, time consuming and troublesome.

The HIV-1-derived resistant gene amplification method using the premix kit of the present invention has a reduced cost and extended detection range compared with the conventional in house method, and even in the case of a sample which is not amplified by the conventional in house method, Of the HIV-1 resistant gene is amplified for some samples.

FIG. 1 shows the result of the conventional in house method and the premix method produced by the present invention for a sample collected from a patient infected with HIV-1, and 4 out of 7 samples Is detected through the method of the present invention.
FIG. 2 shows the results of the 2 ^nd PCR of the present invention using RT-PCR products obtained by the conventional In house method. As a result, only a small part of the target gene (4,5, 1.5 Kb) It is the result that it can not be properly amplified.

Patient origin HIV - From 1 HIV -1 Target gene amplification

Among the samples (plasma) of patients infected with HIV-1, samples which did not amplify the target gene by the conventional in house method were selected. In order to extract HIV-1 RNA from the samples, nucleic acid was extracted from the samples according to the manual using the Abbott molecular mass spectrometer (m2000sp; Abbott molecular Inc., Des Plaines, IL, USA) or QIAamp Viral RNA mini kit (QIAGEN, USA) . Sequence of the primer used for RT-PCR (SEQ ID NO: 1 and 2), and 2 ^nd PCR (SEQ ID NOS: 3 and 4) were represented in Table 2 below.

Primer sequence (existing in house) existing
In house primer Target gene order RT-PCR
MAW26
(RT-F-Outer) 2028-2050; HXB2 5 'TTG GAA ATG TGG AAA GGA AGG AC 3' RT-21
(RT-R-Outer) 3539-3509; HXB2 5'CTG TAT TTC TGC TAT TAA GTC TTT TGA TGG G 3 ' 2 ^nd PCR

PRO1
(PCT-F-Inner) 2147-2166; HXB2 5'CAG AGC AGA CCA GAG CCA ACA GCC CCA CCA 3 ' RT-20
(PCT-R-Inner) 3462-3441; HXB2 5'CTG CCA GTT CTA GCT CTG CTT C 3 '

The primer sequence (the present invention) germinal
invent order
number primer Target gene order RT-PCR
One RT-OJF-Outer 1876-1898; HXB2 5 'TG GCT GAA GCA ATG AGC CAA GT 3 2 RT-OJR-Outer 3544-3521; HXB2 5 'TGC TTC TGT ATT TCT GCT ATT AAG 3' 2 ^nd PCR 3 OJF-Inner 2019-2039: HXB2 5 'AAA GGG CTG TTG GAA ATG TGG 3' 4 OJR-Inner 3474-3454: HXB2 5 'TCT CTC TGT TTT CTG CCA GTT 3'

RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) and 2 ^nd PCR reactions were carried out using KNIH-OJ One-step RT PCR (HIV-1) kit and KNIH-OJ One-step PCR 3 to Table 6. < tb >< TABLE >

One-step RT-PCR reaction conditions step Temperature time Number of cycles One 50˚C 30 min One 2 94˚C 10 min One 3 94˚C 15 sec 40

4 55˚C 20 sec 5 72˚C 2 min 6 72˚C 10 min One 7 4 ° C ∞ One

One-step RT PCR reaction composition KNIH-OJ One-step RT PCR kit (HIV-1) Component volume 4X Reaction Premix (w / enzyme 1 unit) 12.5 μl Primer F (10 pmol / ul) of SEQ ID NO: 2.0 μl Primer R (10 pmol / ul) of SEQ ID NO: 2.0 μl Distilled water 8.5 μl synthesis 25.0 uL KNIH-OJ One-step RT PCR kit (HIV-1) 25 μl The sample (nucleic acid) 15 μl Distilled water 10 μl synthesis 50 μl

2 ^nd PCR reaction conditions step Temperature time Number of cycles One 94˚C 2 min One 2 94˚C 15 sec
35
3 60˚C or 65˚C 30 sec 4 72˚C 2 min 5 72˚C 10 min One 6 4 ° C ∞ One

2 ^nd PCR reaction composition KNIH-OJ One-step PCR kit (HIV-1) 2X PreMIX (w / enzyme 1 unit)
- 10X Reaction Buffer Mix
- 2.5 mM dNTP mixture
- - Polymerase (1 unit) 25.0 uL Primer F of SEQ ID NO: 3
(10 pmol / ul) 2.0 μl The primer R of SEQ ID NO: 4
(10 pmol / ul) 2.0 μl Distilled water 1.0 μl synthesis 30.0 μl KNIH One-step PCR kit (HIV-1) 30 μl RT PCR product 2 μl Distilled water 18 μl synthesis 50 μl

RT PCR and 2 ^nd PCR were performed according to the above Tables 3 to 6 to obtain the same results as in Fig. 1 (M: 1 kb ladder, 1 to 7: clinical sample, N: negative control). As shown in FIG. 1, in the conventional In house method, the target gene of 1.3 kb was not amplified. However, when a premix produced using the modified primer of the present invention (target gene of 1.5 kb) is used, And four of the seven non-amplified samples were amplified. In addition, RT-PCR was performed using the conventional in house method, and 2 ^nd PCR was performed on the synthesized cDNA, and it was confirmed that most of the target gene was not amplified correctly in 7 samples (FIGS. 1 and 2 ).

Table 7 below compares the existing Inhouse method with the method of the present invention.

Comparing the existing in-house method with the present invention division
Original House Invention of the present invention compare 1 ^st PCR





Reverse transcription
45 ° C 30 min 50 ° C 30 min Different Pre-denaturation
94 ° C 2 min 94 ° C 10 min Different denaturalization 40
Cycle

94 ° C 15 sec 94 ° C 15 sec Same Annealing 55 ° C 30 sec 55 ° C 20 sec Different kidney 72 ° C 2 min 72 ° C 2 min Same Final height
72 ° C 10 min 72 ° C 10 min Same keep
4 ℃ 4 ℃ Same 2 ^nd PCR





1 ^st PCR product
3ul 2 ul Different Pre-denaturation
94 ° C 2 min 94 ° C 2 min Same denaturalization 35
Cycle

94 ° C 15 sec 94 ° C 15 sec Same Annealing 63 ° C 30 sec 60 ° C or 65 ° C 30 sec Different kidney 72 ° C 2 min 72 ° C 2 min Same Final height
72 ° C 10 min 72 ° C 10 min Same keep
4 ℃ 4 ℃ Same

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. It will be possible. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

<110> KOREA CENTER FOR DISEASE CONTROL AND PREVENTION <120> Primer set for the amplifying antiretroviral drug          resistance-related HIV-1 gene and Premix Composition with the          primer set <130> IPDB49720 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> HUMAN <400> 1 ttggctgaag caatgagcca agt 23 <210> 2 <211> 24 <212> DNA <213> HUMAN <400> 2 tgcttctgta tttctgctat taag 24 <210> 3 <211> 21 <212> DNA <213> HUMAN <400> 3 aaagggctgt tggaaatgtg g 21 <210> 4 <211> 21 <212> DNA <213> HUMAN <400> 4 tctctctgtt ttctgccagt t 21

Claims (9)

delete delete A primer composition for RT-PCR comprising a primer set for RT-PCR for amplification of an antiretroviral-related resistance gene derived from an HIV-1 infection comprising a sense primer of SEQ ID NO: 1 and an antisense primer of SEQ ID NO: 2; And
A primer composition for secondary PCR comprising a sense primer of SEQ ID NO: 3 and an antisense primer of SEQ ID NO: 4 and a second PCR primer set for cDNA amplification for amplification of an antiretroviral-related resistance gene derived from HIV-1 infection
Wherein the anti-retroviral agent is selected from the group consisting of HIV-1 and HIV-1.
Synthesizing cDNA with the primer set for RT-PCR according to claim 3; And
A method for amplifying an HIV-1 gene comprising the step of amplifying a target gene with a second PCR primer set for cDNA amplification of claim 3.
5. The method of claim 4,
Wherein the target gene is a gag-pol gene.
5. The method of claim 4,
In the cDNA synthesis, the reverse transcription step is carried out at 50 ° C for 30 minutes, pre-denaturation is carried out at 94 ° C for 10 minutes, and the annealing step is carried out at 55 ° C for 20 seconds. Way.
5. The method of claim 4,
Wherein the amplification step of the target gene is performed using 2 1 of the first RT-PCR product, and the annealing step is performed at 60 캜 or 65 캜 for 30 seconds.
Synthesizing cDNA with the primer set for RT-PCR according to claim 3; And
Amplifying the target gene for HIV-1 with a second PCR primer set for cDNA amplification of claim 3,
Wherein the amplified HIV-1 gene is subjected to sequencing analysis to provide a sample for analysis of resistance to an antiretroviral agent.
9. The method of claim 8,
The antiretroviral agent is used as a combination therapy and is any one or more selected from the group consisting of a protease inhibitor, a nucleic acid-based reverse transcriptase inhibitor and a non-nucleic acid-based reverse transcriptase inhibitor. / RTI &gt;

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