CN1384888A - Detection of anti-hepatitis drug resistance - Google Patents

Abstract

The present invention relates to a method for the monitoring of anti-HBV drug resistance in a patient by genetic detection of at least one of the mutations L528M, M552V/I and/or V/L/M555I in the DNA polymerase of the HBV strains present in a biological sample of said patient. The present invention provides new HBV DNA polymerase sequences to be used for the design of new probes allowing a very specific and sensitive detection of anti-HBV drug resistance.

Description

The detection of anti-hepatitis drug resistance

Invention field

The present invention relates to hepatitis B virus (HBV) diagnostic field.More particularly, the present invention relates to the gene monitoring field of anti-HBV drug resistance in the HBV therapeutic process.

Background of invention

Hepatitis B virus is a kind of long parcel film dna virus of about 3200bp that belongs to Hepadnaviridae, it is characterized in that become significantly liver property and specific specificity.The HBV genome is the natural complex with dsdna segment structure, and described dna structure has the eclipsed open reading-frame (ORF) of coded surface antigen, cAg, polysaccharase and X gene.HBV duplicates and adopts reverse transcription (Summers and Mason, 1982) through the RNA intermediate in its replication strategy.The main medical problem that HBV produces is such as chronic hepatopathy and hepatocellular carcinoma (Schroder and Zentgraf, 1990).

Duplicate among the patient of active chronic HBV (HBV) infection at wild-type virus, the feature of successful antiviral therapy is to remove HBV-DNA from blood circulation, then removes hepatitis B e antigen (HBeAg) and sun and transfers anti--HBe antibody to.Unfavorable is that not HBeAg sun commentaries on classics always after HBV-DNA disappears it is believed that quantitative HBeAg detects the value (Heijtink etc., 1997) with prediction antiviral therapy result.

At present, alpha-interferon (IFN ) and lamivudine (3TC) are two kinds of medicines that are approved for the treatment chronic hepatitis B.IFN- has antiviral and immunomodulatory properties.Lamivudine (3TC) is (-) enantiomer of 3 ' sulfo-cytidine (2 ' 3 '-dideoxyribonucleoside analogue), known it by the active effective inhibitor that becomes hbv replication of the reversed transcriptive enzyme (RT) that suppresses the HBV polysaccharase.Lamivudine therapy can cause that the histology of chronic hepatitis patient improves, and when before the liver transplantation with after when giving, can prevent that graft from infecting (Honkoop etc., 1995 again; Naoumov etc., 1995).These two kinds of medicines can give (Lai etc., 1998 at single therapy or in combination therapy; Mutimer etc., 1998).Another kind of compound Famciclovir (9-[4-hydroxyl-3-methylol-Ding-1-yl] guanine) with direct antivirus action is at present just in III phase clinical evaluation.Other antiviral compound lobucavir (Colacino and Staschke, 1998) and adefovir (Heathcote etc., 1998) have shown it is safe, and effectively suppress hepatitis B replication, but yet in the clinical phase.Shown that Penciclovir suppresses the reverse transcriptase activity of described HBV polysaccharase (Shaw etc., 1996).

Yet in some patient, observe hepatitis and break out in treatment or after the treatment, ALT raises and can detect HBV DNA once more.The bounce-back of this HBV DNA is relevant with new quasispecies balance.Several separate report has been described the differentiation (summarize in Bartholmeusz etc., 1998) of Famciclovir resistance and lamivudine resistance HBV virus strain.The definite character that it is believed that this resistance is to be caused by the sudden change accumulation in the RT of described polysaccharase part.The treatment plan of the antiviral compound of employing such as lamivudine and Famciclovir has caused the accumulation of various HBV polymerase mutations.Sequential analysis shows, specific mutant occurs in tyrosine-methionine(Met)-aspartic acid-aspartic acid (YMDD) primitive of HBV pol gene, and methionine(Met) is thus by any alternative (Ling etc., 1996 in Isoleucine or the Xie Ansuan; Tipples etc., 1996).In HIV RT polysaccharase, found similar mechanism, accumulation sudden change (Gao etc., 1993) in described YMDD primitive with lamivudine therapy the time.Another important mutational site is positioned at the 24th amino acid of YMDD primitive upstream, and leucine (L) replaces methionine(Met) (M) (WO98/21317 of Locarnini etc.).V555I (X199 among the HBsAg) sudden change also may be relevant clinically.Pichoud etc. (1999) point out that the V555I mutant has the ability that reduction is duplicated, and it does not produce HBsAg, anti-Penciclovir, but to the lamivudine sensitivity.Can obtain at present about the sudden change after lamivudine or Famciclovir treatment of discovery so far and viral adaptive result's summary (Ling etc., 1996; Tipples etc., 1996; Honkoop etc., 1997; Bartholomeusz etc., 1998; Buri etc., 1998; Chayama etc., 1998; Melagari etc., 1998; Mutimer etc., 1998; Niesters etc., 1998; Pillay etc., 1998; Hunt etc., 2000).With the anti-HBV pharmacological agent of Penciclovir and other time, also detected the sudden change in the HBV polysaccharase.

Because it is generally acknowledged these sudden changes is reasons of viral anergy and treatment failure, has clinical importance for the anti-HBV drug resistance of monitoring in the HBV therapeutic process so detect these sudden changes.Therefore, need a kind of method that detects these sudden changes fast, reliably and very specifically.This will make can be rapidly and accurately monitor HBV drug resistance and will be faster and more effectively design anti-HBV therapeutic strategy.

Goal of the invention

An object of the present invention is to provide quick, the reliable and accurate method of anti-HBV drug resistance among a kind of patient of monitoring.

Another object of the present invention provides a kind of quick, reliable and accurate method of monitoring anti-HBV drug resistance among the patient who accepts anti-HBV treatment.

Another object of the present invention provide a kind of gene test in HBV virus strain archaeal dna polymerase sudden change L528M, M552V/I and/or at least a quick, the reliable and accurate method among the V/L/M555I.

Another object of the present invention provides sudden change L528M, M552V/I in the HBV virus strain archaeal dna polymerase of a kind of gene test in the biological sample that is present in the patient who accepts anti-HBV treatment and/or at least a quick, the reliable and accurate method among the V/L/M555I.

Another object of the present invention provides a kind of quick, reliable and accurate method that detects suddenly change at least L528M and M552V/I in HV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides a kind of quick, reliable and accurate method that detects suddenly change at least L528M and V/L/M555I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides a kind of quick, reliable and accurate method that detects suddenly change at least M552V/I and V/L/M555I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides a kind of L528M that suddenlys change at least, M552V/I in HBV virus strain archaeal dna polymerase and quick, reliable and accurate method of V/L/M555I of detecting simultaneously.

Another object of the present invention provides quick, the reliable and accurate method of lamivudine resistance among a kind of patient of monitoring.

Another object of the present invention provides quick, the reliable and accurate method of Famciclovir resistance among a kind of patient of monitoring.

Another object of the present invention provides quick, the reliable and accurate method of Penciclovir resistance among a kind of patient of monitoring.

Another object of the present invention provides one or more probes that uses in aforesaid method.

Another object of the present invention provides the composition that comprises at least a aforesaid probe.

An object of the present invention is to provide the diagnostic kit of anti-HBV drug resistance among the monitoring patient.

Another object of the present invention provides the diagnostic kit that anti-HBV drug resistance among the patient of anti-HBV treatment is accepted in monitoring.

Another object of the present invention provide gene test in HBV virus strain archaeal dna polymerase sudden change L528M, M552V/I and/or at least a diagnostic kit among the V/L/M555I.

Another object of the present invention provides sudden change L528M, M552V/I in the HBV virus strain archaeal dna polymerase of gene test in the biological sample that is present in the patient who accepts anti-HBV treatment and/or at least a diagnostic kit among the V/L/M555I.

Another object of the present invention provides the diagnostic kit that detects suddenly change at least L528M and M552V/I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides the diagnostic kit that detects suddenly change at least L528M and V/L/M555I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides the diagnostic kit that detects suddenly change at least M552V/I and V/L/M555I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides the L528M that suddenlys change at least, the M552V/I that detect simultaneously in HBV virus strain archaeal dna polymerase and the diagnostic kit of V/L/M555I.

Another object of the present invention provides the diagnostic kit of lamivudine resistance among the monitoring patient.

Another object of the present invention provides the diagnostic kit of Famciclovir resistance among the monitoring patient.

Another object of the present invention provides the diagnostic kit of Penciclovir resistance among the monitoring patient.

An object of the present invention is to provide the line formula probe assay (Line Probe Assay) of anti-HBV drug resistance among the monitoring patient.

Another object of the present invention provides the line formula probe assay that anti-HBV drug resistance among the patient of anti-HBV treatment is accepted in monitoring.

Another object of the present invention provide gene test in HBV virus strain archaeal dna polymerase sudden change L528M, M552V/I and/or at least a line formula probe assay among the V/L/M555I.

Another object of the present invention provides sudden change L528M, M552V/I in the HBV virus strain archaeal dna polymerase of gene test in the biological sample that is present in the patient who accepts anti-HBV treatment and/or at least a line formula probe assay among the V/L/M555I.

Another object of the present invention provides the line formula probe assay that detects suddenly change at least L528M and M552V/I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides the line formula probe assay that detects suddenly change at least L528M and V/L/M555I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides the line formula probe assay that detects suddenly change at least M552V/I and V/L/M555I in HBV virus strain archaeal dna polymerase simultaneously.

Another object of the present invention provides the L528M that suddenlys change at least, the M552V/I that detect simultaneously in HBV virus strain archaeal dna polymerase and the line formula probe assay of V/L/M555I.

Another object of the present invention provides the line formula probe assay of lamivudine resistance among the monitoring patient.

Another object of the present invention provides the line formula probe assay of Famciclovir resistance among the monitoring patient.

Another object of the present invention provides the line formula probe assay of Penciclovir resistance among the monitoring patient.

An object of the present invention is to provide new HBV dna polymerase gene sequence.

Description of drawings

Fig. 1. as described in embodiment 1, derive from the contrast that HBV treats the new HBV archaeal dna polymerase sequence of patient's serum sample in preceding and the treatment.Can be used for the target sequence box indicating of probe design.

The design of Fig. 2 .HBV drug resistance LiPA band.Conj.cont.: the conjugate contrast; Amp.cont.: the amplification contrast.This band comprises 19 line probe altogether, has 38 probes altogether; The n probe equals the probe total amount on each line.Be displayed in Table 6 the specific probe that is used for each line.Illustrate: Polym. or HBsAg: be used for the described polysaccharase on this particular probe line or the amino acid of HBsAg open reading-frame (ORF).

Fig. 3. represent the result of reactive HBV drug resistance LiPA band of the line probe that each is independent.16 recombinant clones with reference sequences figure (reference panel) obtain the result.Reactive line is indicated with the numbering of described recombinant clone.Shown on the right side one show band and the codon that on this line, detects and the amino acid of wired relative position.Below each band, provided the explanation of each codon (cd).

Fig. 4. monitor two HBV infected patients with anti-HBV drug resistance.Left side: patient A; Right side: patient C.The fate that shows tracking in X-axis.Provided the explanation of the pattern of reactivity of three codons (representing) on each band with cd.Top at figure has shown treatment plan.

Fig. 5. have as patient treatment scheme in viral load amount described in the embodiment 4 and ALT data.This figure shows following the tracks of the analysis of sample.Below described data the codon 528,552 that provides and 555 genetic information.Cd: codon; Seq: the data of sequential analysis; LiPA: analyze the data that obtain by LiPA.Represent amino acid with alphanumeric codes.Represent mixture with lowercase: lm=leucine+methionine(Met); Mvi=methionine(Met)+valine+isoleucine; Mi=methionine(Met)+Isoleucine; Vm=Xie Ansuan+methionine(Met).The 3TC=lamivudine; LT: liver transplantation; HBIg: hepatitis B immune globulin.

Fig. 6. as described in the embodiment 4, the LiPA to described patient in 1259 days that follow the tracks of analyzes.Understand the fate of sampling at each band subscript.Also shown treatment plan: the 3TC=lamivudine; Fam: Famciclovir, HBIg: hepatitis B immune globulin.Conj.cont.: the conjugate contrast; The Amp.cont.:HBV contrast of increasing.

Fig. 7. to the detailed analysis of the amplicon on each LiPA probe selected.Understand the fate of following the tracks of at each band subscript.Left figure: the sample of during lamivudine therapy, getting; Right figure: the sample of after the 3TC treatment stops, getting; Middle figure: the genome compound that is used for the correlated series of the position of indicating.At the 524th day, be that codon 551 comprises on the probe of TAA (terminator codon) also to observe weak hybridization.Codon 552I and codon 555 are not used probe.Do not show control line.

Fig. 8. in the analysis of three time points (X-axis) HBV polysaccharase variability.The clone's who on Y-axis, indicates at each time point number.In diagram, pointed out described cloned genes composition.Form table 1.HBV archaeal dna polymerase drug resistance codon is with respect to the genotypic position of different HBV.

Wild-type	Genotype				Mutant
						????A	B, C and F	????D	????E
	L	????528	????526	????515						????525	????M
M					????552	????550	????539	????549	????V/I
	V/M/L	????555	????553	????542						????552	????I

Table 2. is used for the probe of gene test HBV archaeal dna polymerase L528M sudden change.

Title	Sequence	SEQ?ID?NO
			HBPr270	?5′-CCGTTTCTCTTGGCTC-3′	????1
HBPr293	?5′-CCGTTTCTCCTGGCTCA-3′	????2
			HBPr294	?5′-CGTTTCTCTTAGCTCAG-3′	????3
HBPr412	?5′-CGTTTCTCCTAGCTCAG-3′	????4
			HBPr274	?5′-CGTTTCTCATGGCTCA-3′	????5
HBPr355	?5′-CGTTTCTCATGGCCCAG-3′	????6
			HBPr415	?5′-GTTTCTTATGGCTCAG-3′	????7
HBPr461	?5′-GTTTCTCATGACTCAG-3′	????8
			HBPr468	?5′-TTTCCCATGGCTCAG-3′	????9
HBPr468-1	?5′-TTTCCCATGGCTCAGTTT-3′	????10

Table 3. is used for the probe of gene test HBV archaeal dna polymerase M552V/I sudden change.

Title	Sequence	SEQ?ID?NO
			?HBPr308	?5’-CTTTCAGCTATATGGATGA-3′	????11
?HBPr322	?5’-CTTTCAGTTATATGGATGAT-3′	????12
			?HBPr349	?5’-GTGGCTTTTAGTTATATGGAT-3′	????13
?HBPr478	?5’-GCTTTCAGCCATATGGATG-3′	????14
			?HBPr309	?5’-TTTCAGCTATGTGGATGAT-3′	????15
?HBPr318	?5’-TTTCAGCTATGTGGACGA-3′	????16
			?HBPr426	?5’-TTTCAGTTATGTGGATGAT-3′	????17
?HBPr427	?5’-TTTTAGTTATGTGGATGAT-3′	????18
			?HBPr463	?5’-TTTCAGTCATGTGGATGA-3′	????19
?HBPr315	?5’-CTTTCAGCTAAGTGGATGA-3′	????20
			?HBPr363-1	?5’-GCTTTCAGCTATATAGATGA-3′	????21
?HBPr407	?5’-GCTTTCAGCTATATTGATG-3′	????22
			?HBPr488	?5’-TTTCAGCTATATCGATGAT-3′	????23
?HBPr433	?5’-CTTTCAGTTATATTGATGA-3′	????24
			?HBPr433-1	?5’-CTTTCAGTTATATTGATGAT-3′	????25
?HBPr465	?5’-CTCTCAGTTATATTGATGA-3′	????26
			?HBPr456	?5’-TGGTTTTCAGTTATATTGAT-3′	????27
?HBPr380	?5’-GCTTTTAGTTATATCGATG-3′	????28
			?HBPr453	?5’-CTTTCAGTTACATTGATGA-3′	????29
?HBPr485	?5’-TTCGGTTATGTGGATGAT-3’	????97
			?HBPr486	?5’-TTTCGGTTATGTGGATGA-3’	????98
?HBPr487	?5’-CTTTCGGTTATGTGGAT-3’	????99
			?HBPr488	?5’-GCTTTCGGTTATGTGG-3’	????100

Table 4. is used for the probe of gene test HBV archaeal dna polymerase V/L/M555I sudden change.

Title	Sequence	??SEQ?ID?NO
			?HBPr279	?5′-GATGATGTGGTATTGGG-3′	????30
?HBPr338	?5′-GGACGATGTGGTATTGG-3′	????31
			?HBPr341	?5′-TGATGTGGTACTGGGGG-3′	????32
?HBPr345	?5′-TGATGTGGTTTTGGGGG-3′	????33
			?HBPr474	?5′-GATGTGGTGTTGGGGG-3′	????34
?HBPr332	?5′-GATGATGTGATATTGGGGG-3′	????35
			?HBPr328	?5′-GATGATCTGGTATTGGGGG-3′	????36
?HBPr385	?5′-ATGATATTGTATTGGGGGC-3′	????37
			?HBPr289	?5′-GATGATATAGTACTGGGG-3′	????38
?HBPr299	?5′-ATGATATAGTATTGGGGGC-3′	????39
			?HBPr419	?5′-GATGATATGGTATTGGG-3′	????40
?HBPr490	?5’-GATGACGTGGTATTGGGGC-3’	????101
			?HBPr491	?5’-ATGACGTGGTATTGGGGCC-3’	????102
?HBPr492	?5’-TGACGTGGTATTGGGGCC-3′	????103
			?HBPr494	?5’-GATGATTTGGTATTGGGG-3’C	????104
?HBPr495	?5’-ATGATTTGGTATTGGGGCC-3’	????105
			?HBPr496	?5’-ATGATTTGGTATTGGGGCCA-3’	????106

Table 5. be used to the to increase primer of HBV dna polymerase gene or its part.

Title	Sequence	Justice/antisense is arranged	SEQ?ID?NO
				?HBPr134	?5′-TGCTGCTATGCCTCATCTTC-3′	Justice is arranged	????41
?HBPr135	?5′-CA(G/A)AGACAAAAGAAAATTGG-3′	Antisense	????42
				?HBPr135A	?5′-CAGAGACAAAAGAAAATTGG-3′	Antisense	????43
?HBPr135B	?5′-CAAAGACAAAAGAAAATTGG-3′	Antisense	????44
				?HBPr75	?5′-CAAGGTATGTTGCCCGTTTGTCC-3′	Justice is arranged	????45
?HBPr94	?5′-GGTA(A/T)AAAGGGACTCA(C/A)GATG-3′	Antisense	????46
				?HBPr94A	?5′-GGTATAAAGGGACTCACGATG-3′	Antisense	????47
?HBPr94B	?5′-GGTATAAAGGGACTCAAGATG-3′	Antisense	????48
				?HBPr94C	?5′-GGTAAAAAGGGACTCAAGATG-3′	Antisense	????49
?HBPr105	?5′-GG(T/C)A(A/T)AAAGGGACTCA(C/A)GATG-3′	Antisense	????50

Table 6. is set up is used for detecting simultaneously the INNO-LiPA band of the sudden change at the codon 528,552 of HBV archaeal dna polymerase and 555 places.Shown the HBV archaeal dna polymerase codon of indicating and in the HBsAg of correspondence codon detected De amino acid.The production and the use of LiPA band in embodiment 2, have been explained.

Line	Probe	Codon in the archaeal dna polymerase	Codon among the HBsAg
					??1	??HBPr270，293	????L528	???S171	???W172
??2	??HBPr294，412	????L528	???S171	???X172
					??3	??HBPr274，355	????M528	???S171	???W172
??4	??HBPr415	????M528	???L171	???W172
					??5	??HBPr461	????M528	???S171	???X172
??6	??HBPr468-1	????M528	???P171	???W172
					??7	??HBPr308，322，349，478	????M552	???I195	???W196
??8	??HBPr309，318，426，427，463	????V552	???M195	???W196
					??9	??HBPr315	????V552	???K195	???W196
??10	??HBPr363-1	????I552	???I195	???X196
					??11	??HBPr407，433-1，465，456	????I552	???I195	???L196
??12	??HBPr380，488	????I552	???I195	???S196
					??13	??HBPr453	????1552	???T195	???L196
??14	??HBPr279，338，341，345，474	????V555	???M198	???W199
					??15	??HBPr332	????V555	???M198	???X199
??16	??HBPr328	????L555	???I198	???W199
					??17	??HBPr419	????M555	???I198	???W199
??18	??HBPr385	????I555	???I198	???L199
					??19	??HBPr289，299	????I555	???I198	???X199

Table 7. in the HBV therapeutic process in different time points in two patients in codon 528,552 and/or 555 places mutant in vogue.Fate clone overall number has clone's number of codon combination 528/552/555

L/M/V L/M/I L/V/V M/M/V M/V/V patient A 199000 0,360 84400 0,420 93600 0,510 18 18 000 0,570 55 23 32 00 0,630 46 2112 40,750 13 0000 13 sums 158 59 43 12 53 patient C1 55000 0,148 66000 0,325 11 100 10 0,407 90009 0,450 12 100 11 0,519 23 0003 20,542 600006 sums 72 13 00 33 26 tables 8. are in the analysis of the sudden change at codon 528 places of HBV polymerase. The contrast of data that obtain by LiPA and the data that obtain by order-checking.

	Order-checking
				LiPA	M (mutant)	L (wild-type)	Sum
M (mutant)	????23		????23
				L (wild-type)		????44	????44
L/M (mixing)	????15	????1	????16
				Sum	????38	????45	????83

Table 9. is in the analysis of the sudden change at codon 552 places of HBV polysaccharase.The contrast of data that obtain by LiPA and the data that obtain by order-checking.

	Order-checking
					LiPA	I (mutant)	V (mutant)	M (wild-type)	Sum
I (mutant)	????8			????8
					V (mutant)		????17		????17
M (wild-type)			????45	????45
					V/I (mutant)		????2		????2
M/I (mixing)	????4			????4
					M/V (mixing)		????1	????2	????3
Sum	????12	????20	????47	????79

Detailed Description Of The Invention

The present invention relates to a kind of method, at least a by among sudden change L528M, M522V/I in the HBV Strain archaeal dna polymerase of genetic test in being present in patient's biological sample and/or the V/L/M555I of the method, the anti-HBV drug resistance of monitoring in described patient, the method may further comprise the steps:

Discharge, separate and/or be concentrated in the polynucleotide (polynucleic acid) that exists in the described biological sample when (i) needing;

When (ii) needing with at least one suitable primer to HBV dna polymerase gene or its part of amplification in described biological sample;

(iii) polynucleotide that obtains in step (i) and/or (ii) and at least one can with target sequence in the HBV dna polymerase gene or the Probe Hybridization of its complement specific hybrid, described target sequence is selected from a plurality of target sequences that are shown in Fig. 1;

(iv) detect the crossbred that in step (iii), forms;

(v) by the hybridization signal that in step (iv), obtains, infer the possible anti-HBV drug resistance that in described archaeal dna polymerase, exists or do not exist described L528M, M522V/I and/or V/L/M555I to suddenly change and be present in the HBV Strain in the described biological sample.

Method described above is sensitivity or resistance so that can determine the HBV Strain to certain primary antibodie HBV medicine by genetic test at least one sudden change in the codon 528,552 and/or 555 of HBV dna polymerase gene. Isolate a large amount of new HBV dna polymerase gene sequences so that the present inventor can draw the reference sequences figure of target sequence, can develop on this basis very specificity and highstrung hybridization assays for detection of above-mentioned sudden change.

In whole the application, the codon of HBV pol gene numbering adopts the numbering of genotype A. Table 1 has shown the general introduction of in other genotype codon position 528,552 and 555 reference numeral. Sudden change L528M refers to that the leucine genetic code at codon 528 places of HBV dna polymerase gene replaces with the genetic code of methionine. Sudden change M552V/I refers to that the methionine genetic code at codon 552 places of HBV dna polymerase gene replaces with the genetic code of valine or isoleucine. Sudden change V/L/M555I refers to that valine, leucine or the methionine genetic code at codon 555 places of HBV dna polymerase gene replaces with the genetic code of isoleucine.

The term " genetic test sudden change " that uses in the present invention refers to by measuring the sudden change of corresponding nucleotide sequence detection in amino acid sequence.

In the method for the invention, by will be present in nucleic acid in described patient's biological sample and one or more can with the HBV dna polymerase gene that is shown in Fig. 1 in the Probe Hybridization of target sequence specific hybrid, be detected on the codon 528 of HBV archaeal dna polymerase, the sudden change at 552 and/or 555 places. Term " specific hybrid " refers to that the part of described probe and its target sequence under employed experimental condition or its whole target sequence form duplex, but under the described conditions described probe does not form duplex with other sequence that is present in the polynucleotide in the sample to be analyzed.

Refer to sequence in the HBV dna polymerase gene according to " target sequence " of term probe of the present invention, it comprise coding HBV archaeal dna polymerase amino acid 528,552 and/or 555 codon sudden change or the wild-type nucleic acid sequence, and complementary (i.e. the highest 20% mispairing of described probe and its complete complementary or part, more preferably 15%, more preferably 10% or most preferably 5%). It being understood that the complement of described target sequence also is suitable target sequence in some cases. The target sequence that Fig. 1 describes gets the serum and plasma sample of different patients in comfortable follow-up study and the sampling study, and former unexposed mistake. By using these new polymorphic nucleotide sequences of the HBV dna polymerase gene also consist of the present invention's part, very specificity and the probe that detects sensitively anti-HBV drug resistance become possibility so that design new permission. Should be understood that, being used for may be in the scope of described target sequence with the probe of the target sequence specific hybrid of nucleic acid, perhaps may with described target sequence on a large scale overlapping (namely forming has outside the described target sequence and the duplex of nucleotides wherein).

Term " probe " refers to have strand, the sequence-specific oligonucleotides with the sequence of the target complement sequence of HBV dna polymerase gene. Preferably about 5-50 nucleotides is long for described probe, and more preferably about 10-25 nucleotides is long. The particularly preferred length of probe comprises 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 nucleotides. The nucleotides that is used for probe of the present invention can be the nucleotides (such as inosine) of ribonucleotide, deoxyribonucleotide and modification or the nucleotides that comprises the modification group that does not basically change its hybridization characteristic.

In a specific embodiment, the probe that uses in the methods of the invention is selected from table 2,3 and/or 4, wherein

-be selected from following with the probe of L528M target sequence specific hybrid: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468, HBPr468-1 (SEQ ID NO 1 to SEQ ID NO 10);

-be selected from following with the probe of M552V/I target sequence specific hybrid: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433, HBPr433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 (SEQ ID NO 11 to SEQ ID NO 29, SEQ ID NO 97 to SEQ ID NO 100);

-be selected from following with the probe of V/L/M555I target sequence specific hybrid: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBPr492, HBPr494, HBPr495, HBPr496 (SEQ ID NO 30 to SEQ ID NO 40, SEQ ID NO 101 to SEQ ID NO 106).

By with at least one probe, preferably at least 2, more preferably at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or more oligonucleotide probe hybridization, detect the sudden change at codon 528,552 and/or 555 places.

In a preferred embodiment, the present invention relates to aforesaid method, be further characterized in that described probe is suitable for hybridizing simultaneously with its target area most under the same hybridization that allows to detect simultaneously a large amount of polymorphic districts and wash conditions (for example in the LiPA mode, vide infra).

More particularly, the present invention relates to aforesaid method, be further characterized in that in step (iii) probe that uses is at least a in the following probe combinations:

-for detection of the probe of L528M sudden change: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461 and HBPr468-1 (SEQ ID NO 1 to SEQ ID NO 10);

-for detection of the probe of M552V/I sudden change: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr465, HBPr456, HBPr380 and HBPr453 (SEQ ID NO 11 to SEQ ID NO 29);

-for detection of the probe of V/L/M555I sudden change: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299 and HBPr419 (SEQ ID NO 30 to SEQ ID NO 40).

The invention still further relates to the oligonucleotides as the probe that carries out aforesaid any method.

In a preferred embodiment, the present invention relates to the oligonucleotides of description in table 2,3 and 4.

Be shown by 5 ' terminal single stranded DNA oligonucleotides form to 3 ' end in the sequence table of whole specification middle probe. Arbitrary probe of hereinafter enumerating can former state use, or uses with its complement form, or uses with its rna form (wherein T is replaced by U), and this is apparent to those skilled in the art.

Because the application need to detect single base-pair mismatch, so need to be used for the stringent condition of Probe Hybridization, this condition only allows accurately complementary sequence hybridization. Yet, should be noted that, because the central portion of described probe is essential for its hybridization characteristic, so when using long probe sequence, described probe sequence may allow for the end of described probe with respect to the possible deviation of described target sequence. In the time would rather selecting other hybridization conditions, probe therefore may be its terminal add or lack one or more nucleotides adapt. Should be understood that these follow adaptability should produce identical result, namely described probe still with its type specificity target sequence specific hybrid separately. If the material of amplification is RNA rather than DNA (like this being exactly in the NASBA system), then such adaptability also may be necessary. Yet should be always can depart from and make a variation by this area general knowledge imagination described through test evaluation, in order to check the hybridization characteristic that whether they produce and the probe of accurately complementation is equal to.

For design has the probe that needs characteristic, can use the following useful criterion that those skilled in the art will know that. Because the degree of all as described herein those hybridization reactions and specificity are permitted multifactorial the impact, so one or more that control in these factors will determine definite sensitiveness and the specificity of particular probe, be complete complementary or not exclusively complementary to its target. This paper has further set forth the importance and role of various condition determinations:

The stability of-selected [probe: target] nucleic acid hybrids should with described condition determination fit. This can by avoid long AT enrichment sequence, by stopping described crossbred with the G:C base-pair and realizing by the probe that design has a suitable Tm. Should select starting point and the terminal point of described probe, so that the Tm that length and %GC produce is higher than the about 2-10 of the temperature of measuring at last ℃. The base composition of described probe is important, because compare with the A-T base-pair, the G-C base-pair is owing to extra hydrogen bonding has higher heat endurance. Therefore, it is more stable when higher temperature to relate to the hybridization of complementary nucleic acid of higher G-C content.

During-designing probe, the condition of probe application such as ionic strength and heated culture temperature, also should be taken into account. Known hybridization degree will increase along with the ionic strength of reactant mixture and increase, and the heat endurance of described crossbred will increase along with the increase of ionic strength. On the other hand, destroy the chemical reagent of hydrogen bond, such as formamide, urea, DMSO and alcohol reagent, will increase the stringency of hybridization. Make the hydrogen bond instability can significantly reduce Tm with such reagent. In general, the hybridization that is suitable for the long synthetic oligonucleotide probe of about 10-50 base most occurs in below the melting temperature of the duplex that produces about 5 ℃. Therefore temperature incubation below optimum temperature may so that the base sequence of mispairing can be hybridized, and can cause specificity to reduce.

-it is desirable to obtain the probe of only under high stringent condition, hybridizing. The nucleic acid hybrids of a height of formation complementation under high stringent condition. Do not form the crossbred of the complementarity with enough degree. Therefore, the stringency of testing conditions is determining the amount of the complementarity that the formation crossbred needs between two nucleic acid chains. Select strict degree, so that and the stability difference maximum between the crossbred of target nucleic acid and non-target nucleic acid formation. Under existing conditions, need to detect single base-pair and change, this needs very high strict condition.

The length of-described probe sequence may also be important. In some cases, several sequences may be arranged from a given zone, their position is different with length, generation is had the probe of the hybridization characteristic that needs. In other cases, sequence is significantly better than another sequence that base is different only. Although not exclusively complementary nucleic acid may be hybridized, one section the longest common major decision of complete complementary base sequence crossbred stability. Although can use the oligonucleotide probe of different length and base composition, but the length of the preferred oligonucleotide probe of the present invention is between about 5-50 (more particularly being 10-25) base, and in described sequence, have one section enough sequence, with the target nucleic acid sequence complete complementary.

The district of-not too preferred strong the internal structure that known formation inhibition is hybridized in target DNA or RNA. Similarly, should avoid having extensively the probe of self complementarity. As explained above, to form hydrogen-bonded two strands relevant with the strand of two complementary nucleic acids in hybridization. What need not query is that if in described two chains is contained in the crossbred wholly or in part, then it not quite can participate in forming new crossbred. If there are enough self complementarity, the crossbred that then forms in the molecule of one type probe can be crossbred and intermolecular hybrids in the molecule. Can avoid such structure by careful probe design. So that the aim sequence of considerable part is strand, hybridization speed and degree can significantly increase by designing probe. Can utilize computer program to retrieve such interaction. Yet in some cases, such interaction may be inevitable.

-standard hybridization and wash conditions are partly disclosed at the materials and methods of embodiment. Other condition be for example 3X SSC (natrium citricum salt solution), 20% deionization FA (formamide), 50 ℃. Also can use other solution (SSPE (sodium phosphate edta salt water), TMAC (tetramethyl ammonium chloride) etc.) and temperature, prerequisite is specificity and the sensitiveness that keeps described probe. When needing, must carry out slight modification in length or in sequence to described probe, to remain on specificity required under the given environment and sensitiveness.

The recombinant plasmid that can comprise by the clone Insert Fragment that comprises corresponding nucleotide sequence according to probe of the present invention, can use suitable nuclease described nucleotide sequence of excision from clone's plasmid when needing, and they prepare for example to separate recovery by the classification according to molecular weight. Also can chemical synthesis according to probe of the present invention, for example by conventional phosphotriester method.

The term " biological sample " that uses in the present invention refer to or directly taken out by the people who infects or cultivate any biomaterial (tissue or body fluid) that take out, that comprise the HBV nucleotide sequence after (enrichment). Biomaterial can be such as any phlegm, bronchial perfusate (broncheolavage), blood, skin histology, biological tissue, seminal fluid, lymphocyte blood culture, colony, liquid culture, fecal specimens, urine, liver cell etc. More particularly, " biological sample " refers to serum or plasma sample.

By any method known in the art by discharging in the described biological sample, concentrate and/or separating described nucleic acid. At present, can buy various commercial reagent box, such as QIAamp blood kit (Hilden, Germany) and ' the high-purity pcr template prepares kit ' (Roche Diagnostis from the blood sample isolating nucleic acid of being used for of Qiagen, Brussels, Belgium). Can obtain DNA in other separation of biological samples that is widely known by the people or the method (Maniatis etc., 1989) of RNA.

Nucleic acid in sample to be analyzed can be any among DNA or the RNA, for example genomic dna, messenger RNA(mRNA), viral RNA or its amplification form.These molecules also are called polynucleotide.

Be present in HBV dna polymerase gene or its part in the described biological sample, can pass through polymerase chain reaction (PCR; Saiki etc., 1988), ligase chain reaction (LCR) (LCR; Landgren etc., 1988; Wu and Wallace, 1989; Barany, 1991), based on the amplification (NASBA of nucleotide sequence; Guatelli etc., 1990; Compton, 1991), based on the amplification system (TAS that transcribes; Kwoh etc., 1989), strand displacement amplification (SDA; Duck, 1990) or utilize the amplification (Lomeli etc., 1989) of Q β replicative enzyme or the method amplification by any other suitable permission amplified nucleic acid molecule known in the art.Can also use TMA (Guatelli etc., 1990) or bDNA (Sanchez-Pescador etc., 1988 in the method for the invention; Urdea etc., 1991) technology.

Term " primer " be meant with nucleic acid chains complementary primer extension product that will copy or amplified production synthetic in can play the single stranded oligonucleotide sequence of synthetic starting point effect.The length of described primer and sequence must make them can cause the synthetic of described extension products.Preferably about 5-50 the Nucleotide of the length of described primer.The length of described primer is 10-30 Nucleotide more preferably from about.The length of described primer is 20-25 Nucleotide most preferably from about.Concrete length and sequence depend on the complicacy of required DNA or RNA target and the condition that described primer uses, such as temperature and ionic strength.

Statement " primer sets " is meant the primer of a pair of permission HBV dna polymerase gene or the amplification of its part.A primer sets always is made up of forward primer (adopted primer or 5 ' primer are arranged) and reverse primer (antisense primer or 3 ' primer).

In a preferred embodiment, the present invention relates to aforesaid method, its feature is that also at least a primer that uses in (ii) in step is selected from table 5 (HBPr134, HBPr135, HBPr135A, HBPr135B, HBPr75, HBPr94, HBPr94A, HBPr94B, HBPr94C and/or HBPr105; SEQ ID NO 41 to SEQ ID NO 50).More particularly, the present invention relates to aforesaid method, its feature is that also described primer sets is made up of following two kinds of primers:

-as the HBPr134 of forward primer with as the HBPr135 of reverse primer; And/or

-as the HBPr75 of forward primer with as the HBPr94 of reverse primer; And/or

-as the HBPr75 of forward primer with as the HBPr105 of reverse primer.

The technician will recognize that these primers (SEQ ID NO 41 to 50) can be made amendment by adding or lack one or more Nucleotide at its end.If for example amplification condition changes, if the material of amplification is RNA rather than DNA, in the NASBA system this situation for example, then may need such modification.In the document (Kwok etc., 1990) detail record the following fact: promptly amplimer do not need with template in corresponding target sequence accurately coupling guarantee correct amplification.Yet, when described primer and the incomplete complementation of its target sequence, should consider that the fragment that is increased will have the sequence of described primer rather than the sequence of described target sequence.

Can mark primer of the present invention and/or probe.Can use any method well known by persons skilled in the art to carry out mark.The character of described mark can be isotropic substance ( ³²P, ³⁵S etc.) or heterotope (vitamin H, digitoxigenin etc.).

Can also comprise nucleotide analog or form as the oligonucleotide of primer or probe by it, maybe can comprise intercalator (Asseline etc., 1984), described nucleotide analog is such as thiophosphoric acid (Matsukura etc., 1987), thiophosphoric acid alkyl ester (Miller etc., 1979) or peptide nucleic acid(PNA) (Nielsen etc., 1991; Nielsen etc., 1993).In order to influence the characteristic of the reversibility that forms such as hybridization kinetics, crossbred, the biologically stable of described oligonucleotide molecules etc. energetically, it may be favourable introducing these modifications.

About other variation of great majority and the modification in the original DNA sequence that is incorporated into primer and probe, these variations will make the condition that should use with the described oligonucleotide condition of necessitating that adapts, to obtain required specificity and susceptibility.Yet, basic identical with the result that the oligonucleotide of these modifications causes or the end-result of hybridization should obtain with the oligonucleotide with unmodified.

In a preferred embodiment, the present invention relates to aforesaid method, its feature is that also described probe can hybridize with its target area separately simultaneously under suitable hybridization conditions and wash conditions, make to detect more than one wild-type codon and/or sudden change codon simultaneously.

More particularly, the present invention relates to aforesaid method, its feature also is step detection two kinds of sudden changes L528M and M552V/I.

More particularly, the invention still further relates to aforesaid method, its feature also is step detection two kinds of sudden changes L528M and V/L/M555I.

More particularly, the present invention relates to aforesaid method, its feature also is step detection two kinds of sudden changes M552V/I and V/L/M555I.

More particularly, the present invention relates to aforesaid method, its feature also is step detection three kinds of sudden changes L528M, M552V/I and V/L/M555I.

Method of the present invention can be used for screening (promptly the monitoring pharmacological agent) that before beginning anti-HBV treatment, exists and/or produce in anti-HBV pharmacological agent process and give to lamivudine, Famciclovir and/or Penciclovir resistance, in the sudden change at codon 528,552 and/or 555 places.Therefore, the present invention relates to a kind of method, the HBV virus strain that its feature also is to be present in the described biological sample shows anti-lamivudine, Famciclovir and/or Penciclovir.Method of the present invention can also be used for determining the resistance at the anti-HBV medicine outside the above-mentioned medicine, and prerequisite is with relevant with in three sudden changes of this method detection one or several at the resistance of these other medicines.Term " anti-HBV medicine " is meant any anti-HBV nucleoside analog or any other archaeal dna polymerase inhibitor that causes that the described viral DNA among the patient reduces.Other anti-HBV medicine includes but not limited to, adefovir, BMS200475, phosphine formic acid, fiacitabine, FIAU, (-)-FTC, ganciclovir, GEM132, Interferon, rabbit, L-FMAU, lobucavir, just-behenyl alcohol, ribavirin, Sorivudine, vidarabine or the compound of mentioning in WO98/18818.Method of the present invention also can with detect the method may give one or more other sudden changes of other anti-HBV drug resistance and unite use.Therefore, can also add the probe that allows to detect other sudden change in the method for the invention.

The invention still further relates to a kind of composition that comprises at least a probe of the present invention.The term of Shi Yonging " composition " is meant the probe mixture at suitable buffer that is used for implementing the inventive method in the present invention.The invention still further relates to probe and composition as defined above and be used for the purposes of the anti-HBV drug resistance of external monitoring patient.

The invention still further relates at least a by among sudden change L528M, M552V/I in the HBV archaeal dna polymerase of the HBV virus strain of gene test in being present in patient's biological sample and/or the V/L/M555I, monitor the diagnostic kit of anti-HBV drug resistance among the described patient, it comprises following component:

(i) under the suitable situation, be used for discharging, separating or concentrate the material of the polynucleotide that is present in described biological sample;

Under the (ii) suitable situation, at least one primer that is fit to is right;

(iii) at least one aforesaid probe may be fixed on the solid support;

(iv) hybridization buffer or produce the essential component of described damping fluid;

(v) washing soln or produce the essential component of described solution;

(under the vi) suitable situation, be used to detect the material of the crossbred that produces by aforementioned hybridization;

(under the vii) suitable situation, be used for described probe is attached to the material of the known location on the solid support.

Term " hybridization buffer " is meant and allows described probe and be present in polynucleotide in the described sample or hybridization takes place amplified production damping fluid under suitably strict condition.

Term " washing soln " is meant the solution that can wash the crossbred that forms under suitably strict condition.

Can use any measuring method that depends at the polynucleotide of described biological sample and form crossbred between according to oligonucleotide probe of the present invention.For example, use southern blotting technique, RNA trace or Dot blot mode, unlabelled amplification sample is bonded to film, this film is introduced at least a label probe under suitable hybridization and wash conditions, and the existence of monitoring bonding probes, can finish described hybridization.Perhaps use " oppositely " mode, wherein extension increasing sequence comprises mark.In this mode, the probe stationary of selection some position on solid support, the polynucleotide of mark amplification is so that can detect the crossbred of formation.Term " solid support " can refer to any matrix that can the coupling oligonucleotide probe, and prerequisite is the background level that it is possessed its hybridization characteristic and keeps low hybridization.Common described solid phase matrix is microtiter plate (for example microtiter plate in the DEIA technology), film (for example nylon or nitrocotton) or microsphere (bead) or chip.Before being applied to described film or fixing, can modify described nucleic acid probe aptly, to be convenient to fixing or to improve hybridization efficiency.Such modification can comprise homopolymeric tailing; The reactive group that coupling is different is such as aliphatic group, NH ₂Group, SH group, carboxyl; Or coupling vitamin H, haptens or albumen.

Therefore, the present invention relates at least a by among sudden change L528M, M552V/I in the HBV archaeal dna polymerase of the HBV virus strain of gene test in being present in patient's biological sample and/or the V/L/M555I, monitor the line formula probe assay of antiviral resistance among the described patient, it comprises following component:

(i) under the suitable situation, be used for discharging, separating or concentrate the material of the polynucleotide of the biological sample that is present in described patient;

Under the (ii) suitable situation, at least one primer that is fit to is right;

(iii) at least a be shown in L528M target sequence probe specific hybrid, that be fixed in solid support of Fig. 1, be selected from HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461 and HBPr468; And/or

(iv) at least a be shown in M552V/I target sequence probe specific hybrid, that be fixed in solid support of Fig. 1, be selected from HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487 and HBPr488; And/or

(v) at least a be shown in V/L/M555I target sequence probe specific hybrid, that be fixed in solid support of Fig. 1, be selected from HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBPr492, HBPr494, HBPr495 and HBPr496.

(vi) hybridization buffer or produce the essential component of described damping fluid;

(vii) washing soln or produce the essential component of described solution;

(under the viii) suitable situation, be used to detect the material of the crossbred that produces by aforementioned hybridization;

In this embodiment, selected probe groups is fixed in the film band of wire.Described probe can be fixed in described position separately or as mixture.Can detect described crossbred with the on-radiation Color Appearance System through vitamin H-Streptavidin coupling then with HBV archaeal dna polymerase polynucleotide or its part of biotin labeling amplification.

The invention still further relates to new sequence (SEQ IN NO 51-86 and SEQIN NO 107-109) or its fragment as in Fig. 1, describing, wherein said fragment comprises at least 10 of being shown in Fig. 1, preferred 15 even more preferably 20 successive Nucleotide, and described fragment contains one in the amino acid 528,552 of the wild-type of coding HBVDNA polysaccharase or sudden change and/or 555 the codon.Therefore, the present invention relates to comprise aforesaid nucleotide sequence or by its isolating nucleic acid of forming.Term " nucleic acid " is meant strand or double-strandednucleic acid sequence, and it can contain the extremely full nucleotide sequence of 10 Nucleotide that are shown in Fig. 1.Nucleic acid can comprise the Nucleotide of deoxyribonucleotide, ribonucleotide, nucleotide analog or modification.The complement that it being understood that above-mentioned nucleic acid has also constituted part of the present invention.

In whole specification sheets and claims subsequently, unless need in addition in the literary composition, otherwise word " comprises " to be understood to mean and comprises pointed integer or step or pointed integer or step group, but does not also get rid of any other integer or step or integer or step group.

EXAMPLE Example 1: separate new HBV dna polymerase gene sequence a. patient

By collecting serum or plasma sample among 41 patients in the follow-up study process, by collecting serum or plasma sample among 80 patients in sampling study.Described patient in follow-up study is as follows: 18 patients are in the follow-up study of doctor F.Zoulim (INSERM, Lyon, France); 5 patients are in the follow-up study of doctor D.Pillay (Public Health Laboratory Service, Birmingham, Britain); 3 patients in the follow-up study of doctor G.Leroux (Uinversity Hospital, Gent, Belgium) and 15 patients in the follow-up study of doctor D.Lau (NIH, Bethesda, MD, the U.S.).Described sampling study is undertaken by doctor J.Lau (Schering Plough, Madison, NJ, the U.S.).The purifying of b.HBV DNA and amplification

Use commercially available＞high purity pcr template to prepare test kit ' (Boehringer-Mannheim, Brussels, Belgium) by separating HBV DNA in described serum or the plasma sample.Use the HBV polysaccharase district DNA of nested PCR method amplification purification, described nested PCR method is: DNA and 5 μ l 10x damping fluids, 0.4 μ l 10mM dXTP, the 10pmol of 10 μ l purifying are had adopted primer, 10pmol antisense primer, 1 Tap of unit polysaccharase (StratageneEurope, Amsterdam, Holland) mix, and to make final volume with hplc grade water be 50 μ l.PCR comprises 45 ℃ of annealing, 72 ℃ of prolongations and 94 ℃ of sex change, each 30 seconds.Outer PCR comprises 40 circulations; The nested type reaction comprises 35 circulations.With following combination of primers amplification HBV polysaccharase district: adopted primer HBPr134:5 '-TGCTGCTATGCCTCATCTTC-3 ' (SEQ ID NO 41) is arranged outward; Outer antisense primer HBPr135:5 '-CAG/AAGACAAAAGAAAATTGG-3 ' (SEQ ID NO 42); Nested type has adopted primer HBPr75:5 '-CAAGGTATGTTGCCCGTTTGTCC-3 ' (SEQ ID NO45); Nested type antisense primer HBPr94:5 '-GG (T/C) A (A/T) AAAGGGACTCA (C/A) GATG-3 ' (SEQ ID NO 46).The nested type amplified production is analyzed on 2% sepharose and with the ethidium bromide colour developing, is 341bp long (comprising primer).Under the situation of LiPA test, be provided at the primer that its 5 ' end has the vitamin H group.C. plasmid clone and DNA purifying

The pGemT carrier (Promega, Leiden, Holland) of 2 μ l amplified productions and the pretreated EcoRV cutting of 1 μ l is mixed, and utilization＞Ready to Go=T4 ligase enzyme (Pharmacia, Leusden, Holland) connects.After in the competence coli strain, transforming, select one recombinant clone, with＞high purity plasmid separating kit (BorhringerMannheim, Brussels, Belgian) or ' Qia prep 96 Turbo Bio Robot test kits ' (Qiagen, Hilden, Germany) plasmid DNA purification.Utilize or derive from the primer or the nested type HBV primer of plasmid, pcr amplification is from the insertion fragment of recombinant clone.D. reference sequences figure draws and probe design

The reference sequences figure that drafting is used for probe design estimates probe simultaneously.In the initial period, design specific detection sudden change L528M, M552V/I and/or V/L/M555I exist or more non-existent probes, and have obtained described probe after the ionic strength of considering GC percentage, probe length, hybridization buffer and these parameters of heated culture temperature.By with nitrocellulose membrane on these specific probes, follow the reverse hybridized biotinylated PCR fragment that produces by described blood plasma or serum sample (for the LiPA form), described probe is estimated in Streptavidin-alkaline phosphatase incubation and colour developing.About the details of probe optimization phase (probe optimization phase), described in (1998) such as (1996), Stuyver etc. (1997) such as Stuyver and Van Geyt that the LiPA band is produced and reverse hybridized.

With described serum of this initial stage LiPA strip analysis and plasma sample the time, many probe reactions of not selecting with those in their the PCR product at initial stages.The sequential analysis of these unreacted PCR products has shown new primitive, for described new primitive designs corresponding probe.The LiPA band that comprises these newly-designed probes causes the not reactive decline of described sample.

Subsequently, in reactive further analysis,, in described reference sequences figure, add described new primitive, and design new probe the nonreactive PCR product order-checking of all and selected probe groups to new probe groups and described blood plasma and serum sample.At last, kept the clone of 35 selections altogether by reference sequences figure in such a way and to its order-checking.Obtain double-stranded sequence by biotinylation PCR product, or under the situation of recombinant clone, use the sequencing primer that derives from carrier to obtain double-stranded sequence as in (1996) such as Stuyver, describing.The new sequence that obtains is shown in Fig. 1 (SEQ ID NO 51-86 and SEQ ID NO 107-109).Embodiment 2: it is anti-that design and test are used for monitoring the patient's who infects at HBV medicine

The property LiPAa. be designed for the monitoring drug resistance LiPA

The sequence of use in described reference sequences figure, design and checking are to codon (comprising wild-type and mutant primitive) and the different genotype and the polymorphic specific probe of codon of position 528,552 and 555.In the HBV archaeal dna polymerase, detect the ability of different wild-types and sudden change codon according to it, and, merge probe and also be splined on band according to the relevant information of overlapping HBsAg frame.Several different IPs thuja acids that are intended for use in are polymorphic but do not introduce the probe that amino acid changes and combine and a last line.This is final to form one and has 19 different probe lines, band (Fig. 2 of 38 specific probes altogether; Table 6).The details of producing with the LiPA band mutually about the probe optimization has been described in (1998) such as (1996), Stuyver etc. (1997) such as Stuyver and Van Geyt.B. use clone to test described LiPA from described reference sequences figure

Then with described LiPA band and the biotinylated PCR product that gets the recombinant clone among comfortable described reference sequences figure incubation together.In (1998) such as (1996), Stuyver etc. (1997) such as Stuyver and Van Geyt, described about reverse hybridized details.Fig. 3 has shown the specific reaction of some PCR products and the probe on described band.The probe of selecting is very specific for detecting corresponding amplicon.The wild-type or the sudden change codon that detect simultaneously in an independent experiment at codon 528,552 and 555 places are possible.Embodiment 3: use two of LiPA monitorings to accept anti-HBV among the patient of HBV treatment

Drug resistance a. analyzes the drug resistance that the patient follows the tracks of sample with HBV LiPA

Use described LiPA test before begin treatment, in the treatment neutralization treatment but during the virus bounce-back by the tracking sample that takes out among 2 patients.Evidence with viral load amount and ALT level (Fig. 4) conduct treatment failure.Patient A has infected A genotype HBV, and patient C infects C genotype HBV.Also be shown in Fig. 4 by these LiPA reactivities of following the tracks of the sample acquisition.With among the patient A of lamivudine therapy, of short duration existence in the 360th, 420,570 day and at the 630th day the faint mixture (line 14 and 19) that has V555I.Can infer that by Fig. 2 online 19 reactivity is with relevant at the Transcription Termination of HBsAg codon 199.This primitive is that disappearing after the mutant appears in codon 552 and 528.Still at the 630th day, observe the mixture on the two at codon 528 (line 1+3) and 552 (line 7+8).Wild-type primitive L528 and M552 disappear when virus broke through in the 750th day.Using Famciclovir in the patient C of lamivudine therapy, observe select progressively M528 mutant, be the M528+V552 double-mutant afterwards.In the 325th day wild-type L528 (line 1) and mutant M528 (line 3) coexistence, but no longer detected this wild-type primitive from the 407th day.Arised from codon 552 observed mutated viruses (line 8) and replaces wild-type virus (line 7) gradually from the 450th day.From the 542nd day backward, pure M528+V552 double-mutant appears.In patient C, do not observe codon in codon 555 places and change.As if selected at present probe useful to the drug resistance that monitoring occurs in antiviral therapy.B. clonal analysis is followed the tracks of sample

Obtain 7 plasma samples altogether in by patient A being dispersed throughout different time points in 750 day cycle of antiviral therapy.The clonal expansion product keeps 158 recombinant clones and is used for LiPA analysis (table 7).Except I555, in 99 clones altogether, be not chosen in the evidence of codon 528 and 552 places sudden change at the 1st day to the 570th day.At the 630th day, described clone's major part belonged to M528+V552 double-mutant group.Yet, there are single mutant L/V/V and M/M/V more or less, they can be interpreted as the vestiges of the intermediate forms of double-mutant selection.This shows that the appearance of resistance occurred in 60 days period (the 570th day to the 630th day).

In patient C, other 72 clones of 7 different time point analysiss that in the HBV therapeutic process, scatter.Owing to adopt the treatment plan of Famciclovir, exist as main group at the 325th day single mutant M528.Just after introducing lamivudine (the 325th day), detecting the M528+V552 mutant becomes main group (Fig. 4 and table 7).The selection of M552 mutant occurred in maximum 69 days period (the 450th day to the 519th day).The pressure to single M528 mutated viruses (rising by the 325th day) that is produced by lamivudine therapy causes the quick selection to described double-mutant in the 450th day.Embodiment 4: use the relevant sudden change of LiPA monitoring lamivudine in hepatitis B virus

The kinetics a. patient medical history of appearing and subsiding

When a Caucasia male sex of 52 years old in being found in the lab investigation in routine in 1985 when having abnormal serum transaminase (ALT) level, he at first is diagnosed as suffers from chronic hepatitis B.It is asymptomatic always that this patient keeps, and nineteen ninety-five just begins to seek medical advice.Find that at that time he slightly rises at the ALT level.Detect with solution hybridization test (Abbott Diagnostics, Chicago, IL, the U.S.), serum HBV DNA is 0.7pg/ml.This patient's thrombopenia (thrombocyte=64000), and the sign of slight liver synthesizer dysfunction is arranged, serum albumin reduces (3.1g/dl), and prothrombin time prolongs (17.1 seconds) a little.This patient's of routine test HBV serology distributes, and monitors this patient's some months closely.In December nineteen ninety-five, he the HBV DNA that becomes is highly positive, and shows abnormal Serum ALT levels (Fig. 5).This patient begins lamivudine (3TC) treatment (150mg every day) (the 331st day), and this causes HBV DNA to rapidly disappear, serum ALT activities normalizing.The 633rd day (treating back 302 days at 3TC), Serum ALT and viral load amount sharply rose, and detect HBV DNA again.Stop lamivudine therapy at the 633rd day, replace Famciclovir (FCV) (500mg, every day three times).This treatment is invalid to the HBV dna level, stops in the 779th day.After three months (the 856th day), this patient develops into ascites, coagulopathy worsens and varix is hemorrhage.He is put into the liver transplantation list in August, 1997 (about the 950th day).This patient does not accept antiviral therapy when transplanting although wait, and has experienced spontaneous, the of short duration decline of HBV DNA.When the HBV reactivate (the 1105th day), restart 3TC treatment, cause HBV DNA be dropped rapidly to detection less than level.This patient accepted donor livers at the 1223rd day, and began to infect preventing with hepatitis B immune globulin (HBIg) treatment again.At the 1259th day, detect less than the HBV dna level by routine test (non-PCR), and the horizontal normalizing of ALT.The b.DNA operation

HBV purifying and amplification.Use is used for the three reagent LS methods of DNA and extracts HBV DNA by 50 μ l blood plasma.Use nested PCR amplification HBV polysaccharase district A-E.First round PCR combination comprises: the dna profiling of 3 μ l purifying, 10 μ l 10X contain MgCl ₂Damping fluid, 2 μ l dNTP (120mM stock solution), forward primer (HBV-8For 5 '-CATCAGGATTCCTAGGACC-3 '; SEQ ID NO 87) and reverse primer (HBV-9Rev 5 '-ATACTTTCCAATCAATAGGCC-3 '; SEQ ID NO 88) each 1 μ l (30pmol/ μ l stock solution), 0.5 μ l Taq archaeal dna polymerase, and be supplemented to 100 μ l with hplc grade water.Amplification program comprises: 94 ℃ of sex change (45 seconds), 49.8 ℃ of annealing (1 minute), 72 ℃ of prolongations (2 minutes), 40 circulations.With the product of Qiagen PCR purification kit purifying 810bp, and be eluted in the 30-50 μ l water.The nested PCR reactant comprises: the PCR product of the 5 μ l first round, 5 μ l 10X damping fluid II, 3.7 μ l MgCl ₂, 1 μ l dNTP (10mM stock solution), forward primer (HBV-2For 5 '-CGCTGGATGTGTCTGCGGCG-3 '; SEQID NO 89) and reverse primer (HBV-R3 5 '-CCAACTTCCAATTACATAACCC-3 '; SEQ ID NO 90) each 1 μ l (30pmol/ μ l stock solution), 0.5 μ l Taq archaeal dna polymerase, and be supplemented to 50 μ l with hplc grade water.Amplification program comprises: 94 ℃ of sex change (45 seconds), 55 ℃ of annealing (1 minute) and 72 ℃ of prolongations (2 minutes), 35 circulations.Final segmental size is 533bp.

Order-checking.Using the fluorescently-labeled following primer of collecting to carry out Pharmacia (Upsala, Sweden) A.L.F. checks order: HBV-8ALF (5 '-CGTTCCACCAAACTCTTCAAG-3 '; SEQ ID NO 91), HBV-10ALF (5 '-CAAGGTATGTTGCCCGTTTGTCCTC-3 '; SEQ ID NO 92), HBV-12ALF (5 '-CCCATCCCATCATCTTGGGC-3 '; SEQ ID NO 93), HBV-14ALF (5 '-GGGTATGTAATTGGAAGTTGG-3 '; SEQ ID NO 94), HBV-3ALF (5 '-GCACTAGTAAACTGAGCCA-3 '; SEQ ID NO 95) or HBV-6ALF (5 '-AGTCTAGACTCGTGGTGGAC-3 '; SEQ ID NO 96).Adopt standard A .L.F. sequencing, and set the A.L.F. routine analyzer, suddenly change at the highest stringent condition (homology) scanning sequence.

Clonal analysis.2 μ l HBPr75-94 amplified productions (referring to embodiment 1) are connected in the pretreated pGem plasmid vector (Promega, Leusden, Holland), and are transformed in the competence Bacillus coli cells.Select single recombinant chou, and with high purity plasmid separating kit (Boehringer Mannheim, Brussels, Belgium) plasmid DNA purification.With or derive from the primer of described plasmid or the amplification of nested type HBV primer PCR insertion fragment from recombinant clone.Utilize LiPA to analyze all products.C. in the variability of HBV archaeal dna polymerase drug resistance codon 528,552 and 555

(at the 331st, 338,345,633,715,726,738,752,779 and 802 day) selects sample before lamivudine therapy, after the process neutralization, measures the sequence in zone between their the HBV polysaccharase amino acid (aa) 473 to 561.This district comprises territory B (HBV polysaccharase amino acid 508 to 530) and territory C (HBV polysaccharase amino acid 548 to 558).Than the 331st day sequence, observed variability from the 633rd day backward, detect M528 and V552, but from the 779th day backward, wild-type primitive L528 and M552 reappear (Fig. 5).Not observing other amino acid in these tracking samples changes.

All obtainable samples (Fig. 5 and Fig. 6) also on LiPA, have been analyzed.Just detected lamivudine resistant mutation (L 528 and M as compounding mixture as far back as the 524th day; M 552, V and I).Can not detect the I552 varient again at the 548th day, but keep all other mixture.Detected pure mutant from the 633rd day.This increase with the viral load amount is consistent.Until the 835th day, all detect the mixture of wild-type and mutant at two codon positions.At last, obviously had only pure wild-type virus at the 1041st day.Restarted lamivudine therapy at the 1105th day, and after 139 days, occur at codon 552 places wild-type and mutant strain compounding mixture (with the 524th day much at one).But because can not detect M528, so L528+M552 and L528+I552 combination are the selecteed combination of most probable (the 1244th days and the 1259th day).D. the virus in the antiviral therapy process develops: the quasispecies analysis

For the better evolution (Fig. 6) of understanding the codon position that described virus changes in early stage demonstration Nucleotide in described therapeutic process, carry out another cross experiment.Therefore, the LiPA probe that will merge (as before described in the embodiment 2) is now in addition gone up band separately, and with by the viral biotinylated amplicon incubation (Fig. 7) that obtains of described plasmid.The result has two significantly to find: the M528 and the V552 varient of selection (i) occur in the intermediate nucleotide sequence, and do not impact on amino acid levels; (ii) above-mentioned intermediate forms has been crossed in the disappearance of described mutated viruses.Develop into L (CTG) for codon 528: wild-type L (TTG), finally produce resistance varient M (ATG).After stopping the 3TC treatment, observe by mutant M (ATG) and return back to L (TTG) through L (CTG).For codon 552: 550 places observe intermediate forms at codon, occur Serine simultaneously and change (becoming AGT by AGC).Yet as V552 (GTG) when occurring, AGT disappears.With suitable with the treatment of termination 3TC subsequently,, temporarily detect the variability of codon 550 (AGT) simultaneously again in the answer of codon 552 places appearance to wild-type to the observation of codon 528.

For further describing these intermediate forms, the clone and further analyzes 54 single clones (Fig. 8) altogether from the HBV amplicon of the 442nd day, 468 days and 524 days in plasmid.Detect 6 kinds of clones, wherein have different primitive combinations with 552 places at codon 528,550.Before selecting the resistance primitive, the clone with L528 (CTG)+M552 (having AGT on 550) exists as important group.And, find two clones with M528+M552, show the intermediate forms that this single mutant group also is a double-mutant.Yet, can not find single mutant I552 among isolating 19 clones by the 524th day amplicon.Although this clones results abnormity, it clearly is present among the described group, as (line 11, the 524 days) as shown in Fig. 6.Embodiment 5: contrast is with the data of the LiPA acquisition of monitoring drug resistance with by order-checking

The data a. patient sample that obtains

Collect blood plasma or serum sample at following two centers: doctor S.Locarnini professor, research and molecule progress, Victorian Infectious Diseases Reference Laboratory, North Melboume, Australia (9 patients) and doctor A.S.F.Lok professor, Michigan University Medical Center, Ann Arbor, MI, the U.S. (9 patients).All patients are the HBV infected individuals under lamivudine therapy.First included sample must be consistent with ' baseline ' (just time point before the beginning lamivudine therapy).Second with the 3rd sample must be respectively with can to detect the viral load water gaging more consistent with time point afterwards before flat.For some patient, also obtained extra sequential sample.The b.DNA operation

Carry out HBV separation and amplification as mentioned above.Use standard method (Sambrook etc., 1989) to check order.As described in embodiment 2, using LiPA.C. result

Be displayed in Table 8 and used LiPA and order-checking analysis in the possible sudden change in codon 528 places.For most of samples, data that obtain with LiPA and the data that obtain by order-checking are consistent: 44 samples show and have wild-type (L) virus, and there is mutant (M) virus in 23 samples demonstrations.In 15 samples, LiPA detects wild-type/mutant (L/M) mixture, and order-checking only detects described mutant (M).In 1 sample, LiPA shows wild-type/mutant (L/M) mixture, and order-checking only detects wild-type (L).

Be displayed in Table 9 and used LiPA and order-checking analysis in the possible sudden change in codon 552 places.For most of samples, data that obtain with LiPA and the data that obtain by order-checking are consistent: 45 samples show and have wild-type (M) virus, and 17 samples show and have mutant (V) virus, and there is mutant (I) virus in 8 samples demonstrations.In 1 sample, LiPA detects wild-type/mutant (M/V) mixture, and order-checking only detects described mutant (V).In 2 samples, LiPA detects wild-type/mutant (M/V) mixture, and order-checking only detects described wild-type (M).In 4 samples, LiPA detects wild-type/mutant (M/I) mixture, and order-checking only detects described mutant (I).In 2 samples, LiPA detects mutant V/I, and order-checking only detects described mutant V.

The analysis to carrying out in the possible sudden change in codon 555 places with LiPA and order-checking is presented at and has wild-type codon V555 in all samples.

Can know from this research and to see that data that obtain with the LiPA of monitoring drug resistance and the data that obtain by order-checking are consistent.In addition, detect the mixed sequence of comparatively high amts at codon 528 and/or 552 places with LiPA, and do not detect these mixtures with order-checking, this shows that forcefully LiPA has than hypersensitivity.

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Claims (16)

1. at least a by among sudden change L528M, M522V/I in the HBV virus strain archaeal dna polymerase of gene test in being present in patient's biological sample and/or the V/L/M555I, the method of the anti-HBV drug resistance of monitoring in described patient, this method may further comprise the steps:

When (i) needing, discharge, separate and/or be concentrated in the polynucleotide that exists in the described biological sample;

When (ii) needing, with HBV dna polymerase gene or its part of at least one suitable primer to increasing in described biological sample;

The polynucleotide of (iii) will be and/or obtaining (ii) in step (i) and at least a can with the target sequence in the HBV dna polymerase gene or the probe hybridization of its complement specific hybrid, described target sequence is selected from a plurality of target sequences that are shown in Fig. 1;

(iv) detect the crossbred that forms in (iii) in step;

(v) infer the possible anti-HBV drug resistance that in described archaeal dna polymerase, has or do not exist described L528M, M522V/I and/or V/L/M555I sudden change and be present in the HBV virus strain in the described biological sample by the hybridization signal that obtains in (iv) in step.

2. according to the method for claim 1, the feature of this method also is to be selected from table 1,2 and/or 3 at the probe that step is used in (iii), wherein:

-be selected from following with the probe of L528M target sequence specific hybrid: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468, HBPr468-1 (SEQ ID NO 1 to SEQ ID NO 10);

-be selected from following with the probe of M552V/I target sequence specific hybrid: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433, HBPr433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488 (SEQ ID NO 11 to SEQ ID NO 29 or SEQ ID NO 97 to SEQ ID NO100);

-be selected from following with the probe of V/L/M555I target sequence specific hybrid: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBPr492, HBPr494, HBPr495, HBPr496 (SEQ ID NO 30 to SEQ IDNO 40, SEQ ID NO 101 to SEQ ID NO 106).

3. according to each method in the claim 1 to 2, the feature of this method is that also at the probe that step is used in (iii) be at least a in the following probe combinations:

-be used to detect the probe of L528M sudden change: HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468 and HBPr468-1;

-be used to detect the probe of M552V/I sudden change: HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433, HBPr433-1, HBPr465, HBPr456, HBPr380 and HBPr453;

-be used to detect the probe of V/L/M555I sudden change: HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299 and HBPr419.

4. according to each method in the claim 1 to 3, the feature of this method also is to be selected from following (table 5): HBPr134, HBPr135, HBPr135A, HBPr135B, HBPr75, HBPr94, HBPr94A, HBPr94B, HBPr94C and/or HBPr105 (SEQ ID NO 41 to SEQ ID NO50) in the primer sets that step is used in (ii).

5. according to the method for claim 4, the feature of this method is that also described primer sets is made up of following two kinds of primers:

-as the HBPr134 of forward primer with as the HBPr135 of reverse primer; And/or

-as the HBPr75 of forward primer with as the HBPr94 of reverse primer; And/or

-as the HBPr75 of forward primer with as the HBPr105 of reverse primer.

6. according to each method in the claim 1 to 5, the feature of this method is that also a step detects two kinds of sudden change L528M and M552V/I.

7. according to each method in the claim 1 to 5, the feature of this method is that also a step detects two kinds of sudden change L528M and V/L/M555I.

8. according to each method in the claim 1 to 5, the feature of this method is that also a step detects two kinds of sudden change M552V/I and V/L/M555I.

9. according to each method in the claim 1 to 5, the feature of this method is that also a step detects three kinds of sudden change L528M, M552V/I and V/L/M555I.

10. according to each method in the claim 1 to 9, the HBV virus strain that the feature of this method also is to be present in the described biological sample shows anti-lamivudine, Famciclovir and/or Penciclovir.

11. as in each of claim 1 to 10 defined probe, be used for monitoring patient's antiviral resistance and be used for sudden change L528M, M552V/I and the V/L/M555I of gene test in being present in the HBV virus strain archaeal dna polymerase of described patient's biological sample.

12. comprise the composition of at least a probe that in claim 11, defines.

13. be used for purposes in external monitoring patient antiviral resistance according to the probe compositions of claim 12.

14. diagnostic kit, be used for by gene test in the HBV archaeal dna polymerase of the HBV virus strain that is present in patient's biological sample sudden change L528M, M552V/I and/or V/L/M555I at least a, monitor antiviral resistance among the described patient, described diagnostic kit comprises following component:

(i) under the suitable situation, be used for discharging, separating or concentrate the material of the polynucleotide that is present in described biological sample;

Under the (ii) suitable situation, at least one primer that is fit to is right;

(iii) at least a probe according to claim 11 may be fixed on the solid support;

(iv) hybridization buffer or produce the essential component of described damping fluid;

(v) washing soln or produce the essential component of described solution;

(under the vi) suitable situation, be used to detect the material of the crossbred that produces by aforementioned hybridization;

(under the vii) suitable situation, be used for described probe is attached to the material of the known location on the solid support.

15. line formula probe assay (1ine probe assay), be used for by gene test in the HBV archaeal dna polymerase of the HBV virus strain that is present in patient's biological sample sudden change L528M, M552V/I and/or V/L/M555I at least a, monitor antiviral resistance among the described patient, described line formula probe assay comprises following component:

(i) under the suitable situation, be used for discharging, separating or concentrate the material of the polynucleotide of the biological sample that is present in described patient;

Under the (ii) suitable situation, at least one primer that is fit to is right;

(iii) at least a be shown in L528M target sequence probe specific hybrid, that be fixed in solid support of Fig. 1, be selected from HBPr270, HBPr293, HBPr294, HBPr412, HBPr274, HBPr355, HBPr415, HBPr461, HBPr468; And/or

(iv) at least a be shown in M552V/I target sequence probe specific hybrid, that be fixed in solid support of Fig. 1, be selected from HBPr308, HBPr322, HBPr349, HBPr478, HBPr309, HBPr318, HBPr426, HBPr427, HBPr463, HBPr315, HBPr363-1, HBPr407, HBPr488, HBPr433-1, HBPr465, HBPr456, HBPr380, HBPr453, HBPr485, HBPr486, HBPr487, HBPr488; And/or

(v) at least a be shown in V/L/M555I target sequence probe specific hybrid, that be fixed in solid support of Fig. 1, be selected from HBPr279, HBPr338, HBPr341, HBPr345, HBPr474, HBPr332, HBPr328, HBPr385, HBPr289, HBPr299, HBPr419, HBPr490, HBPr491, HBPr492, HBPr494, HBPr495, HBPr496;

(vi) hybridization buffer or produce the essential component of described damping fluid;

(vii) washing soln or produce the essential component of described solution;

(under the viii) suitable situation, be used to detect the material of the crossbred that produces by aforementioned hybridization.

16. be included in described in Fig. 1 sequence (SEQ ID NO 50-86 and SEQ IDNO 107-109) or by its isolating nucleic acid of forming or its fragment, wherein said fragment is made up of 10 successive Nucleotide that are shown in Fig. 1 at least, and comprises a kind of in the amino acid 528,552 of coding HBV archaeal dna polymerase or 555 the codon.

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