KR102085634B1 - Methods for detecting drug resistance of Hepatitis B virus - Google Patents

Abstract

The present invention relates to a kit for detecting a drug-resistant HBV variant and a detection method using the same. According to the present invention, it is possible to accurately and quickly detect a mutation of drug-resistant HBV and has a high sensitivity. Even very low concentrations of HBV variants can be detected accurately. Therefore, it is expected that the development of the kit for detecting drug-resistant HBV variants of the present invention will greatly contribute to confirming drug resistance and treatment effect of HBV through monitoring of drug treatment.

Description

Methods for detecting drug resistance of Hepatitis B virus

The present invention relates to a method for detecting hepatitis B virus (HBV) variant with drug resistance using the kit according to the present invention.

Around two billion people worldwide are infected with Hepatitis B virus (hereinafter referred to as HBV), and about 690,000 people die every year from HBV complications. In particular, about 400 million people are infected with chronic hepatitis B virus (hereinafter referred to as CHB). Viral treatment with nucleos (t) ide analogs (hereinafter referred to as 'NAs') drugs has been shown to inhibit the progression of CHB to hepatic fibrosis and hepatocellular carcinoma. Purpose. High replication levels of HBV DNA are risk factors for liver cancer (Non-Patent Document 1), and viral inhibition by NAs reduces progression to liver cancer (Non-Patent Document 2).

Currently, several NAs have been used for the treatment of CHB, which act on reverse transcriptase (RT) to inhibit replication of HBV, but does not kill the virus itself. Therefore, NAs must be administered continuously for a long time in order to maintain complete virus inhibition. However, long-term administration of NAs risks developing drug resistant mutants. In particular, in the case of lamivudine (LMV), resistance incidence reached 70% after 5 years of administration to CHB patients (Non Patent Literatures 3 to 5). In addition, high-potency NAs such as entercavir (ETV) and tenofovir (TDF), which are currently in use, have a very low risk of genetic resistance in patients not treated with NAs. Previously, exposure to low-potency antiviral agents has been reported to increase the likelihood of developing ETV resistance. In addition, many CHB patients developed multidrug resistant mutants by early antiviral treatment with low-efficiency NAs and subsequent drug use. The emergence of such drug resistant mutations remains a problem even with the development of NAs.

Fast and accurate determination of genetic resistance is critical for immediate and proper treatment. Methods for detecting HBV mutations include, for example, sequencing, dot blot, real time-PCR, line probe assay, pyrosequencing, Peptide nucleic acid (PNA) analysis, restriction fragment mass polymorphism (RFMP) and DNA microarrays. However, most of these methods are time-consuming and difficult, and there is no optimized method for detecting drug-resistant HBV mutations.

Drug-resistant HBV mutations are the result of single nucleotide polymorphism (SNP) mutations in viral polymerases. Sequences with SNP mutations are difficult to distinguish from wild type (WT) sequences when using existing techniques. DNA microarrays examine mutations in DNA sequences to detect SNP mutations in HBV, but conventional DNA microarrays have high background signals and are difficult to identify SNPs that induce drug resistance. The surface of the microarray slide is important for the performance of the DNA microarray. Existing slides have too many functionalities and irregular spaces.

In order to solve the space problem, the present inventors have developed a tool for diagnosing the resistance of the HBV gene using a dendron-coupled slide, which has a regular and wide probe spacing called mesopacing. Mesospace of the dendron-coupled slides can provide selectivity and sensitivity than other microarray slides and can distinguish SNPs (Non-Patent Document 6).

In the present invention, we propose the accuracy and specificity of the dendron-bound slide for detecting the genetic resistance of the HBV to NAs.

Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006; 295: 65-73. Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004; 351: 1521-1531. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B: summary of a clinical research workshop. Hepatology 2007; 45: 1056-1075. Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos (t) ide analogues. Gastroenterology 2009; 137: 1593-1608 e1591-1592. Yim HJ, Hwang SG. Options for the management of antiviral resistance during hepatitis B therapy: reflections on battles over a decade. Clin Mol Hepatol 2013; 19: 195-209. Oh SJ, Ju J, Kim BC, et al. DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene. Nucleic Acids Res 2005; 33: e90.

It is an object of the present invention to provide a kit for detecting HBV mutation with drug resistance and a method for detecting the mutation.

The present inventors have been studying to develop a method for detecting drug-resistant HBV mutations, and by attaching probes at regular intervals on dendron-bound slides, HBV-resistant HBV is more effective than conventional detection methods. It was confirmed that only mutations can be accurately detected.

As a result of the study, the present invention confirmed that drug-resistant HBV mutations can be accurately and quickly detected from a dendron-fixed slide and a probe bound to the dendron without separating and purifying HBV DNA from blood. To complete.

Accordingly, the present invention relates to a technique for detecting HBV mutations with drug resistance.

Hereinafter, the structure of this invention is demonstrated concretely.

The present invention,

Probe set for detecting HBV variants resistant to lamivudine, including oligonucleotides having an oligonucleotide of SEQ ID NO: 1 to 4 or a complementary nucleotide sequence thereof;

Probe set for detecting HBV variants resistant to entecavir, including oligonucleotides having an oligonucleotide of SEQ ID NO: 5 to 10 or a complementary nucleotide sequence thereof, and

At least one probe set selected from the group consisting of a set of probes for detecting HBV variants resistant to adefovir, including oligonucleotides having an oligonucleotide of SEQ ID NOs: 11 to 13 or a complementary sequence thereof;

Primer sets comprising oligonucleotides having the oligonucleotides of SEQ ID NOs: 24 and 25 or complementary sequences thereof; And

Provided is a kit for target amplification capable of detecting hepatitis B virus (HBV) variant with drug resistance, comprising a dendron molecule coupled to the probe set.

In the present invention, "probe" refers to a single chain nucleic acid molecule comprising a nucleic acid sequence complementary to the target nucleic acid sequence. In the present invention, the probe is designed by analyzing the nucleotide sequence of HBV having resistance (resistance) to a total of three drugs (lamibudine, entecavir and adefovir). Specifically, the probe set in the present invention consists of thirteen probes each capable of specifically detecting variants of HBV that are resistant to three drugs.

The probe set is composed of oligonucleotides of SEQ ID NOs: 1 to 4, or oligonucleotides having complementary nucleotide sequences thereof, which is a probe set for detecting HBV variants resistant to lamivudine, or for detection of HBV variants resistant to enticavir. Oligonucleotides SEQ ID NOs: 5 to 10 oligonucleotides or complementary sequences thereof, or a probe set for detection of HBV variants resistant to adefovir, SEQ ID NOs: 11 to 13 oligonucleotides It may be composed of oligonucleotides having complementary sequences thereof, or one or more probe sets for detecting HBV variants for each agent.

More specifically, the HBV variant detection probe set having drug resistance of the present invention,

For HBV variants resistant to lamivudine, valine (V) is substituted for leucine (L) at the 173th amino acid residue of the reverse transcriptase (RT) of HBV or leucine (L) is replaced with methionine (M) at the 180th amino acid residue. A substitution occurs in which the methionine (M) is replaced with isoleucine (I) or valine (V) at the 204th amino acid residue,

For HBV variants resistant to entecavir, valine (V) is substituted with leucine (L) at the 173th amino acid residue of RT of HBV, or leucine (L) is substituted with methionine (M) at the 180th amino acid residue, or 204th Methionine (M) is substituted with isoleucine (I) or valine (V) at the amino acid residue, isoleucine (I) is substituted with threonine (T) at the 169th amino acid residue, or threonine (T) is substituted with threonine (T) at the 184th amino acid residue. ) Or a substitution in which leucine (L) is substituted or serine (S) is substituted for glycine (G) or valine (V) at amino acid residue 202 or methionine (M) is substituted for valine (V) at 250th amino acid residue Happened,

For HBV variants resistant to adefovir, alanine (A) is substituted with threonine (T) or valine (V) at the 181th amino acid residue of RT of HBV or asparagine (N) is threonine (T) at the 236th amino acid residue. The mutation may be replaced with.

In one embodiment, the oligonucleotide of the probe set may be one having an amine group (3 'or 5'). The dendron molecule that binds to the probe set may be in a form attached to a slide. The dendron slide is coated with a dendron molecule, and a probe having an amine group is attached to the end of the dendron molecule. Accordingly, mesopacing can be maintained to reduce steric hindrance between HBV variants bound to the probe. The mesospace can reduce noise in gene sequencing.

In the following examples, HBV mutations having drug resistance were detected using a probe having an amine group at 5 ', but the present invention is not limited thereto.

Probes of the invention can be used to diagnose or detect mutations in the virus through hybridization-based assays that hybridize to target sequences of DNA of HBV.

In the present invention, “hybridization” refers to a process of specifically combining two single-stranded nucleic acid molecules including complementary sequences to form double-stranded nucleic acid molecules (hybrids).

The probe is bound to a dendron molecule fixed to a support. The support may be, but is not limited to, a glass slide, silica, fused silica, or oxidized silicon wafer. In the present invention, the support may be applied to any type of solid support capable of attaching a dendron.

In one embodiment, the support can be a glass slide.

In the present invention, "Dendron molecule" means a dendrimer type. Dendrimers are highly branched polymers of uniform size, molecular weight, and well-defined structures. Dendrimers consist of a central multifunctional core, a multifunctional repeat unit attached around the core and a terminal or terminator. Dendrimers are classified into two types according to their form. The dendrimer of the first type has a circular or elliptical shape in which the repeating unit extends regularly from the core, and the dendrimer of the second type has a conical shape with the repeating unit extending directionally from the core. In the present invention, dendron means a second type of dendrimer.

In the present invention, "immobilized" may be used in the same sense as attached to the support. In the present invention, the dendron molecule is arranged on the support and immobilized. Such immobilization can be performed according to methods well known in the art. In addition, in the present invention, the dendron molecules fixed to the support may be regularly arranged such that intervals between the dendron and the dendron are constant intervals, such as 1 nm to 6 nm, 2 nm to 5 nm, and 3 nm to 4 nm. In one embodiment, the support may be a regular array of dendron so that the spacing between the dendron and the dendron is 3nm to 4nm. In the above interval range, the detection efficiency of the variant of the dendron molecule is the highest.

The kits of the invention also comprise a primer set comprising oligonucleotides of SEQ ID NOs: 24 and 25. Specifically, the primer set may be included to amplify HBV DNA in a sample, and the primer set may include a forward primer comprising an oligonucleotide of SEQ ID NO: 24 and a reverse primer comprising an oligonucleotide of SEQ ID NO: 25. It may include a primer. The oligonucleotides of SEQ ID NOs: 24 and 25 may comprise a set of oligonucleotide primers capable of amplifying the reverse transcriptase portion of the polymerase of HBV.

In the present invention, the kit includes a linker, a deoxynucleotide triphosphate (dNTP), a control, an amplification buffer, a detection reagent, and the like, in addition to a detection probe set, a primer set, and a dendron for detecting drug-resistant HBV variants. can do.

The present invention may further comprise a linker or linker molecule for linking the probe set with the dendron molecule. The linker or linker molecule is used to mean a molecule that binds a dendron and a probe. For example, the linker may select, but is not limited to, a molecule capable of attaching a probe capable of attaching a probe capable of detecting HBV mutations to drug resistance to the dendron. In the present invention, a representative example of the linker or linker molecule is di- (N-succinimidyl) carbonate (Di- (N-succinimidyl) carbonate), disuccinimidyl glutarate (DSG), 1, 5-difluoro-2,4-dinitrobenzene (1,5-difluoro-2,4-dinitrobenzene (DFDNB), bis (sulfosuccinimidyl) suberate (bis (sulfosuccinimidyl) suberate (BS3), Tris- (succinimidyl) aminotriacetate (TSAT), pegylated bis (sulfosuccinimidyl) suberate (BS (PEG) 5), pegyl PEGylated bis (sulfosuccinimidyl) suberate (BS (PEG) 9), dithiobis (succinimidyl propionate); DSP, 3,3 '-Dithiobis (sulfosuccinimidyl propionate) (3,3'-dithiobis (sulfosuccinimidyl propionate); DTSSP), disuccinimidyl tart rate; DST), bis (2-succinimidooxycarbonyloxy) ethyl) sulfone (bis (2- (succinimidooxycarbonyloxy) ethyl) sulfone; BSOCOES), ethylene glycol bis (succinimidyl succinate) (ethylene glycol bis ( succinimidyl succinate; EGS), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobis Dimethyl 3,3'-dithiobispropionimidate; DTBP; Wang and Richard's Reagent).

Kits according to the invention may further comprise an internal control probe. As the internal control probe, for example, wild-type of amino acid residues of 169, 173, 180, 181, 184, 194, 202, 204, 236, and 250th amino acids of RT of HBV. WT) may be used to detect a sequence, and more specifically, may be an oligonucleotide having a nucleotide sequence of SEQ ID NOs: 14 to 23. Through the internal control probe, when PCR is performed using the kit of the present invention, when the HBV variant is found to be absent in the sample, the result is an experimental mistake or an actual HBV mutation does not occur. This is necessary to ensure accurate identification of HBV mutations.

In addition, the kit of the present invention may further include a positive control probe. The positive control probe indicates that there is no problem in PCR itself when PCR is performed using the kit of the present invention.

In one embodiment, the control probe may be one having an oligonucleotide of SEQ ID NO: 14 to 23, but is not limited to the sequence range.

The invention may further comprise a deoxynucleotide triphosphate (dNTP) with a detectable label to aid in the detection of the amplified nucleic acid. Since the dNTP is introduced into the nucleic acid material amplified by the polymerase used, the amplified material may contain a detectable label. Amplification of the nucleic acid material and addition of dNTPs can increase the detection efficiency when performing PCR. The detectable label may be a fluorescent material, but is not limited thereto. The fluorescent material may be applied to any material that can be used in the art as it is.

In addition, the kit of the present invention may further include a hybridization reaction solution, a non-hybridization reaction DNA washing solution, or an instruction manual in addition to the aforementioned materials.

In the present invention, “detection” includes both quantitative and qualitative detection.

In addition, the present invention,

Provided are methods for detecting drug resistant HBV variants comprising contacting and hybridizing a biological sample taken from an HBV infected patient.

In the present invention, the biological sample may be a blood sample, for example, a whole blood, plasma or serum sample.

The method for detecting the drug-resistant HBV variant may be to amplify the HBV DNA in the biological sample, and then not to separate (extract) and purify the amplified HBV DNA.

According to the present invention, there is no process of isolating (extracting) and purifying amplified HBV DNA from a biological sample, thereby minimizing gene loss occurring during purification of the gene amplification product, thereby improving detection efficiency.

In the present invention, the method may provide a method for detecting drug resistant HBV variants in a biological sample. This includes amplifying the HBV DNA in the presence of a probe for detecting the HBV variant, which may be bound to the support by a dendron.

The use of samples in PCR in amplification reactions using PCR can be carried out by any means known to those skilled in the art. Novagen, for example, described a buffer mixture that can directly perform PCR using blood samples. The mixture of Novagen Company includes BloodDirect Buffer 1 and BloodDirect Buffer A (for human blood), or BloodDirect Buffer B (for mouse blood) and the blood for amplification of the desired DNA by PCR. Samples, anticoagulants, Taq thermostable DNA polymerase, primers and deoxynucleotide triphosphate were mixed together (see user protocol TB404 from Novagen).

In the present invention, the probe set may be used as a "capture probe" by being bonded to a support on which a dendron is fixed. The present invention may include a step of preparing amplified DNA by amplifying HBV DNA in a sample by performing PCR in the presence of the capture probe. The amplification reaction described in the present invention is to label the nucleic acid material amplified by the inclusion of a detectable label attached deoxynucleotide triphosphate in the reaction mixture, the detectable label may be a fluorescent material, but is not limited thereto.

The amplified HBV DNA hybridizes with the probe bound to the dendron after denaturation to allow the formation of the double stranded complex. The complex can be detected and analyzed by any suitable means known to those skilled in the art.

In the present invention, the analysis can be carried out by any suitable means known to those skilled in the art. In DNA analysis after hybridization, confocal laser scanners for image acquisition and quantitative microarray analysis software for fluorescence intensity analysis can be used, but are not limited thereto.

Advantages and features of the present invention and methods of achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Using the kit for detecting drug resistance of the HBV variant of the present invention and the detection method using the same, it is possible to accurately and quickly detect the mutation of drug resistance HBV, and the sensitivity is very high in HBV variants present in the sample. Even can be detected accurately. Therefore, it is expected that the development of the kit for detecting drug-resistant HBV variants of the present invention will greatly contribute to confirming drug resistance and treatment effect of HBV through monitoring of drug treatment.

1 is a comparison of the results of the dendron slide and the conventional slide of the present invention in the specificity test for HBV drug resistance mutations, which verifies the PCR amplification efficiency of the probe binding region and wild type polymerization with mutations at rt180 and rt204 Plasmids encoding enzymes or polymerases are shown. Below is a comparison of the specificity of dendron and ethoxy or two aldehyde slides.
Figure 2 shows the sensitivity and selectivity of the dendron slide for detecting drug-resistant HBV mutations, A shows the sensitivity test results of the dendron slide compared to the aldehyde 2 slide. B is a comparison of the scan signal intensities obtained using the quantitative mode of the software used for ScanArray scanner operation, and C and D are the selectivity of the dendron slides (W: wild-type plasmid, M: mutant plasmid).
FIG. 3 shows signal enrichment on dendron slides following addition of cy3-labeled dNTPs during hybridization, with A representing PCR amplified HBV plasmids (10 or 10 ² copies) and B representing dendron slides. The relative signal intensities of the HBV plasmids on the phases are compared.
Figure 4 shows the results of detecting drug resistance mutations from the serum of the patient using sequencing and RFMP without validation of virus purification and dendron slides. A compares microarray detection of PCR products amplified directly from serum with amplified from purified HBV DNA, and B represents the clinical course (left) of two CHB patients and serum using dendron slides, RFMP and sequencing. This is the result of analyzing the HBV mutation from (right).
FIG. 5 shows the design and verification of dendron-coupled microarrays capable of simultaneously detecting multiple drug resistance variations in a single slide, where A represents the design of the microarray and B is the known resistance for each drug. The combination of mutations is shown and C is the specificity of the dendron microarray.
FIG. 6 shows the results of direct detection of various drug resistance mutations in the serum of a patient in a single dendron microarray and verified using sequencing and RFMP. A is the result of using the sera of 6 CHB patients directly for PCR, and the PCR product was analyzed using a dendron slide, and B is the result of confirming HBV variation of the patient serum identified on the dendron slide by RFMP and sequencing.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[ Example ]

Example  1: Experimental Materials and Methods

<1-1> HBV  DNA preparation

Ten plasmids (replicable HBV1.2mer) were constructed that encode wild-type (WT) HBV sequences or encode HBV sequences with mutations in the polymerase gene. They were constructed by inserting the reverse transcriptase portion of the polymerase using XhoI and NcoI restriction enzyme sites to insert HBV DNA into the PGEM-4z vector to express HBV with wild type and polymerase mutations. HBV DNA was extracted using a virus spin kit (Qiagen GmbH, Hilden, Germany) from 200 μl serum of HBV infected patients. PCR was performed using viral DNA isolated from serum as well as serum samples without DNA extraction. At this time, PCR was performed using 5 × EzWay PCR buffer (KOMA Biotech Inc., Seoul, Korea).

<1-2> experimental patient

13 chronic hepatitis B patients (CHB) who developed viral breakthrough during treatment with lamivudine (LMV), adefovir dipivoxil (ADV), or entercavir (ETV) at the Konkuk University Medical Center DNA microarrays were tested. The study was approved by the Institutional Review Board or Konkuk University Medical Center (IRB number; KUH1010277), Konkuk University Medical Center, and was conducted in accordance with the Helsinki Declaration of Ethics Guidelines in 1975.

The average duration of LMV treatment was 12 months before LMV resistance appeared during LMV treatment. Serum (P6-1, P6-2, and P6-3, respectively) collected from Patient # 6 in 2006, March 2007, and March 2008 were transferred to DNA microarray using dendron slides, RFMP, and sequencing. Analyzed. Patient # 6 was prescribed LMV as the initial treatment for HBV, and viral breakthrough occurred 15 months after LMV treatment.

ADV was added to LMV treatment and viral DNA levels steadily decreased with this combination therapy. Serum (P7-1, P7-2 and P7-3, respectively) collected from October 2006, November 2007 and February 2009 from Patient # 7 was analyzed in the same manner as Patient # 6. Patient # 7 was prescribed LMV as the initial treatment for HBV, but showed viral breakthrough after 13 months of LMV treatment. The patient had reduced HBV DNA levels after switching to ADV, but another viral breakthrough occurred eight months after ADV treatment.

ETV was added to ADV and viral DNA levels were steadily decreasing. In total, eleven serum samples from seven patients were analyzed and six serum samples were received from Konkuk University Hospital (FIG. 6).

<1-3> take Probe Combined  Slide authoring

Dendron molecules are a second type of dendrimer and have a conical shape with repeating units directionally extending from the core. In the present invention, the dendron molecule means that the conical shape is bonded to the bottom of the slide and is arranged at regular intervals on the support to be fixed to the slide. The immobilized at regular intervals is called mesopacing, and a linker molecule between the dendron molecule and the capture probe is a di- (N-succinimidyl) carbonate (Di -(N-succinimidyl) carbonate) was used. The dendrimer-attached slides were reacted for 30 minutes in a buffer made of Di- (N-succinimidyl) carbonate of 10mM or more, so that the amine group at the conical vertex of the dendrimer was combined with one side of the di- (N-succinimidyl) carbonate. The capture probe 20 pM was then attached to the dendron glass slide. The probe was attached with an amine group in the 5 'direction to bind with the other side of the linker molecule. Capture probes in spotting buffer were attached onto slides using a Q-array microarray (Genetix Ltd., UK). After removing the unattached capture probe, the slides were dried with nitrogen steam and stored at 4 ° C. The capture probes used at this time are shown in Tables 1 and 2 below. In this case, the probes of Tables 1 and 2 below are amine groups bonded to the 5 'end. N is the Degenerate (IUB) base code, which means that A, C, G, and T are randomly inserted during probe synthesis. That is, the N region has one nucleotide of A, C, G or T, and when the final synthesized probe is received, four types of rt180 probes (A, C, G, and T are mixed differently in the N position, If you receive 100 probes, it means that you have a heterogeneous probe of 25 probes each). Where R has G or A nucleotides, K has G or T nucleotides, and Y means T or C nucleotides.

Probe SEQ ID NO Probe name Sequence (5 '→ 3') length Lamivudine resistance One rtV173L GCA AGA TTC CTA TGG GAT TGG 21 2 rtL180M CTC AGT CCG TTT CTC ATG G 19 3 rtM204I TGT TTG GCT TTC AGT TAT ATT GAT G 25 4 rtM204V GTT TGG CTT TCA GTT ATG TGG 21 Entecavir resistant 5 rtI169T GGG CTT TCG CAA GAC TCC 18 6 rtT184A CTC CTG GCT CAG TTT GCT AG 20 7 rtT184L TTT CTC ATG GCT CAG TTT CTT AG 23 8 rtS202I ACT GTT TGG CTT TCA TTT A 19 9 rtS202G CTG TTT GGC TTT CGG TT 17 10 rtM250V GGC TAC TCC CTT AAC TTC GTG G 22 Adefovir
tolerance 11 rtA181T CAG TCC GTT TCT CCT GAC TCA 19 12 rtA181V AGT CCG TTT CTC CTG GTT CA 20 13 rtN236T CTT TGG GTR TAC AKT TR ACC CCT 23

Probe SEQ ID NO Probe name Sequence (5 '→ 3') length Wild-type 14 rtI169 CCC GGG CTT TCG CAA GAT TCC 21 15 rtV173 GCA AGA TTC CTA TGG GAG TGG 21 16 rtL180 TCA GTC CGT TTC TCC TNG CTC 21 17 rtA181 GTC CGT TTC TCC TGG CTC A 19 18 rtT184 TTT CTC CTG GCT CAG TTT ACT AG 23 19 rtA194 TTC AGT GAT TCG YAG GGC TT 20 20 rtS202 CCC ACT GTT TGG CTT TCA GTT AT 23 21 rtM204 GTT TGG CTT TCA GTT ATA TGG AT 23 22 rtN236 GTC TTT GGG TAT ACA TTT GAA CCC 24 23 rtM250 GCT ACT CCC TTA ACT TCA TGG G 22

<1-4> target  DNA amplification

PCR primers for amplifying HBV DNA were constructed to contain sequences encoding amino acids that determined drug resistance and generated 440 base pairs. The 5 'end of all primers was marked cy3 (Table 3). 30 μl of the reaction mixture used was 3 μl of 10 × buffer, 4 dNTPs (10 mM, 1 μl each), 0.6 μl of Taq polymerase (5 units / μl), 1 μl of cy3-forward primer (BVF, 5 pmol / Μl), 1 μl reverse primer (BVR, 5 pmol / μl), 1 μl sample (plasmid or HBV DNA), and 22.4 μl purified water. PCR reaction mixtures for direct PCR amplification using serum without purification of viral DNA were 6 μl of 5 × PCR buffer (KOMA Biotech Inc.), 4 dNTPs (10 mM, 1 μl each), 0.6 μl of Taq polymerase. (5 units / μl), 1 μl BVF (5 pmol / μl), 1 μl BVR (5 pmol / μl), 1 μl serum and 19.4 μl purified water. PCR was performed as follows: heating at 94 ° C. for 5 minutes; 1 minute at 94 ° C, 1 minute at 52 ° C, 1 minute at 72 ° C, last 7 minutes at 72 ° C, 40 cycles.

SEQ ID NO Primer name Sequence (5 '→ 3') length Primer 24 BVF Cy3-TTT CCC TCT TGT TGC TGT ACA AAA CC 26 25 BVR Cy3-TGA CAT ACT TTC CAA TCA ATA GGT CTA 27

<1-5> Hybridization  And scanning

After PCR, the mixture was heated at 94 ° C. for 5 minutes and cooled on ice. PCR mixtures were transferred to glass slides and incubated in heat blocks at 50 ° C. for 2 hours. After hybridization, the slides were washed with distilled water, dried with nitrogen gas and scanned with a laser scanner (ScanArray PLUS; PerkinElmer, Inc., USA). Quantitative data analysis was performed using QantArray software (PerkinElmer, Inc.).

Example  2: results

<2-1> drug resistance HBV  For detecting mutations Dendron  Compare slides with conventional slides

In order to compare the dendron slide and the conventional slide of the present invention, first, four HBV plasmids encoding the WT and the mutated polymerase were prepared to confirm that the PCR of the clone was similar (FIG. 1A).

The dendron slide of the present invention was designed as shown in FIG. 1B, and five capture probes were fixed to each slide by three. As shown in FIG. 1B above, only the dendron slides showed a perfectly matched signal for WT and three mutations without non-specific binding. In contrast, the rtL180L and rtL180M mutations could not be distinguished from the epoxy slides used as conventional slides, and rtM204V could not be detected. Similarly, aldehyde and aldehyde 2 slides could not distinguish between rtM204I and rtM204M, and rtL180M could not be distinguished from rtL180L. Non-specific signals were more frequent in aldehyde slides. These results indicate that dendron slides are superior in specificity and sensitivity in detecting drug-resistant HBV mutations compared to conventional slides.

<2-2> drug resistance HBV  To detect mutations Dendron  Check sensitivity and selectivity of slides

Since the aldehyde 2 slide and the dendron slide showed similar signal intensities, the sensitivity and selectivity of the two slides were compared. First, the sensitivity was determined by serial dilution of the plasmid encoding the rtL180M / rtM204I double mutant. The detection limit of the dendron slide was HBV 1 copy / μl, whereas the aldehyde 2 slide was 10 copies / μl (FIG. 2A). Nonspecific signals, especially at rt180, increased with the ratio of copy number in the aldehyde 2 slide (Figures A and 2B). These results indicate that the dendron slide is about 10 times more sensitive than the aldehyde 2 slide and has excellent selectivity.

Next, template DNA mixed with rtL180 and rtL180M, rtM204 and rtM204I at known ratios was used to determine the selectivity of the dendron slides (FIGS. 2C and 2D). The quantified signal of the mixtures showed a linear change and fit well with the expected data. These results indicate that dendron slides can be applied for semi-quantitative detection of drug resistant HBV mutations.

<2-3> Hybridized  During cy3 -Labeled of dNTPs  Drug resistance by addition HBV  Enhanced sensitivity of detection

It was confirmed that signal amplification for detection of HBV mutations increased by the formation of branched DNA during hybridization.

The signal intensity of rtL180 on the dendron slide was five times that of aldehyde 2 slide without cy3-dNTP (FIG. 3A). However, addition of cy3-dNTPs during hybridization markedly increased signal intensity on dendron slides (two-fold increase of rtL180 detection at 10 copies). However, there was no significant increase in signal intensity in aldehyde 2 slides when cy3-dNTP was added (FIG. 3B). This difference may be due to the nature of the regular wide probe space of the dendron slide. The results indicate that the addition of cy3-dNTP upon hybridization can significantly increase signal strength in dendron slides.

<2-4> Direct Detection of Drug-Resistant Mutations in Patient Serum Without Purifying Viral DNA

Purified HBV DNA and patient sera were compared as PCR templates to determine if viral purification was required on dendron slides. When dendron slides were used the results were found to be similar to the patient's serum and purified HBV DNA. However, aldehyde 2 slides showed significant non-specific binding and low detection signals in both purified HBV DNA and patient serum (FIG. 4A).

To verify the results of detecting mutations using the dendron slides of the patient's serum, RFMP and sequencing were compared. Six serum samples of two CHB patients (P6-1, P6-2, P6-3, P7-1, P7-2 and P7-3) were analyzed using dendron slides, RFMP and direct sequencing (FIG. 4B). For dendron slides, the occurrence of rtL180M and rtM204I / V mutations in the P6-2 sample was found. This is a clinical viral breakthrough due to LMV. This result matches the sequencing data but not the RFMP.

The occurrence of rtL180M and rtM204I / V mutations in P7-2 samples was also confirmed. This is also a clinical viral breakthrough with LMV. LMV-resistant mutations were no longer found when serum HBV DNA levels were reduced by ADV and ETV combination therapy. This result was also consistent with RFMP and sequencing data.

The HBV mutations identified in the dendron slides using 11 CHB serum samples were in close agreement with RFMP and sequencing results (Table 4). The results of P6-2, P6-3, and P7-2 partially matched the RFMP data but completely matched the sequencing data. Given that the sequencing results are the most reliable, it can be seen that the dendron slide can be used as a diagnostic technique for the detection of drug-resistant HBV.

Sample
No. Viral titer
(copy / ml) Analysis method RT position rtL180 rtM204 P1 1.80 × 10 ⁷ Dendron chip - - RFMP - - Sequencing - - P2 3.88 × 10 ⁷ Dendron chip L, M M, I RFMP L, M M, I Sequencing L, M M, I P3 2.43 × 10 ⁷ Dendron chip M V RFMP M V Sequencing M V P4 4.50 × 10 ⁶ Dendron chip - I RFMP - I Sequencing - I P5 8.21 × 10 ⁶ Dendron chip M V RFMP M V Sequencing M V P6-1 3.04 × 10 ⁴ Dendron chip - - RFMP - - Sequencing - - P6-2 3.13 × 10 ⁷ Dendron chip L, M V, I RFMP M V, I Sequencing L, M V, I P6-3 6.77 × 10 ⁴ Dendron chip M V, I RFMP M V Sequencing M V P7-1 1.43 × 10 ⁵ Dendron chip - - RFMP - - Sequencing - - P7-2 1.35 × 10 ⁷ Dendron chip L, M V, I RFMP - I Sequencing L, M V, I P7-3 2.53 × 10 ⁶ Dendron chip - - RFMP - - Sequencing - -

<2-5> single Dendron  Of patient serum in a modified array chip LMV -, ADV Simultaneous and direct detection of-and ETV-resistant mutants

After confirming that the dendron slide was able to detect LMV resistant mutant HBV in the serum of CHB patients (FIG. 4), it was confirmed that LMV-, ADV- and ETV- resistant mutations could be simultaneously identified on the same slide. A total of 22 probes (9 WTs, 4 LMV resistant mutations, 3 ADV mutations, 6 ETV resistant mutations) were obtained (FIG. 5A). To verify the chips and probes according to the present invention, six HBV mutants were constructed comprising various combinations of drug resistance mutations (FIG. 5B). As can be seen in FIG. 5C, the dendron chip completely distinguished each mutation without any non-specific binding. The WT area on the left side of the chip shows which position on the HBV RT caused the mutation, and each drug resistant region in the center of the chip shows what the specific drug mutation is.

Therefore, the dendron chip was verified using the serum of CHB patients (FIG. 6A). As a result of the chip analysis, serum sample B1247 contained a complete WT HBV (both positive signals were detected in the left box and no signals in the middle and right boxes). HBV detected in serum B5656, B5454 and B8373 showed an ETV resistance pattern (positive in the right box). All sera except B1247 showed LMV resistance mutations (rtL180M and rtM204I / V). To confirm this result, chip data was compared with HBV sequences analyzed using RFMP and sequencing (FIG. 6B). The detection of rtM204V by RFMP was inconsistent with the chip and sequencing data of the two patients, but the sequencing and chip results were in perfect agreement. This indicates that dendron modified microarrays can be used for diagnostic purposes for detecting all known drug resistant HBV variants.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Methods for detecting drug resistance of Hepatitis B virus <130> P17U10C1439 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtV173L <400> 1 gcaagattcc tatgggattg g 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtL180M <400> 2 ctcagtccgt ttctcatgg 19 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtM204I <400> 3 tgtttggctt tcagttatat tgatg 25 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtM204V <400> 4 gtttggcttt cagttatgtg g 21 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtI169T <400> 5 gggctttcgc aagactcc 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtT184A <400> 6 ctcctggctc agtttgctag 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtT184L <400> 7 tttctcatgg ctcagtttct tag 23 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtS202I <400> 8 actgtttggc tttcattta 19 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtS202G <400> 9 ctgtttggct ttcggtt 17 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtM250V <400> 10 ggctactccc ttaacttcgt gg 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtA181T <400> 11 cagtccgttt ctcctgactc a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtA181V <400> 12 agtccgtttc tcctggttca 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtN236T <400> 13 ctttgggtrt acakttracc cct 23 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtI169 <400> 14 cccgggcttt cgcaagattc c 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtV173 <400> 15 gcaagattcc tatgggagtg g 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtL180 <400> 16 tcagtccgtt tctcctngct c 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtA181 <400> 17 gtccgtttct cctggctca 19 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtT184 <400> 18 tttctcctgg ctcagtttac tag 23 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtA194 <400> 19 ttcagtgatt cgyagggctt 20 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtS202 <400> 20 cccactgttt ggctttcagt tat 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtM204 <400> 21 gtttggcttt cagttatatg gat 23 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe_rtN236 <400> 22 gtctttgggt atacatttga accc 24 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe_M250 <400> 23 gctactccct taacttcatg gg 22 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer_BVF <400> 24 tttccctctt gttgctgtac aaaacc 26 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer_BVR <400> 25 tgacatactt tccaatcaat aggtcta 27

Claims (14)

A probe set comprising an oligonucleotide represented by SEQ ID NOs: 1 to 4 or a complementary sequence thereof, wherein the probe set comprises valine (L) leucine (L) at the 173rd amino acid residue of reverse transcriptase (RT) of HBV. To detect a mutation wherein leucine (L) is substituted with methionine (M) at the 180th amino acid residue, or methionine (M) isoleucine (I) or valine (V) at the 204th amino acid residue Probe set for detecting HBV variants resistant to lamivudine for
A probe set comprising an oligonucleotide represented by SEQ ID NO: 5 to 10 or a complementary sequence thereof, wherein the probe set is substituted with leucine (L) by valine (V) at the 173th amino acid residue of RT of HBV Leucine (L) is substituted with methionine (M) at the 180th amino acid residue, or methionine (M) is substituted with isoleucine (I) or valine (V) at the 204th amino acid residue, or isoleucine (I) is threonine at the 169th amino acid residue (T), threonine (T) is substituted with alanine (A) or leucine (L) at the 184th amino acid residue, or serine (S) is replaced with glycine (G) or valine (V) at the 202th amino acid residue A set of probes for detecting HBV variants that are resistant to entercavir for detecting a substituted or substituted 250-amino acid residue with methionine (M) substituted with valine (V), and
A probe set comprising an oligonucleotide represented by SEQ ID NOs: 11-13 or a complementary sequence thereof, wherein the probe set comprises a threonine (T) or a valine (A) at the 181th amino acid residue of RT of HBV. At least one member selected from the group consisting of a set of probes for detecting HBV variants which are resistant to adefovir for detecting a mutation substituted with V) or asparagine (N) with threonine (T) at the 236th amino acid residue. Probe sets;
A primer set comprising an oligonucleotide represented by SEQ ID NOs: 24 to 25 or a complementary nucleotide sequence thereof; And
A kit for detecting hepatitis B virus (HBV) variant having drug resistance, comprising a dendron molecule coupled to the probe set. The method of claim 1,
The probe set is a kit for detecting HBV variants having an amine (amine) group 3 'or 5' of the probe. The method of claim 1,
The kit kit for detecting HBV variants further comprises a linker for connecting the probe set and the dendron molecule. The method of claim 1,
The kit is a kit for detecting a HBV variant further comprises dNTP with a detectable label. The method of claim 1,
The kit is a kit for detecting a HBV variant further comprises a probe set comprising a wild-type oligonucleotide corresponding to the position where the mutation appears in drug-resistant HBV as a control. The method of claim 1,
The dendron molecule is fixed to the support HBV variant kit for detection. The method of claim 6,
Dendron molecules fixed to the support is a kit for detecting HBV variants having a distance between the dendron 1nm to 6nm. The method of claim 6,
The support is a glass slide, silica, fused silica or oxidized silicon wafer kit for detecting HBV variants. delete A method of detecting drug resistant HBV variants comprising contacting and hybridizing a biological sample taken from an HBV infected patient with a kit according to any one of claims 1-8. The method of claim 10,
A method for detecting drug resistant HBV variants further comprising adding a detectable labeled deoxynucleotide triphosphate (dNTP) prior to hybridizing the biological sample and probe set. The method of claim 10,
A method for detecting drug resistant HBV variants wherein the biological sample is blood. The method of claim 12,
The method of detecting drug resistant HBV variants, wherein the blood is whole blood, plasma or serum. The method of claim 10,
A method for detecting drug resistant HBV variants, wherein the variants are detected by measuring a fluorescence signal from a label of the hybridized oligonucleotide.

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